Note: Descriptions are shown in the official language in which they were submitted.
CA 02249103 1998-09-30
B&P File No. 9157-004/MG
Title: Novel Hyaluronic Acid Produced from Algae
BACKGROUND OF THE INVENTION
Technical Field
This invention relates to a novel hyaluronic acid and the
production of the hyaluronic acid from algae.
Brief Description of the Prior Art
Algae have been used as culturable sources of biomedically
useful compounds (Parker, Bruce C. (ed.). Journal of Applied Phycology, 6:
10 pp. 91-98 (1994)). The synthesis of such compounds, which may be
secondary metabolites, depends on the culture growth conditions. If
optimal growth conditions with respect to nutrient availability, pH vaIue,
irradiance, light quality, aeration and temperature are maintained,
microalgal cells double or quadruple their cellular components and
15 divide. This can result in the continuous production of healthy growing
and reproductive generations, as demonstrated in Chlamydomonas segnis
Ettl (Badour, S.S., et al., Canadian Journal of Botany, 51:67-72 (1973); Foo,
S.K., and Badour, S.S. Canadian Journal of Botany, 55:2178-2185 (1973);
Badour, S.S., et al. Journal of Phycology, 13:80-86 (1977); Badour, S.S.
20 Journal of Phycology, 17:293-299 (1981); Harris, Elizabeth (ed). The
Chlamydomonas source book, Academic Press Inc. (1989)~.
In nature, numerous microalgae have been shown to
produce mucopolysaccharide cell coverings in the form of highly
hygroscopic capsules (Kinzel, Helmut. Unterschungen uber die Chemie
25 und Physikochemie der Gallertbildungen von SuI~wasser algen.
Ostereichische Botanische Zeitschrift, 100:25-79 (1953)). These capsules are
metachromatic, i.e. they exhibit a colour different from that of the basic
dye (e. g. toluidine blue) used to stain them, because they are negatively
charged. The precise culture conditions that induce the formation of
30 capsules and the production of mucopolysaccharides in green microalgae,
however, have never been specified (Badour, S.S. Excess light evokes cell
encapsulation in Chlamydomonas segnis Ettl. Abstracts of the
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International Meeting on Molecular Biology, Biochemistry and Physiology
of Chloroplast Development. Philipps Universitat, Marburg, Germany
(1995); Allard, B., and Tazi, A. Phytochemistry, 32:41-47 (1993); Starr, R.C.,
and Zeikus, J.A. Journal of Phycology (Supplement) 23: 8(1987)).
ChZamydomonas species are haploid, biflagellate unicellular
organisms. Chlamydomonas segnis has been used as an experimental
organism by Badour et al, who have described its morphology and
ultrastructure, and studied its growth physiology, biochemistry and
photobiology (e. g. Badour, S.S., et al., Canadian Journal of Botany,
10 51:67-72 (1973); Foo, S.K., and Badour, S.S. Canadian Journal of Botany,
55:2178-2185 (1973); Badour, S.S., et al. Journal of Phycology, 13:80-86 (1977);Badour, S.S. Journal of Phycology, 17:293-299 (1981); Badour, S.S. et al.,
Journal of Phycology (supplement) 8:16 (1972); Badour, S.S., and Irvine,
B.R. Botanica Acta, 103: 149-154 (1990); Badour, S.S., and Tan, C.K.
15 Zeitschrift fur Pflanzenphysiologie, 112: 287-295 (1983); Badour, S.S., and
Tan, C.K. Plant and Cell Physiology, 28: 1485-1492 (1987); Badour, S.S.
Ribulose-bis-phosphate carboxylase in Chlamydomonas. In: Handbook of
Phycological Methods. Vol. II, pp. 209-216. Edited by Hellebust, J.A. and
Craigie, J.S. Cambridge University Press (2978)).
Hyaluronic acid, also known as hyaluronan, is a
mucopolysaccharide made up of alternating 1,4-linked residues of
hyalobiuronic acid, forming a gelatinous material. Hyaluronic acid is a
naturally occuring heteropolysaccharide consisting of alternating residues
of D-glucuronic acid and N-acetyl-D-glucosamine. It is a linear polymer of
25 high molecular weight, up to about 8 to 13 million.
Hyaluronic acid is used as a fluid replacement to correct
pathological conditions in the eye and in the joint, and has been used to
facilitate wound protection and healing (Urman, B. and Gomel, V., Fertil.
Steril. 56(3):568-570, (1991); Avid, A.D. and Houpt, J.B., J. Rheumatol.
30 21(2):297-301 (1994); and Adams, M.K. et al., Osteoarthritis Cartilage
3(4):213-225 (1995); King et al., in Surgery 109:76-84, (1991)). Hyaluronic
acid and fractions thereof have been used in ophthalmic surgery or as
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therapeutic, auxilliary and substitutive agents for natural organs and
tissues. It has been found to be particularly useful in the keatment of
arthropathies, wound healing and cystisis. Hyaluronic acid is also used as
a gum in food industry and pharmaceuticals, and as a source of mucilage
in cosmetics and skin-care products (See for example U.S. Patents Nos.
4,141,973 and 5,442,053 reviewing the technical literature describing
isolation, characterization and uses of hyaluronic acid).
Hyaluronic acid and its salts, hereafter collectively referred to
as hyaluronic acid, has been obtained from various sources including
10 human umbilical cords, rooster combs, bovine joints, whale cartilages and
certain bacterial cultures, such as various Streptococcus, as well as
Pasteurella multocida and Pseudomonas aeruginosa. Various methods of
culturing microorganisms to prepare hyaluronic acid have been taught
(see for example: Japanese laid-open patent application No. 58-56692; Kjem
15 and Lebech, Acta. Path. Microbiol. Scand. Sect. B. 84:162-164, 1976). For
example, enriching oxygen in the culture medium (U.S. Patent No.
4,897,394), inhibiting hyaluronidase in the microorganisms (U.S. Patent
No. 4,782,046), or adding aromatic compounds to the culture medium
(U.S. Patent No. 4,885,244), have been described to facilitate the yield of
20 hyaluronic acid from microorganims.
However, the above-mentioned methods of extracting high
hyaluronic acid from animal tissues or microorganisms have various
disadvantages, making it difficult to obtain hyaluronic acid effectively and
in abundance.
For example, hyaluronic acid present in animal tissues is
present only in trace amounts, and it forms a complex with proteins or
other mucopolysaccharides. According to Dorfman and Cifonelli,
Methods in Enzymology, III, pgs 20-27 (1957), the various procedures
employed in the preparation of acid mucopolysaccharides from
30 mammalian tissues involve the following basic steps: (1) extraction, (2)
removal of protein, (3) precipitation, and (4) final purification.
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Complicated and delicate purification and extraction processes may
degrade the hyaluronic acid.
Purifying hyaluronic acid from cultured microorganisms
which are capable of producing hyaluronic acid has an advantage in that
the purification process tends to be simpler than the aforementioned
methods for extraction from animal tissues. This is because a protein-free
medium may be used for culturing microorganisms to obtain hyaluronic
acid. However, these methods have a disadvantage that the amount of
hyaluronic acid produced per culture volume is low, and the use of
10 bacteria can give rise to the need for the removal of toxins as pointed out
in the Biology of Hyaluronan, Ciba Foundation Symposium, 143, pgs
265-280(1989).
In view of the above, there is a need for a non-bacterial and
non-animal source for production of a hyaluronic acid or hyaluronic acid-
like compound in order to reduce purification costs associated withpreparing hyaluronic acid by bacteria or by the extracellular matrix of
tissues from animals.
SUMMARY OF THE INVENTIC)N
The present invention relates to a novel hyaluronic acid
derived from a green alga, termed chlamyhyaluronic acid, or
chlamyhyaluronan, and a method for preparing same.
Broadly stated, the present invention provides an isolated
chlamyhyaluronic acid produced by a green alga.
The chlamyhyaluronic acid may be characterized by having
various properties. In one embodiment, a solution of the sodium salt of
chlamyhyaluronic acid has (a) has an acetyl group peak of about 1.7599
ppm on a 500 MHz nuclear magnetic resonance spectra when in a solution
with deuterium oxide at 300K; (b) has carbohydrate peaks of about 3.7052
ppm and about 3.6008 ppm on a 500 MHz nuclear magnetic resonance
spectra when in a solution with deuterium oxide at 300K; (C) forms a
complex with bivalent copper and said complex shows an absorption band
at 232 nm; (d) develops turbidity with serum at an acid pH and forms
CA 02249103 1998-09-30
stable colloidal suspensions; and (e) is degraded by the specific enzyme
hyaluronate lyase (EC 4.2.2.1) from Streptomyces hyalurolyticus as well as
by hyaluronidase (EC 3.2.1.35) from bovine testes.
The present invention further comprises algal cell
encapsulations containing chlamyhyaluronic acid.
The present invention also provides a method for the
production of chlamyhyaluronic acid comprising culturing an alga of the
phylum Chlorophyta under stressful conditions for a period of time
sufficient for the alga to produce extracellular capsules containing
10 chlamyhyaluronic acid.
The term "stressful conditions" as used herein means
conditions that alter the normal growth of the algae and causes the cells to
divert from the regular metabolic pathways to adjust for survival. Certain
cells, such as algal cells described herein, produce extracellular protective
15 structures or capsules in response to stressful conditions.
The term "capsule" as used herein means a layer or layers of
mainly mucopolysaccharides external to but contiguous with the algal cell
wall. Such capsules may reach 50 fold to over 200 fold of the algal cell
volume. These capsules contain compounds that protect the cells from
20 excessive light (particularly blue light and UV), catalyze the breakdown of
oxygen free radicals, and stop invasion by predators. The present inventor
has found that chlamyhyaluronic acid can be a major component of these
capsules, and in particular can be a major component of Chlamydomonas
segnis capsules.
These capsules can be extracted from the cultures in the form
of partially dried thin layers of caked encapsulated Chlamydomonas
segnis Ettl (CENC). Because of its hygroscopic nature, CENC may impart
moisture to the skin and protect it from reactive oxygen radicals due to its
chlamyhyaluronic acid and catalase contents.
In one embodiment, an alga of the phylum Cholorphyta is
cultured at a temperature of from about 15~C to about 35~C, and exposed to
excessive light. In response to the stress induced by the excessive light, the
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chlorophyte produces capsules containing chlamyhyaluronic acid, for the
purposes of protecting the organism from such light.
The term "excessive light" as used herein means the light
intensity, i.e., irradiance, at which the inception of cell encapsulation in
5 algal cultures exposed to air, aerated with either air or air enriched with
carbon dioxide, is detectable by metachromasy.
In a preferred embodiment, the excessive light has a blue:red
spectral ratio of higher than one. The term "blue light" as used herein
means irradiances with spectral range between 320 nm and 500 nm which
10 includes bluish green light, blue light, violet light and ultraviolet A of 320
nm to 380 nm. The term "red light" as used herein means irradiances
with a spectral range from 600 nm to 700 nm and does not include far red
light.
In another embodiment, stressful conditions are imposed on
15 the algae to facilitate the production of chlamyhyaluronic acid by the use
of boron deficient nutrient medium. In another embodiment, stressful
conditions are imposed on the algae to facilitate the production of
chlamyhyaluronic acid by the use of low salt (2-6 mM) nutrient medium.
In another embodiment, stressful conditions are imposed on the algae to
20 facilitate the production of chlamyhyaluronic acid by the use of the
application of carbon dioxide enriched air (0.1% to 5% CO2 by volume),
during the exponential phase of growth and the encapsulation phase.
In one embodiment of the invention, the alga selected for
culture is of the genus ChZamydomonas. In a preferred embodiment, the
25 alga selected is Chlamydomonas segnis. In a further preferred
embodiment, the alga is Chlamydomonas segnis Ettl. The alga may be
obtained from the stock cultures of UTEX, culture No. 1919.
In an embodiment of the invention the algal cells are
cultured to produce large extracellular capsules in mass cultures generally
30 within a relatively short period of time. Under specified physical and
cultural conditions described herein, the photoautotrophic growth of algae
capable of producing chlamyhyaluronic acid can be regulated. As a result,
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immobile encapsulated cells with high yields of chlamyhyaluronic acid in
their capsules may be obtained.
The present invention further comprises the method for
extracting the chlamyhyaluronic acid from the culture. A partially
5 purified chlamyhyaluronic acid may be obtained from the extracellular
capsules of the alga. The extracted algal chlamyhyaluronic acid may be
partially purified for use as substitute or supplement for animal and
bacterial hyaluronic acid, which has well established therapeutic and
cosmetic utility.
The present invention also provides pharmaceutical
compositions comprising chlamyhyaluronic acid. The pharmaceutical
composition can be used in various applications where hyaluronic acid is
normally used. For example, the composition can be used as a fluid
replacement to correct pathological conditions in the eye and in the joint,
to facilitate wound protection and healing, as therapeutic, auxiliary and
substitutive agents for natural organs and tissues, particularly in the
treatment of arthropathies, ophthalmic conditions, and cystitis. The
pharmaceutical composition can also be used in the cosmetic industry for
example in skin-care products. Additionally, the invention includes a
composition comprising chlamyhyaluronic acid for use in the food
industry, for example as a gum.
The present invention further comprises the use of the algal
cell encapsulations containing chlamyhyaluronic acid as a compound in a
topical preparation to protect skin from reactive oxygen radicals or to
moisturize skir .
BRIEF DES~RIPTION OF THE DRAWINGS
Figure 1 depicts the changes in the levels of blue, red, and far
red spectra with increasing irradiances from a combination of cool-white
fluorescent and standard incandescent lamps.
Figure 2 shows the absorbance spectra of acetone extracts from
shallow cultures of ChZamydomonas segnis Ettl, at (a) late exponential
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phase of growth, (b, c and d) early and mid encapsulation phase, and (e)
late encapsulation phase.
Figure 3 shows the growth curves of deep cultures of
Chlamydomonas segnis Ettl produced in the low salt boron-deficient
medium, and in the high salt nutrient medium but without adding boric
acid.
Figure 4 shows photomicrographs of encapsulated cells of
Chlamydomonas segnis Ettl at the end of encapsulation phase, stained
either with toluidine blue (a and b) or alcian blue (c).
Figure 5 depicts growth curves of Chlamydomonas segnis Ettl
in shallow cultures.
Figure 6 depicts the relationship between the dilution index
and the level of irradiance required for the production of encapsulated
algal biomass.
Figure 7 shows photomicrographs of Chlamydomonas segnis
Ettl stained with toluidine blue: (a) as capsule free cells at mid exponential
phase of growth, (b) as cells with metachromatic extracellular matrix at the
beginning of encapsulation, (c) as encapsulated cells with polymorphic
capsules, and (d) as spaced cells due to capsule enlargement.
Figure 8 shows transmission electron micrographs, of
Chlamydomonas segnis Ettl cell sections; (a) at mid exponential phase of
growth showing a chloroplast with interthyalkoidal starch grains in a
capsule free cell, and (b) starch sheath around the pyrenoid, (c) at late
encapsulation phase showing the stratified structure of the capsule
25 surrounding a cell with large starch grains within a fenestrated chloroplast
and large dark lipid droplets, and (d) the relative thickness of the capsule.
Figure 9 shows scanning electron micrographs of desiccated
cells of Chlamydomonas segnis Ettl at mid-encapsulation phase, (a) coated
with thick mantles of mucopolysaccharides, (b) in a fractured layer of dried
30 extracellular mucopolysaccharide material released in the culture
medium, and (c and d) embedded in the thick matrix of this material.
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Figure 10 shows photomicrographs of: (a) immobilized spaced
cells of Chlamydomonas segnis Ettl at mid encapsulation phase, (b) the
autofluorescence of the capsule material and (c) encapsulated cells stained
with alcian blue, or (d) toluidine blue to visualize the capsule.
Figure 11 depicts the retarding effect of the presence of one
,uM boric acid (+ Boron) on the inception of the encapsulation phase.
Figure 12 depicts the inhibitory effect of aerating
Chlamydomonas segnis Ettl cells with 5% CO2 in N2 instead of 5% CO2 in
air (volume by volume) on the inception of the encapsulation phase.
Figure 13 shows photomicrographs of: (a) a sample of caked
encapsulated Chlamydomonas segnis (CENC) homogenate, (b) a two-gram
slightly moist pellet of CENC, (c) the size increase of the moist pellet of
CENC during water imbibition, and (d) three samples of CENC during
hydration exhibiting colour shades.
rigure 14 depicts the apparent viscosity of ;% C~NC
homogenate in water, compared to 1% solution of either xanthan or guar
in water, as a function of shear rate.
Figllre 15 shows photomicrographs of samples from CENC
homogenate stained with toluidine blue, (a) before extraction, (b) after
20 extraction with the buffered saline solution, and (c) exhibiting the
acapsular appearance of CENC cells after extraction.
Figure 16 depicts the increase in absorption around 230 nm of
chlamyhyaluronic acid after incubation with about 70 units of hyaluronate
lyase (upper graph), compared to a control sample without the said
25 enzyme (lower graph).
Figure 17 depicts the increase in absorption around 230 nm of
chlamyhyaluronic acid after incubation with about 980 units of
hyaluronidase (upper graph), compared to a control sample without the
enzyme (lower graph).
Figure 18 shows a print-out of a cellulose thin layer
chromatogram of the products formed by the degradation of
chlamyhyaluronic acid (c and d), and hyaluronic acid from human
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- 10 -
umbilical cord (e and fl with hyaluronate lyase at 37~C for six hours, and
the absence of such products (a and h) or their reduced level (b and g) in
samples without enzyme or with heated enzyme, respectively.
Figure 19 shows increases in the degradation products
indicated in Figure 18, as a result of extending the incubation period to
twelve hours at 37~C.
Figure 20 shows a print-out of a chromatogram produced as
described in Figure 18, except that hyaluronidase is the enzyme used.
Figure 21 depicts the reduced viscosity of chlamyhyaluronic
10 acid (potassium salt) as a function of concentration, using a Ubbelohde
viscometer, and the intrinsic viscosity is determined by extrapolating to
zero concentration with an asymptotic model.
Figure 22 depicts the reduced viscosity of hyaluronic acid
(sodium salt) from human umbilical cord, as a function of concentration.
15 DETAILED DES~RIPTION OF THE INVENTION
As hereinbefore mentioned, the present invention relates to
a novel hyaluronic acid derived from a green alga, termed
chlamyhyaluronic acid, and a method for its production.
Chlamyhyaluronic Acid
In one aspect, the present invention provides isolated
chlamyhyaluronic acid. The present inventor extracted, partially purified,
and identified the capsular metachromatic mucopolysaccharide in the
caked encapsulated Chlamydomonas segnis Ettl. The partially purified
mucopolysaccharide (referred to as Chlamyhyaluronic acid) was found to
25 depolymerize in a similar fashion as the hyaluronic acid from human
umbilical cord by the action of hyaluronate-lyase from Steptomyces
hyalurolyticus (EC 4.2.2.1), as well as hyaluronidase from bovine testes
(EC3.2.1.35). Chlamyhyaluronic acid is metachromatic, ultraviolet-
absorbent, and gives fairly stable colloidal suspensions, i.e. turbidity, when
30 mixed with serum albumin at pH 4. The turbidity is reduced or prevented
upon depolymerization by either one of the aforenamed enzymes, with
simultaneous production of reducing low molecular weight molecules
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- 11 -
and increase in absorbance at 230 nm. The products of chlamyhyaluronic
acid degradation, visualized on thin layer chromatograms match those
from human umbilical cord hyaluronic acid.
The chlamyhyaluronic acid may be characterized by having
5 various properties. In one embodiment, a solution of the sodium salt of
chlamyhyaluronic acid (a) has an acetyl group peak of about 1.7599 ppm on
a 500 MHz nuclear magnetic resonance spectra when in a solution with
deuterium oxide at 300K; (b) has carbohydrate peaks of about 3.7052 ppm
and about 3.6008 ppm on a 500 MHz nuclear magnetic resonance spectra
10 when in a solution with deuterium oxide at 300K; (c) forms a complex
with bivalent copper and said complex shows an absorption band at 232
nm; (d) develops turbidity with serum at an acid pH and forms stable
colloidal suspensions; and (e) is degraded by the specific enzyme
hyaluronate lyase (EC 4.2.2.1) from Streptomyces hyalurolyticus as well as
15 by hyaluronidase (EC 3.2.1.35) from bovine testes.
In another aspect, the present invention provides capsules
containing chlamyhyaluronic acid.
The term "capsule" as used herein means a layer or layers of
mainly mucopolysaccharides external to but contiguous with the algal cell
20 wall. Such capsules may reach 50 fold to over 200 fold of the algal cell
volume, due to the high water content of the capsule.
Capsules such as those produced by Chlamydomonas segnis
Ettl are easily visible by light microscopy after staining wet smears from
liquid cultures or agar slants with a metachromatic dye, i.e. they are
25 chromotropes. These capsules contain compounds that protect the cells
from excessive light (particularly blue light and UV), catalyze the
breakdown of oxygen free radicals, and stop invasion by predators. The
present inventor has found that chlamyhyaluronic acid can be a major
component of these capsules, and in particular can be a maJor component
30 of Chlamydomonas segnis capsules. In contrast, well characterized
Chlamydomonas reinhardtii (Harris, Elizabeth (ed). The Chlamydomonas
source book, Academic Press Inc. (1989)), which is widely used in genetic,
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cytological and biochemical research, does not produce capsules under the
stressful cultural conditions designed for Chlamydomonas segnis.
These capsules can be extracted from the cultures in the form
of partially dried thin layers of caked encapsulated Chlamydomonas segnis
5 Ettl (CENC). Because of its hygroscopic nature, CENC may impart
moisture to the skin and protect it from reactive oxygen radicals due to its
chlamyhyaluronic acid and catalase contents.
Preparation of Chlamyhyaluronic Acid
In another aspect, the present invention provides a method
10 for the production of chlamyhyaluronic acid comprising culturing an alga
capable of providing extracellular capsules containing chlamyhyaluronic
acid under stressful conditions for a period of time sufficient for the alga to
produce extracellular capsules containing chlamyhyaluronic acid.
The term "stressful conditions" as used herein means
15 conditions that alter the normal growth of the algal cells and causes the
cells to divert from the regular metabolic pathways to adjust for survival.
For photoautotrophic alga, stressful conditions include culture conditions
under which algal cultures at the lag phase, exponential phase, or
stationary phase of photoautotrophic growth are unable to utilize all the
20 absorbed irradiance for biosynthetic activity directed to cell growth and
division, but are capable of using the excessive irradiance to synthesize
protective secondary metabolites for preserving cell viability. Examples of
stressful conditions include nutrient deficiency, low or high nutrient
concentration, increased acidity or alkalinity, dim or excessive light with
25 unbalanced spectral coll.position, suboptil..al or relatively high
temperature and/or carbon dioxide tension. These conditions force the
cells to adjust for survival and divert the regular metabolic pathways to
biosynthetic routes that lead to the active production of secondary
metobolites in order to protect the cells from the harmful effects of the
30 stressful environment. Certain cells, such as algal cells described herein,
produce extracellular protective structures or capsules in response to
stressful conditions.
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In one embodiment, an alga of the phylum Cholorphyta is
cultured at a temperature of from about 15~C to about 35~C, and exposed to
excessive light. In response to the stress induced by the excessive light, the
chlorophyte produces capsules containing a secondary metabolite, for the
purposes of protecting the organism from such light. The present
inventor has found that this secondary metabolite is chlamyhyaluronic
acid.
The term "excessive light" as used herein means the light
intensity, i.e., irradiance, at which the inception of cell encapsulation in
10 algal cultures exposed to air, aerated with either air or air enriched with
carbon dioxide, is detectable by metachromasy.
In a preferred embodiment, the excessive light has a blue:red
spectral ratio of higher than one. The term "blue light" as used herein
means irradiances with spectral range between 320 nm and 500 nm which
includes bluish green light, blue light, violet light and ultraviolet A of 320
nm to 380 nm. The term "red light" as used herein means irradiances
with a spectral range from 600 nm to 700 nm and does not include far red
light.
In another embodiment, stressful conditions are imposed on
the alga to facilitate the production of chlamyhyaluronic acid by the use of
boron deficient nutrient medium. In another embodiment, stressful
conditions are imposed on the alga to facilitate the production of
chlamyhyaluronic acid by the use of low salt (2-6 mM) nutrient medium.
In another embodiment, stressful conditions are imposed on the alga to
facilitate the production of chlamyhyaluronic acid by the use of the
application of carbon dioxide enriched air (0.1% to 5% C ~2 by volume),
during the exponential phase of growth and the encapsulation phase.
The alga used to prepare the chlamyhyaluronic acid includes
the species Tetraspora, Gloeomonas, Chlamydomonas segnis,
Chlamydomonas gymnogama, Chlamydomonas pallidostigmatica,
Chlamydom onas augustae, Chlamydom onas intermedia,
Chlamydomonas sajao Levin and Chlamydomonas corrosa. These are
CA 02249l03 l998-09-30
-14-
eukaryotic unicellular green algae containing chlorophyll a and b, with 2
flagella (motile) or without flagella (non-motile), or colonial (coenobia),
possessing a pyrenoid or pyrenoids associated with the chloroplast and
storing starch.
In one embodiment of the invention, the alga is selected
from the genus Chlamydomonas. In a preferred embodiment, the alga is
Chlamydomonas segnis. In a further preferred embodiment, the alga is
Chlamydomonas segnis Ettl. The alga may be obtained from the stock
cultures of UTEX, culture No. 1919.
In an embodiment of the invention the algal cells are
cultured to produce large extracellular capsules in mass cultures generally
within a relatively short period of time. Under specified physical and
cultural conditions described herein, the photoautotrophic growth of algae
capable of producing chlamyhyaluronic acid can be regulated. As a result,
15 immobile encapsulated cells with high yields of chlamyhyaluronic acid in
their capsules may be obtained.
The present invention further comprises the method for
extracting the chlamyhyaluronic acid from the culture. A partially
purified chlamyhyaluronic acid may be obtained from the extracellular
20 capsules of the algae.
Applications of Chlamyhyaluronic Acid
The chlamyhyaluronic acid of the invention may be used in a
variety of applications, including applications for which hyaluronic acid is
known to have utility.
For example, chlamyhyaluronic acid may be used in
pharmaceutical applications, including use in wound treatment, replacing
or supplementing biological fluids, such as fluids in the eye or the joints,
retarding cancer development, relieving pain, or treating cystitis.
Chlamyhyaluronic acid also has cosmetic applications, including use as an
30 ultra-violet ray screening agent or as a moisturizer.
Further, some applications of chlamyhyaluronic acid may be
utilized without isolating chlamyhyaluronic acid from its cellular
CA 02249103 1998-09-30
capsules. Caked encapsulated algae containing chlamyhyaluronic acid
may be used in some therapeutic or industrial, pharmaceutical or cosmetic
applications which do not require a high degree of purity of
chlamyhyaluronic acid. On hydration, cell encapsulations containing
5 chlamyhyaluronic acid produces highly viscous solutions with rheological
properties comparable to that described for high polymeric hyaluronic
acids used for medical, pharmaceutical, or cosmetic purposes.
The use of chIamyhyaluronic acid or cell encapsulations
containing chlamyhyaluronic acid as a non-bacterial and non-animal
10 source for production of hyaluronic acid-like agents may reduce
purification costs by eliminating the need for the removal of toxins from
bacterial capsules as pointed out in the Biology of Hyaluronan, Ciba
Foundation Symposium, 143, pgs 265-280 (1989), or for the fermentation of
various components associated with hyaluronic acid in the extracellular
15 matrix of tissues from human, bovine or ovine organs. This is because
the algal capsule components may be almost completely extracted by saline
phosphate buffer solutions, leaving intact cells inside ghost capsules.
The novel algal hyaluronic acid extracted by the method
described herein may have a range of molecular weights and may not be
20 ultrapure. Its molecular weight can be increased to match the commercial
animal or bacterial hyaluronic acid, by improving the extraction
methodology to increase the yield of the high molecular weight fraction in
cell encapsulations containing chlamyhyaluronic acid extracts. Moreover,
the physicochemical properties of cell encapsulations containing
25 chlamyhyaluronic acid extract can be modified for medical and cosmetic
purposes.
(1) Pharmaceutical Compositions
The chlamyhyaluronic acid of the invention and capsules
containing the chlamyhyaluronic acid may be formulated into
30 pharmaceutical compositions for adminstration to subjects in a
biologically compatible form suitable for administration in vivo. By
biologically compatible form suitable for administration is meant a form
CA 02249l03 l998-09-30
-16-
of the substance to be administered in which any toxic effects are
outweighed by the therapeutic effects. The substances may be
administered to living organisms including humans, and animals in a
therapeutically effective amount. Administration of a therapeutically
5 effective amount of the pharmaceutical compositions of the present
invention is defined as an amount effective, at dosages and for periods of
time necessary to achieve the desired result. For example, a
therapeutically effective amount of a substance may vary according to
factors such as the disease state, age, sex, and weight of the individual, and
10 the ability of peptide to elicit a desired response in the individual. Dosage regime may be adjusted to provide the optimum therapeutic response.
For example, several divided doses may be administered daily or the dose
may be proportionally reduced as indicated by the exigencies of the
therapeutic situation.
The active substance may be administered in a convenient
manner such as by topical or transdermal application, injection
(subcutaneous, intravenous, etc.), oral administration, inhalation, or rectal
administration. Depending on the route of administration, the active
substance may be coated in a material to protect the compound from the
20 action of enzymes, acids and other natural conditions which may
inactivate the compound.
The compositions described herein can be prepared by per se
known methods for the preparation of pharmaceutically acceptable
compositions which can be administered to subjects, such that an effective
25 quantity of the active substance is combined in a mixture with a
pharmaceutically acceptable vehicle. Suitable vehicles are described, for
example, in Remington's Pharmaceutical Sciences (Remington's
Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA
1985). On this basis, the compositions include, albeit not exclusively,
30 solutions of the substances in association with one or more
pharmaceutically acceptable vehicles or diluents, and contained in
CA 02249l03 l998-09-30
-17-
buffered solutions with a suitable pH and iso-osmotic with the
physiological fluids.
Compositions for injection include, albeit not exclusively,
chlamyhyaluronic acids in association with one or more pharmaceutically
5 acceptable vehicles or diluents, and contained in buffered solutions with a
suitable pH and iso-osmotic with the physiological fluids. Any
pharmaceutically suitable diluent can be used in the composition for
injections: distilled water, physiological or a salt solution, and/or a buffer
solution. The composition for injections may be prepared by
10 conventional volume-weight procedures. A certain amount of
chlamyhyaluronic acid is diluted to the necessary volume with a diluent
or solvent. The solution is then filtered through sterilized filters, bottled
or ampouled. The resultant solution is a stable transparent liquid, and
does not contain any chemical or other impurities.
Solid form preparations for oral administration can be made
in the form of tablets, powders, or capsules. It may contain a medium for
the active substance and other additives, including dyes, aromas, etc.
The compositions and treatments are indicated as therapeutic
agents or treatments either alone or in conjunction with other therapeutic
20 agents or other forms of treatment.
In one embodiment, chlamyhyaluronic acid of the invention
or capsules containing the chlamyhyaluronic acid may be formulated into
pharmaceutical compositions for adminstration to subjects for wound
healing. Chlamyhyaluronic acid facilitates the formation of stable
25 colloidal suspensions in pharmaceuticals. As with other known
hyaluronic acids, chlamyhyaluronic acid reacts with proteins to form
either a granular precipitate or a stable "mucin" clot, i.e. it forms stable
colloidal suspensions with acidic serum. This property, as well as its
antibacterial activity, point to the efficacy of chlamyhyaluronic acid as an
30 agent for wound healing. Chlamyhyaluronic acid may be used to reduce
tissue fibrosis in injured tissue caused by, for example, skin burns, ulcers,
ruptured tympanic membranes and abraded cornea.
CA 02249103 1998-09-30
Accordingly, the present invention provides a
pharmaceutical composition for reducing tissue fibrosis comprising
chlamyhyaluronic acid in admixture with a suitable diluent or carrier.
The present invention also provides a method for reducing tissue fibrosis
comprising administering a therapeutically effective amount o~ a
chlamyhyaluronic acid to a patient in need thereof.
In another embodiment, chlamyhyaluronic acid of the
invention and capsules containing the chlamyhyaluronic acid may be
formulated into pharmaceutical compositions for adminstration to
10 subjects for the treatment of cystitis.
Accordingly, the present invention provides a
pharmaceutical composition for reducing cystitis comprising
chlamyhyaluronic acid in admixture with a suitable diluent or carrier.
The present invention also provides a method for reducing cystitis
15 comprising administering a therapeutically effective amount of a
chlamyhyaluronic acid to a patient in need thereof.
In another embodiment, the chlamyhyaluronic acid of the
invention and capsules containing the chlamyhyaluronic acid may be
formulated into pharmaceutical compositions for adminstration to
20 subjects as a biological fluid replacement or biological fluid supplement.
For example, chlamyhyaluronic acid or preparations containing
chlamyhyaluronic acid may be used as a fluid replacement to correct
pathological conditions in the eye or the joints. Like hyaluronic acid,
chlamyhyaluronic acid polymers may be very large and can displace a large
25 volume of water. This property makes them excellent lubricators and
shock absorbers. Chlamyhyaluronic acid or preparations containing
chlamyhyaluronic acid may be used in ophthalmic surgery or as
therapeutic, auxiliary and substitutive agents for natural organs and
tissues, and may be particularly useful in the treatment of arthropathies.
Accordingly, the present invention provides a
pharmaceutical composition for biological fluid replacement or biological
fluid supplement comprising chlamyhyaluronic acid in admixture with a
CA 02249103 1998-09-30
- 19 -
suitable diluent or carrier. The present invention also provides a method
for replacing or supplementing biological fluids comprising administering
a therapeutically effective amount of a chlamyhyaluronic acid to a patient
in need thereof.
In another embodiment, chlamyhyaluronic acid of the
invention and capsules containing the chlamyhyaluronic acid may be
formulated into pharmaceutical compositions for administration to
subjects to treat cancer. Research indicates that hyaluronic acid may play a
role in cancer development and progression. Hyaluronic acid is involved
10 in both cell transformation and cancer cell metastasis. Paradoxically, in
large quantities it can also retard cancer development. Thus
chlamyhyaluronic acid may be particularly useful as a cancer treatment,
and adjunct to cancer treatment, or a drug delivery vehicle for cancer
treatment. Hyaluronic acid, and by extension, chlamyhyaluronic acid, may
15 have particular utility in treating basal cell skin carcinoma, actinic
keratoses, precancerous lesions resulting from sun exposure, melanoma,
ovarian cancer and breast cancer .
Accordingly, the present invention provides a
pharmaceutical composition for cancer treatment comprising
20 chlamyhyaluronic acid in admixture with a suitable diluent or carrier.
The present invention also provides a method for treating cancer
comprising administering a therapeutically effective amount of a
chlamyhyaluronic acid to a patient in need thereof.
In another embodiment, chlamyhyaluronic acid of the
25 invention and capsules containing the chlamyhyaluronic acid may be
formulated into pharmaceutical compositions for adminstration to
subjects for the purposes of pain relief. Thus in this embodiment,
chlamyhyaluronic acid may have pain relief utility, for example, in
arthritis treatment.
Accordingly, the present invention provides a
pharmaceutical composition for pain relief comprising chlamyhyaluronic
acid in admixture with a suitable diluent or carrier. The present
CA 02249l03 l998-09-30
-20-
invention also provides a method for relieving pain comprising
administering a therapeutically effective amount of a chlamyhyaluronic
acid to a patient in need thereof.
In any of the above-noted pharmaceutical uses,
chlamyhyaluronic may be particularly useful when used in conjunction
with a biologically active protein.
(2) Cosmetics
In another embodiment, chlamyhyaluronic acid of the
invention and capsules containing the chlamyhyaluronic acid may be
10 formulated into cosmetic compositions. Because of its hygroscopic nature,
thin layers or films of cell encapsulations containing chlamyhyaluronic
acid impart moisture to the skin. As chlamyhyaluronic acid facilitates the
formation of stable colloidal suspensions, it may also be used as a source of
mucilage in cosmetics and skin-care products.
Accordingly, the present invention provides a cosmetic
composition comprising chlamyhyaluronic acid in admixture with a
suitable diluent or carrier.
Like hyaluronic acid, chlamyhyaluronic acid is extremely
hydrophilic, and may be able to mix with and hold in place several
hundred times its weight in liquid. Thus chlamyhyaluronic acid may
facilitate the ability of skin to hold its moisture, making it a useful
moisturizing ingredient in cosmetics.
Accordingly, the present invention provides a moisturizing
composition for moisturizing mammalian skin, hair or nails, comprising
chlamyhyaluronic acid in admixture with a cosmetically suitable vehicle,
diluent or carrier.
The chlamyhyaluronic acid of the invention and capsules
containing the chlamyhyaluronic acid may be formulated into ultra violet
ray blocking compositions suitable for topical administration.
30 Chlamyhyaluronic acid and catalase components of the capsules protect
the skin from reactive oxygen radicals. Furthermore, chlamyhyaluronic
CA 02249103 1998-09-30
acid's skin adhesive characteristics contribute to its cosmetic utility as a
potential ultra-violet ray screening agent.
Accordingly, the present invention provides a sunscreen
composition for the photoprotection of mammalian skin or hair,
5 comprising chlamyhyaluronic acid in admixture with a suitable vehicle,
diluent or carrier.
(3) Foodstuffs
The chlamyhyaluronic acid of the invention and capsules
containing the chlamyhyaluronic acid may be formulated into
10 compositions in an edible form for use in foodstuffs.
In one embodiment, chlamyhyaluronic acid of the invention
and capsules containing the chlamyhyaluronic acid may be formulated
into gum compositions for use as a gum additive to foodstuffs.
Chlamyhyaluronic acid may be particularly useful as a gum for the food
15 industry because it facilitates the formation of stable colloidal suspensions.
Accordingly, the present invention provides a composition
for gelling a foodstuff comprising chlamyhyaluronic acid in admixture
with a suitable diluent or carrier. The present invention also provides a
method for gelling a foodstuff comprising contacting the foodstuff with an
20 effective amount of chlamyhyaluronic acid.
In another embodiment, chlamyhyaluronic acid of the
invention and capsules containing chlamyhyaluronic acid may be
formulated into compositions for use as a preservative in foodstuffs. The
present inventors have found that homogenates of cell encapsulations
25 containing chlamyhyaluronic acid prepared at room temperature in open
air and stored over a year at 4-6~C have not been the subject of fungal or
bacterial contamination. Hence, the homogenates are bacterio- and/or
fungi-static and may be of benefit for the food industry as a preservative.
Accordingly, the present invention provides a composition
30 for inactivating enzymes, microorganisms or spores in a foodstuff
comprising chlamyhyaluronic acid in admixture with a suitable diluent or
carrier. The prf~sent invention also provides a method for inactivating
CA 02249l03 l998-09-30
-22-
enzymes, microorganisms or spores in a foodstuff comprising contacting
the foodstuff with an effective amount of chlamyhyaluronic acid.
(4) Biosensor
The chlamyhyaluronic acid of the invention and capsules
containing the chlamyhyaluronic acid may be formulated for use in
electronic applications. Because of its photonic property,
chlamyhyaluronic acid may also be used as a matrix biopolymer and/or a
biosensor for future electronic applications as recently suggested by Angell,
Science, 267, pgs 1924- 1935 (1995) and Meerholz, K. et al., The Spectrum, 8,
10 pgs 1-6(1995).
Accordingly, the present invention provides a matrix
biopolymer or a biosensor comprising chlamyhyaluronic acid in
admixture with a suitable matrix or solution. The present invention also
provides a method for sensing occurrences photonically, comprising
detecting changes in a detectable amount of chlamyhyaluronic acid.
EXAMPLES
EXAMPLE 1
Selection of Algae
The phylum Chlorophyta, also known as the division
Chlorophycota or the class Chlorophyceae, includes Tetraspora,
Gloeomonas, Chlamydomonas segnis, Chlamydomonas gymnogama,
Chlamydomonas pallidostigmatica, Chlamydomonas augustae,
Chlamydomonas intermedia, Chlamydomonas sajao Levin and
Chlamydomonas corrosa. These are eukaryotic unicellular green algae
containing chlorophyll a and b, with 2 flagella (motile) or without flagella
(non-motile), or colonial (coenobia), possessing a pyrenoid or pyrenoids
associated with the chloroplast and storing starch.
Chlamydomonas segnis, isolated by S. Badour in 1969, was
deposited in 1971 under # 1919 in The Culture Collection of Algae at
Indiana University, which subsequently moved to the University of Texas
(UTEX) at Austin (Starr, R.C., and Zeikus, J.A. Journal of Phycology
(Supplement) 23: 8(1987); Starr, R.C. and Zeikus, J.A., Journal of Phycology
CA 02249103 1998-09-30
(supplement) 29:19-20 (1993)). Another culture of ChZamydomonas segnis
Ettl was deposited in 1985 under #1.79 in the Center of Algal Collection at
the Institute of Plant Physiology, University of Gottingen, Germany
(Schlosser, U.G. SAG- Sammlung von Algenkulturen at the University of
5 Gottingen, Botanica Acta, 107:129 (1994)). Chlamydomonas segnis Ettl is
also formerly known as Tetraspora species having accession No. ATCC
30631, Gloeomonas species, Chlamydomonas gymnogama,
Chlamydomonas pallidosfigmatica, Chlamydomonas intermedia,
Chlamydomonas sajao Levin, which have been assigned accession No.
10 1919, 1638, 1343, 1905, 222, and 2277 according to the list of Richard, C., Starr
and Jeffrey, A., Zeikus in Journal of Phycology supplement to volume 29
of April 1993. Because of nomenclatural uncertainty, two
Chlamydomonas species named Chlamydomonas augustae and
Chlamydomonas corrosa, which release extracellular polysaccharides into
15 the surrounding media, as reported by Allard, B. and Tazi, A.,
Phytochemistry, 32,pgs 41-47 (1993), may later be identified as
Chlamydomonas segnis Ettl. In 1980, two cultures of Chlamydomonas
segnis Ettl, the composition of a high salt nutrient medium and
description of a method used for producing synchronous cultures of this
20 alga were sent to the Ecotoxicology group of the National Research
CounciI of Canada, Ottawa. (Weinberger, P., et al., Canadian Journal of
Botany 65: 696-702 (1987); Dechacin, C., et al., Exotoxicology and
Environmental Safety 21: 25-31 (1991)).
In nature, Chlamydomonas segnis Ettl grows in shade (i.e. at
25 irradiance between 10-25 Wm-2), in soil ditches and roadside pools with
relatively high salt concentrations (15-30 mM). For active growth in the
laboratory, it requires all the essential macro- and micro-nutrients
common to most land plants (Salisbury, F.B., and Ross, C.W. Plant
Physiology, Wadsworth Publishing Company, Belmont California (1992)
30 pp.l16-135). Therefore, low-salt nutrient media coupled with high
irradiances would constitute stressful cultural conditions for this
particular microalga.
CA 02249103 1998-09-30
- 24 -
UTEX culture No.l919, provided on proteose agar slants was
selected for use in the examples described herein. UTEX culture No.1346, a
Chlamydomonas segnis Ettl isolate of H. Ettl appears identical to UTEX
culture No.l919, as it produces encapsulated cells when grown under the
culture conditions for the production of chlamyhyaluronic acid described
herein.
Secondary stock cultures were aseptically prepared for the
inoculation of the low salt boron-deficient nutrient medium used herein
for the mass cultivation of Chlamydomonas segnis Ettl. For this purpose,
10 inocula from the stock cultures of UTEX culture No.l919 were transferred
under sterile conditions to 1.6% agar slants of the inorganic low salt
boron-deficient nutrient medium in 16-20 ml capacity test tubes with
loosened screw plugs to allow the diffusion of air-CO2. These stock
cultures are then kept either at room temperature facing diffused day light
15 from west-located windows, or placed in a growth chamber at 18-20~C and
irradiance of 8-10 Wm-2 using cool white or day light fluorescent lamps
400-700 nm)-
Liquid precultures of about 150 mls, prepared by inoculating
the Woods Hole MBL pH 7.2 nutrient medium with Chlamydomonas
20 segnis Ettl from the agar slants described above, were usually used as the
stock cultures. These precultures were maintained at 18-20~C and low
irradiances of 8-10 Wm-2, and diluted every two to three weeks to a cell
density of about 2X106 cells ml-l. Suspensions of the algae between 25 mls
and 50 mls secured from a liquid stock preculture with a cell number of
25 about 4X106 ml-l were used to inoculate one litre of the low salt
boron-deficient nutrient medium adapted in this novel method for the
production of CENC.
EXAMPLE Z
Selection of Nutrient Medium
Chlamydomonas segnis Ettl, like many other unicellular
green microalgae, grows photoautotrophically in any mineral nutrient
medium as long as it contains all the essential macro- and
CA 02249l03 l998-09-30
-25-
micro-elements, particularly in high salt nutrient media as those listed in
Methods in Enzymology (edited by A. San Pietro), 23 part A, pgs 29-96
(1971), or recommended by Kuhl, A. and Lorenzen, H., Methods in Cell
Physiology (edited by D. M. Presscott), 1, pgs 159-187(1964).
In laboratory cultures, synthetic nutrient media generally
contain the 18 essential elements oxygen, carbon, hydrogen, nitrogen,
potassium, calcium, phosphorus, magnesium, sulfur, chlorine, iron,
manganese, copper, boron, zinc, molybdenum, silicon, and nickel for
higher plants or cobalt for microalgae. A balanced nutrient medium
10 containing the essential macro- and micro-elements required for cell
growth and division, even for a short period of time is generally at a pH
value between 6.0 and 7.5, and osmotic potential between about -0.126 MPa
and about -0.01 MPa, at temperatures within about 20~C to about 30~C
[Salisbury, F.B., and Ross, C.W. Plant Physiology, Wadsworth Publishing
15 Company, Belmont California, pp. 116-135, 226-233 and 240-241 (1992);
Nichols, H. Wayne. In: Handbook of Phycological methods Vol. I, pp. 8-23.
Edited by Stein, R. Janet. Cambridge University Press (1973); Starr, Richard
C. In: Methods in Enzymology, Vol. 23, Part A, pp. 29-52. Edited by San
Pietro, A. Academic Press (1971)]. Water soluble salts are used to provide a
20 variety of balanced nutrient media which become exhausted at the end of
the exponential phase of the algal growth.
A low salt nutrient medium with a total salt concentration
lower than 6.0 mM either during the lag phase, exponential phase, or the
stationary phase of the algal culture supports the algal growth and cell
25 division in cultures, even for a short period of time, or sustains the
photosynthetic capacity of the algal cells in the culture to synthesize and
accumulate secondary metabolites. The use of low-salt nutrient media
would shorten the time required for the algal culture to attain the
stationary phase of growth, during which the cells neither grow nor divide
30 due to the lack of one or more essential elements. Photoassimilation of
inorganic carbon, however, continues so that polysaccharides as well as
organic acids are synthesized and then excreted (Badour, S.S. Excess light
CA 02249l03 l998-09-30
-26-
evokes cell encapsulation in Chlamydomonas segnis Ettl. Abstracts of the
International Meeting on Molecular Biology, Biochemistry and Physiology
of Chloroplast Development. Philipps Universitat, Marburg, Germany
(1995); Allard, B., and Tazi, A~ Phytochemistry, 32:41-47(1993))
Boron-deficient cells are expected to have an inelastic cell
wall that cannot undergo normal cell-stretching growth as reported for
higher plants (Hu, Hening, and Brown, Patrick H. Plant Physiology, 105:
681-689 (1994)). Cell wall expansion and plasticity which are associated
with cell growth and division, require the formation of borate-ester
10 cross-links with mucopolysaccharides (e. g. pectin ) in the cell wall.
Inhibition of cis-diol boric acid complexes due to boron deficiency would
lead to decreases in cell plasticity and cell growth and may result in the
formation of mucilaginous thickened cell walls (Hu, Hening, and Brown,
Patrick H. Plant Physiology, 105: 68l-689 (1994); Raven, J.A. New
15 Phytologist, 84: 231-249(1980)).
The nutrient medium designated Woods Hole MBL pH 7.2
(Nichols, H. Wayne. Growth media- freshwater. In: Handbook of
Phycological methods Vol. I, pp. 8-23. Edited by Stein, R. Janet. Cambridge
University Press (1973) pp.l6-18 and table I-3),is low in salt (5.725 mM, or
20 1.725 mM without added Tris-HCl buffer) and boron deficient. The term
boron-deficient medium or boron-deficiency in the context of mass
cultivation of algae is used herein to mean a nutrient medium in which a
generation time shorter than 48 hours is obtained, or one in which
inhibited algal cell growth is completely restored and cell encapsulation
25 may be retarded by adding only either boric acid or its sodium or
potassium salts to the medium at a final concentration of one millimolar
or less.
This medium was selected and modified to produce cultures
of ChZamydomonas segnis Ettl with reduced exponential growth, and cells
30 with thick mucilaginous matrix (Handbook of Phycological Methods
(edited by Janet R. Stein), 1, pgs 16-18 (1973)).
CA 02249103 1998-09-30
- 27 -
In particular, the medium was modified by excluding
vitamins, sodium carbonate, sodium silicate, and reducing the
concentration of Tris-HCI from 4000 ,uM to 500 ,uM as seen in TABLE 1. As
a result, the initial total salt and buffer concentration of the modified
nutrient medium becomes 1.975 mM instead of 5.725 mM. The decrease
in the concentration of the Woods Hole MBL pH 7.2 nutrient medium
brings about a drop in its osmotic potential from about -0.029 MPa to about
-0.01 MPa at temperatures between 20~C and 30~C; thereby increases the
rate of water movement into the algal cell.
0 Chlamydomonas segnis Ettl is considered a soil microalga
adapted to relatively high salt surrounding media. The use of the
modified low salt boron-deficient nutrient medium, designated B in
TABLE 1, was selected to add more stress to the algal cells already exposed
to excessive light and accelerate cell encapsulation. In constrast, the high
15 salt nutrient medium of Kuhl and Lorenzen, which is slightly modified by
Badour, S. and Waygood, E. R., Phytochemisltry, 10, pgs 967-976 tl971),
shown in TABLE 1, column C, and has almost 10-fold the total salt
concentration of the modified Woods Hole MBL pH7.2 nutrient medium,
will exert its effect as a stress factor for the said microalga in light only
20 when salt becomes depleted.
Figure 3 shows the growth curves of deep cultures of
Chlamydomonas segnis Ettl produced at 25~C in the low salt
boron-deficient rnedium given in TABLE 1, column A, and the high salt
nutrient medium given in TABLE 1, column C, but without adding boric
25 acid. All cultures were bubbled with air and exposed to 30 Wm-2 for 72
hours, followed by aeration with 5% CO2 in air and increasing irradiance
to 60 Wm-2 for the low and high salt-A cultures, and to 135 Wm-2 for the
high salt-B culture. The term deep culture is used herein to mean a
culture in which the growing algal suspension has a depth between about
30 10cmandaboutl2cm.
The results presented in Figure 3 show that deep cultures of
Chlamydomonas segnis Ettl grown in the high salt boron-deficient
CA 02249l03 l998-09-30
-28-
medium with the initial salt concentration of 19.350 mM due to boric acid
exclusion, require at least twice as much irradiance as those grown in the
low salt boron-deficient medium to induce cell encapsulation. The
pronounced decline of cell number in the low salt boron-deficient culture
after being exposed to 60 Wm-2, compared to the slight decrease of cell
number in the high salt (B) boron-deficient culture which receives 135
W m -2, indicate that the algal cells' encapsulation rate in the former
culture is 2.5 times greater than in the latter culture. The term
"encapsulation rate" is used herein to mean the decrease in the cell
10 number, determined by a hemacytometer, per unit time, i.e. per hour(s),
that takes place in algal cultures at early or late stationary phases of
growth, as a result of cell encapsulation and capsule enlargement that
causes progressive cell-spacing and consequently decreases in cell counts
within the hemacytometer chamber.
Importantly, the phase of cell number decrease represents the
encapsulation phase. During this phase the enlargement of the algal
capsule, which is only visl~ali7etl by metachromatic dyes, causes the spread
of the greenish algal cells within the chamber of the hemacytometer used
for counting the cells. As a result, fewer cells are counted due to the
increase in capsule size, and a comparison of the ascending slopes seen in
Figure 3 would reflect the rate of encapsulation and the relative average
size of the capsule.
Figure 4 shows photomicrographs, with a scale bar of 20 ,um,
of encapsulated cells of Chlamydomonas segnis Ettl at the end of
encapsulation phase, stained either with toluidine blue (a and b) or alcian
blue (c), from (a) low salt (5.725 mM) boron-deficient culture at pH 7.2
enriched with 10 mM sodium nitrate and 4.5 mM dipotassium hydrogen
phosphate, and (b) high salt (19.35 mM) boron-deficient culture at pH 4.5,
and (c) low salt (5.725 mM) boron-deficient culture at pH 5Ø As seen from
the photomicrographs in Figure 4, the encapsulated cells of
Chlamydomonas segnis Ettl vary in the size and structure of the capsule,
CA 02249l03 l998-09-30
-29-
which are influenced by the salt concentration and composition of the
nutrient medium as well as the level and duration of irradiance.
Low salt nutrient media can be prepared by diluting high salt
nutrient media with water. For example, when the high salt nutrient
medium given in TABLE 1, column C, is diluted by a factor of five, its
initial salt concentration drops from 19.352 mM to 3.87 mM and the initial
boric acid concentration becomes 0.3,uM instead of 1.5,uM.
In an alternate embodiment, one can select glucose as an
additional source or as the only source of carbon for phototrophic cultures
10 of the alga for the production of encapsulated cells in low salt boron-
deficient nutrient medium. Provision of glucose may lower the activity of
Rubisco, the enzyme which catalyzes the fixation of carbon dioxide and the
formation of the organic material needed for cell encapsulation. As a
result, the blue: red spectral ratio greater than one will not be required.
Instead, a blue: red spectral ration less than one would be appropriate for
active glucose uptake and chlamyhyaluronic acid production. However,
the use of glucose may require one to maintain sterile conditions
throughout the culturing process.
Under the stressful conditions of the low salt boron-deficient
nutrient medium when glucose alone (i.e. in carbon dioxide-free air) is
used for the production of CENC in liquid, semi-solid, or solid cultures in
light, the cultures are photoorganic cultures. When air (i.e. with about
0.03% carbon dioxide) is provided to these cultures instead of carbon
dioxide-free air, the cultures are photomixokophic cultures.
EXAMPLE 3
Selection of Light
When photoautotrophic plants such as algae are exposed to
excessive light, the absorbed but not utilized quanta can destroy the
photosynthetic apparatus due to over-excitation (Long, S.P., and
Humphries, S. Annual Review of Plant Physiology and Molecular
Biology, 45: 633-622 (1994)). Utilization of quanta will decrease when an
essential element is deficient or unavailable for the growing cells.
CA 02249103 1998-09-30
-30 -
However, the detrimental effect of high, i.e. excessive, light is minimized
through energy dissipation and photoprotection (Deemmig-Adams, B. and
Adams, W.W. III, Annual Review of Plant Physiology and Molecular
Biology, 43: 599-625 (1992)).
As previously stated, "excessive light" is the light intensity at
which the inception of cell encapsulation in algal cultures exposed to air,
aerated with either air or air enriched with carbon dioxide, is detectable by
metachromasy.
There is no standard minimal level of irradiance for all
10 cultures. Cell encapsulation usually occurs in dim day-light in aged
cultures, as for example in the immobilized algal cells which are
maintained on agar slants for relatively long period of time in presence of
air. Dim light, e.g. irradiances of 2 to 5 Wm-2, may be excessive for
cultures deprived of some essential macro- or micro-elements.
Imposition of intense light on low-salt cultures of
Chlamydomonas segnis Ettl, which normally thrives at relatively low
irradiances in a high salt medium (Badour, S.S., et al. Journal of
Phycology, 13:80-86 (1977); Badour, S.S. Journal of Phycology, 17:293-299
(1981); Badour, S.S., and Irvine, B.R. Botanica Acta, 103: 149-154 (1990)),
was found to encourage the cells to develop mechanisms for
photoprotection. The ability of the alga to produce the compounds
required for such mechanisms may be linked to the carboxylase activity of
ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) and the levels
of its substrates (Salisbury, F.B., and Ross, C.W. Plant Physiology,
Wadsworth Publishing Company, Belmont California, pp. 226-233 (1992)).
In Chlamydomonas segnis Ettl, the carboxylase function of Rubisco
reaches its highest level of specific activity at the stationary phase of
growth (Badour, S.S. In:Handbook of Phycological Methods. Vol. II, pp.
209-216. Edited by Hellebust, J.A. and Craigie, J.S. Cambridge University
Press (1978)), and blue rather than red light increases such activity (Badour,
S.S. et al., Journal of Phycology (supplement) 8:16 (1972)). In other algae,
blue light also enhances the synthesis of photosynthetic pigments and
CA 02249l03 l998-09-30
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proteins (Laudenbach, B., and Pirson, A., Archiv fur Microbiology, 67:
226-242 (1969); Wallen, D.G., and Geen, G.H.. Marine Biology, 10: 44-51
(1971)). The protein that activates rubisco, i.e. rubisco activase, functions
in light and loses activity in darkness or dim light (Salisbury, F.B. and
5 Ross, C.W., Plant Physiology, Wadsworth Publishing Company, Belmont,
California, pp.240-241 (1992)). Therefore, the present inventor selected
excessive light with higher blue:red spectral ratios to ascertain its effect on
the developmental changes in Chlamydomonas segnis Ettl for
photoprotection.
The term "blue light" is used herein to mean irradiances with
spectral range between about 320 nm and about 500 nm, which includes
bluish green-light, blue-light, violet-light and ultraviolet A of about
320 nm to about 380 nm, and the term "red light" is used herein to mean
irradiances with spectral range between about 600 nm to about 700 nm,
15 which does not include far red.
EXA MPLE 4
Regulation of Carbon Dioxide
Carbon dioxide is the inorganic substrate used by Rubisco to
form the organic acids which provide the carbon skeleton for the
20 biosynthesis of cellular compounds (Badour, S.S., and Tan, C.K. Zeitschrift
fur Pflanzenphysiologie, 112:287-295 (1983)). These compounds are
required for the growth of the algal cells as well as for developmental
changes that would occur in response to stressful environments such as
excessive light. For the enhancement of carbohydrate and protein
25 accumulations, CO2 tensions higher than those at air levels should be
continuously supplied to the cells by bubbling the algal cultures with
0.1-5% CO2 (by volume) enriched air (Badour, S.S., et al., Canadian
Journal of Botany, 51:67-72 (1973); Badour, S.S., et al. Journal of Phycology,
13:80-86 (1977); Badour, S.S. Journal of Phycology, 17:293-299 (1981)). To
30 minimize the escape of CO2 from the nutrient medium at relatively
higher temperature, ChZamydomonas segnis Ettl cultures were kept at
20-30~C. To avoid increases in bicarbonate and carbonate ions which
CA 02249l03 l998-09-30
-32-
prevail in alkaline media bubbled with CO2, the culture medium must be
maintained at around pH 7. At such pH and temperature range,
Chlamydomonas segnis Ettl cells exhibit maximal growth and
biosynthetic activity (Badour, S.S., et al. Journal of Phycology, 13:80-86
(1977); Badour, S.S. Journal of Phycology, 17:293-299 (1981); Badour, S.S.,
and Irvine, B.R. Botanica Acta, 103:149-154 (1990); Badour, S.S., and Tan,
C.K.) Zeitschrift fur Pflanzenphysiologie, 112:287-295 (1983); Badour, S.S.,
and Tan, C.K. Plant and Cell Physiology, 28:1485-1492 (1987)).
Continuous aeration of Chlamydomonas segnis Ettl cultures
10 from the start, by air enriched with CO2 (0.1 to 5% C Oz)~ compared to
aeration only by air (0.03% C ~2)~ enhances the accumulation of
carbohydrates and proteins within the relatively large algal cells as
reported by Badour et al., J. of Phycology, 13:80-86 (1977) and Badour, J. of
Phycology, 17:293-299 (1981). The carbohydrates and proteins, which start
to accumulate in such cultures at early exponential phase of growth are
probably used at late exponential phase of growth for the synthesis of
intracellular carotenoids among other components, e.g. lipids, instead of
extracellular mucopolysaccharides within the cell capsules for
photoprotection. An example is shown in Figure 2, which depicts the
increase in carotenoid content as indicated by the absorbances of acetone
extracts between 420 nm and 480 nm from shallow cultures aging in low
salt boron-deficient nutrient medium bubbled with 5% C ~2 in air at 25~C
and about 40 Wm-2. Figure 2 shows the absorbance spectra (normalized at
662 nm) of acetone extracts from shallow cultures of Chlamydomonas
segnis Ettl, using Hewlett Packard Diode Array Spectrophotometer Model
8452 A, at (a) late exponential phase of growth, (b, c and d) early and mid
encapsulation phase, and (e) late encapsulation phase. In contrast, their
counterparts which are continuously bubbled with air, i.e. 0.03% C ~2~
produce relatively small cells with significantly less carbohydrate and
protein contents, but with higher catalase activity and ascorbate/
dehydroascorbate metabolism than the large cells produced in 5% C ~2
CA 02249103 1998-09-30
probably for photoprotection against oxygen free radicals generated in light
at air levels of CO2, as pointed out by Raven, J. A. et al.,Biological
Reviews, 69, pgs 61-94 (1994). As the air-bubbled shallow cultures age, the
cells show little increases in their low carotenoid content and slowly enter
5 the encapsulation phase. These cultures exhibit a greenish hue. In 5%
CO2 -bubbled shallow cultures, the cells continue to actively increase their
carotenoid content as they age and quickly enter the encapsulation phase.
These cultures exhibit a yellowish hue. The term shallow culture is used
herein to mean a culture in which the growing algal suspension has a
10 depth between about 3 cm and about 4 cm.
Accordingly, for the production of large capsules and
regardless of the cell size of Chlamydomonas segnis Ettl, the mineral
nutrient medium, after being inoculated, is aerated with only air until
about mid-exponential phase of growth, i.e. at cell numbers between 3 and
15 4 X106 cells ml-l depending on the inoculum size, and then 5% CO2 in air
is introduced to lower the oxygen tension and stimulate the accumulation
of the mucopolysaccharides in the algal capsule.
Exposure of Chlamydomonas segnis Ettl cells growing in a
low-salt medium to high CO2 concentrations will lead to an initial
20 enhancement of cell growth and division concomitant with a rapid
progressive reduction of the nutrients in the culture medium. At later
stages when the cells enter the stationary phase of growth due to nutrient
depletion, CO2 will be actively photoassimilated as long as the carboxylase
function of Rubisco is maintained, e.g. by blue light. In other words, the
25 continuous provision of high CO2 will enable Chlamydomonas segnis Ettl
to utilize more of the absorbed excessive light to synthesize intra- and
extra-cellular compounds, and thereby minimize over-excitation and
mitigate the destruction of the chloroplast.
CA 02249103 1998-09-30
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EXAMPLE 5
Selection of Apparatus
The apparatus required for the cultivation of
photoautotrophic microalgae is basically a combination of four
5 constituents, namely culture vessels, a temperature controlled space (e.g. a
chamber or tank), lighting source and aeration equipment. The process of
mass cultivation of Chlamydomonas segnis Ettl described herein specifies
the physical and cultural conditions under which the aforenamed
microalga produces algal biomass of encapsulated cells at low cost within a
10 relatively short period of time.
Routinely four-litre deep cultures of Chlamydomonas segnis
Ettl were grown in the modified low salt boron-deficient nutrient medium
given in TABLE 1, column B, using round vessels of 6 litres capacity at
25~C, exposed to irradiance of about 30 Wm-2 and bubbled with air for 72
15 hours, followed by increasing irradiance to about 60 Wm-2 and switching
aeration to 5% CO2 in air for 72 hours. Shallow cultures of aged
Chlamydomonas segnis Ettl were produced under the same cultural
conditions, using low form Fernbach flasks. Shallow cultures of
Chlamydomonas segnis Ettl were produced under the same cultural
20 conditions, but with continuous aeration from the start with either air or
5% CO2 in air for 150 hours.
Round glass vessels (6 likes capacity) fitted with inlet and
outlet tubing for aeration are used for producing deep cultures of four
litres algal biomass per vessel. In order to demonstrate that under the
25 same physical and cultural conditions, Chlamydomonas segnis Ettl cells
grow and age faster in shallow than in deep cultures, low-form Fernbach
flasks of about 3 litres capacity were also used to obtain one to one and a
quarter litre of algal biomass per flask.
In an alternate embodiment, encapsulated cells of
Chlamydomonas segnis Ettl can be produced on solid nutrient substrates,
as for example on 5 to 10 mm thick layers of 1.6-2.0% agar dissolved in low
CA 02249103 1998-09-30
- 35 -
salt boron-deficient nutrient medium, using flat trays exposed to natural
day-light or cool fluorescent light in air.
Any of the temperature-controlled chambers with circulated
filtered air and fluorescent lamps designed for growing green plants may
5 be employed in the process of the present invention. For example,
Conviron growth chambers from Controlled Enviroments Limited,
Winnipeg, Canada, equipped with cool-white 1500 General Electric (GRO
and SHO Wide Spectrum), or cool-white Sylvania GTE
(GRO-LUX-WS-very high output) fluorescent lamps, and standard
10 incandescent lamps (40-60 W) were used. Alternately, cool-white or
day-light fluorescent lamps (CRI 75, 6500K) from DURO-TEST
Corporation, North Bergen, New Jersey 07047, may be used. Figure 1
depicts the changes in the levels of blue, red, and far red spectra with
increasing irradiances from cool-white fluorescent and standard
15 incandescent lamps.
In order to check that the excessive light applied to
Chlamydomonas seg~is Ettl cultures for the production of CENC provides
blue:red spectral ratios higher than one, a plant growth photometer, IL 150
from International Light Inc. (Dexter Industrial Green, Newburyport,
20 Massachusettes 01950) was used to measure the levels of blue (400-500
nm), red (600-700 nm) and far red (700-800 nm) spectra radiating from the
fluorescent and incandescent lamps at various irradiances. Irradiances
(Wm-2) were measured with a LI-185B radiometer using a LI-COR, Inc.
pyranometer sensor from LI-COR, Inc., Lincoln, Nebraska 68504. As
25 shown in Figure 1, the cool-white fluorescent and incandescent lamps of
the Conviron chamber provide irradiances of up to 145 Wm-2, and the
high irradiances between 90 Wm-2 and 145 Wm-2 are due to small
increases in the red and far red spectra rather than the blue spectrum.
Since standard incandescent lamps, e.g. with CRI 98 and 3200 K have a
30 blue:red ratio of about 0.5 including the violet and ultraviolet spectra as
part of the blue, their use is limited to radiating dense cultures with algal
suspensions more than 4 cm in depth. Thus, incandescent lamps are used
CA 02249l03 l998-09-30
-36-
to supplement dense Chlamydomonas segnis Ettl deep cultures with
irradiances higher than 90 Wm-2, which offset cells' self-shading by
furnishing sufficient red light for active photosynthesis without virtually
affecting the preexisting blue:red ratio of four.
Conventionally, compressed air is used for the aeration of
microalgal cultures. The air pressure is regulated to allow gentle agitation
of the algal suspension, and thereby maximize the cells' exposure to
incident light. The air current is filtered to prevent culture contamination
by dust particles and air borne fungal and bacterial spores. Continuous
10 aeration provides about 0.03% (volume by volume) CO2, which is the only
source of carbon for photoautotrophic algal cells bubbled with air. Gas
regulators, gauges and gas needle valves for regulating and measuring gas
pressure from Matheson of Canada, Ltd. (Whitby, Ontario) and Union
Carbide (Linden Division) were employed in the present invention.
15 Suitable flow rates of air or gas mixtures were judged by minimal water
evaporation from the culture nutrient medium, and measured by
Matheson flowmeters Model 665 with a tube Model No. 603 and steel float
for air and nitrogen gas, and a tube Model No. 601 and steel float for CO2.
To obtain, for example, 5% C ~2 enriched air, or nitrogen gas (volume by
20 volume), CO2 at a flow rate of 100 ml/min was mixed with air or nitrogen
gas at a flow rate of 2000 ml/min using the Matheson flowmeter Model
665. The regulated gas or gas mixture was directed to the culture vessel
inlets via rubber or plastic tubing, and distributed to each culture vessel by
a manifold. Air, CO2 enriched air, or nitrogen gas was introduced into 4
25 litres of the nutrient medium at a flow rate of about 1000 ml/min.
EXAMPLE 6
Culturing Chlamydomonas segnis Ettl for the Production of CENC
The efficient production of CENC is facilitated by the use of
minimal concentrations of the nutrients in the medium coupled with
30 high inocula and low levels of irradiance to limit the microalgal growth
and cell division. This approach accelerates cell entry into the
CA 02249103 1998-09-30
encapsulation phase, i.e. the production of encapsulated algal biomass at
small cost. The example shown in Figure 5 provides strong evidence in
support of this thesis.
In contrast, when shallow cultures of Chlamydomonas segnis
Ettl were grown in the high salt nutrient medium given in TABLE 1,
column C (19.352 mM), and continuously exposed either to low irradiance
of 30-35 Wm-2 or high irradiance of 58-65 Wm-2, and aerated with air for
72 hours followed by 150 hours aeration with 5% CO2 in air, the resulting
algal biomass with a greenish hue was virtually deprived of encapsulated
10 cells.
However, algal growth in the high salt nutrient medium
after being diluted to 3.87 mM and receiving the same type of aeration and
level of irradiances as the high salt cultures, resulted in yellowish algal
biomass. Cells' encapsulation occurs in such diluted high salt cultures
15 after relatively longer period~ of time, i.e. 22q hours or more, compared to
about 120 hours in cultures produced with the modified Woods Hole pH
7.2 nutrient medium given in TABLE 1, column B. Furthermore, the
encapsulation rate is comparatively low, and the encapsulated algal
biomass is of poor quality due to predominance of large cells with small
20 capsules.
Greenish algal samples, secured from various cultures of
Chlamydomonas segnis Ettl during the encapsulation phase, settled after
standing at 4-6~C within 48-72 hours, compared to the yellowish algal
samples which settled after 120-240 hours depending on their oil-content
25 levels.
Therefore, the greenish encapsulated algal biomass rather
than the yellowish ones can easily settle and separate from the
surrounding liquid medium; thereby allowing the removal of the clear
supernatant by suction using vacuum devices.
Figure 5 depicts growth curves of Chlamydomonas segnis Ettl
in shallow cultures, inoculated from a growing liquid precultures to give
the initial cell density of about 0.1 x 106 cells ml-l, and bubbled with air for
CA 02249103 1998-09-30
- 38 -
48 hours followed by aeration with 5% CO2 in air for 48 hours at irradiance
of (a) 18 Wm-2, (b) 42 Wm-2, and (c) 60 Wm-2, at 25~C in Woods Hole MBL
pH 7.2 medium (5.725 mM) as given in TABLE 1, column A (closed
circles), and in the said nutrient medium after modification (1.975 mM) as
given in TABLE 1, column B (open circles).
Thus, regardless of the level of irradiance, the use of the
modified Woods Hole MBL pH 7.2 nutrient medium with a concentration
of 1.975 mM instead of 5.725 mM has little effect on the onset time (around
the 72nd hour) and rate of encapsulation, as indicated by the rate of
10 decrease in cell number between the 72nd and the 96th hour and
highlighted by the use of a polynomial curve fit. At the high irradiances
of 42 Wm-2 and 60 Wm-2, the number of cells in the nutrient medium
with the initial concentration of 5.725 mM is about 133% greater than that
with the lower concentration of 1.975 mM, probably due to the strong
15 buffering action of the high concentration of Tris-HCL used as given in
TABLE 1, column (A). However, there appears to be a relationship
between the level of irradiance and the initial concentration of the
nutrient medium. This is because cultures produced in the modified
nutrient medium (1.975 mM) are not significantly affected by increasing
20 irradiance from 18 Wm-2 to 42 Wm-2 and 60Wm-2, whereas those
produced in the Woods Hole MBL (5.725 mM) show about 20 to 25%
increases in cell number after 72 hours. In other words, relatively high
irradiances are required to promote Chlamydomonas segnis Ettl growth
and particularly cell encapsulation in low salt boron-deficient nutrient
25 media with initial salt/nutrient concentration greater than 2.00 mM.
Figure 6 depicts the inverse relationship between the dilution
index and the level of irradiance required for efficient (i.e. within a
relatively short period of time), production of encapsulated algal biomass
in shallow and deep cultures of Chlamydomonas segnis Ettl.
From the example shown in Figure 6, the dilution index, i.e.
the ratio of cell number (in 106 ml-l) at the onset of the encapsulation
phase to the initial salt/nutrient concentration in mM of the medium, is
CA 02249103 1998-09-30
-39-
used in the present invention to adjust irradiance level for the efficient
production of encapsulated algal biomass in shallow and deep mass
cultures of Chlamydomonas segnis Ettl. When the dilution index is about
0.52, i.e. the cell number ml-l (x106) is about half that of the initial nutrient
5 concentration (mM), the relatively high irradiance of 150 Wm-2 will be
effective for the rapid induction of cell encapsulation in deep cultures of
the said microalga. On the other hand, if the cell number ml-l (x106) of the
same culture increases such that the dilution index becomes about 6.00, 40
Wm-2 will be sufficient to induce cell encapsulation.
The increase in the cell number occurs at the expense of
nutrient utilization, which leads to a decrease in the salt/nutrient
concentration of the medium. The resulting nutrient depleted medium
acts as a stress factor, and under these stressful conditions the
photoautotrophic algal cells become incapable of utilizing all the energy
absorbed from incident light for ~rowth and cell division. Instead~ the
microalgal cells undergo encapsulation and synthesize
mucopolysaccharides within their capsules to offset the effect of excessive
light, even at the low irradiances of 10 Wm-2 to 15 Wm-2 in shallow
cultures or 20 Wm-2 to 25 Wm-2 in deep cultures.
Chlamydomonas segnis Ettl cells with large extracellular
capsules are produced photoautotrophically in shallow and deep cultures
within 90 to 150 hours from the time of inoculating a low salt
boron-deficient nutrient medium, with an initial total salt concentration
between 2 mM and 6 mM, and pH value from 6 to 7.5. (The term
photoautotrophic is used herein to mean that the growth and cell division
is achieved under illumination by providing only inorganic salts, i.e.
mineral nutrients which are assimilated by the algal cells at the expense of
solely light energy via photosynthesis.)
The inoculated nutrient medium with initial cell density
between 0.1x106 cells and 0.2x106 cells ml-l was supplied with air, i.e. about
0.03% C ~2~ during the lag and mid-exponential phases of growth. At
mid-exponential phase, air enriched with 0.1 to 5% CO2 (volume by
CA 02249103 1998-09-30
-40-
volume) is provided to the cultures, which are placed after inoculation in
a growth chamber at a temperature between 20~C and 30~C, and exposed to
irradiances from fluorescent lamps with blue:red spectral ratios greater
than one. During the first 72 hours of incubation and depending on the
initial cell density, the inoculated nutrient medium was exposed to
irradiances between 15 Wm-2 and 30 Wm-2 in case of shallow cultures, and
to irradiances between 25Wm-2 and 30Wm-2 in case of deep cultures.
During the exponential phase of growth, when the cell
density is between 3X106 cells and 4X106 cells ml-l, irradiances are increased
10 to a level within the 60 Wm-2 to 150 Wm-2 range for the induction of cell
immobilization and encapsulation. Irradiances greater than 60 Wm-2 are
applied to the cultures when the ratio of the cell number (in 106 ml-l) at
the onset of the encapsulation phase to the initial salt concentration of the
nutrient medium (in mM), i.e. the dilution index, is less than three.
15 The Encapsulation Phase
The encapsulation phase is the stationary phase of growth or
the second stationary phase of growth followed by a phase of decline in cell
number. This is akin to the death phase discussed in some microbiology
and phycology teachings. Thus, the early- and mid-encapsulation phases
20 discussed herein correspond to early and late stationary phases of growth.
However, the decline of cell number at the end of the stationary phase of
growth in Chlamydomonas segnis Ettl cultures is due solely to
enlargement of the capsules which encase viable cells.
Figure 7 shows photomicrographs, with a scale bar of 40 ,um,
25 of Chlamydomonas segnis Ettl stained with toluidine blue; (a) as capsule
free cells at mid exponential phase of growth, (b) as cells with
metachromatic extracellular matrix at the beginning of encapsulation, (c)
as encapsulated cells with polymorphic capsules, and (d) as spaced cells
due to capsule enlargement.
The encapsulation phase in Chlamydomonas segnis Ettl
cultures commences when the capsule-free cells, with diameters between 6
~m and 8 ,um at the end of the exponential phase of growth, as seen in
CA 02249l03 l998-09-30
-41-
Figure 7, excrete layers of metachromatic polysaccarides that form
relatively small capsules with diameters between 12 ,um and 18 ,um. Such
capsules enlarge and attain various shapes depending on the density of the
algal biomass. Polymorphic and large capsules with diameters ranging
from 30 ,um to 80 ,um prevail by the end of the encapsulation phase. The
volume of the capsule may reach over 200-fold that of the algal cells due to
their high water content. When samples of the encapsulated algal
biomass are dehydrated during the fixation process required for the
preparation of ultra-thin sections for transmission electron microscopy (as
10 described by Badour et al, Canadian J. of Botany, 51, pgs 67-72 (1973)), the
size of the capsule decreases by almost 98% as shown from the capsule in
Figure 8 (c) and (d), with a diameter between 9 ,um and 10 ,um.
Figure 8 shows transmission electron micrographs, with a
scale bar of 4 ,um, of Chlamydomonas segnis Ettl cell sections; (a) at mid
15 exponential phase of growth showing a chloroplast with interthylakoidal
starch grains in a capsule free cell, and (b) starch sheath around the
pyrenoid, (c) at late encapsulation phase showing the stratified structure of
the capsule surrounding a cell with large starch grains within a fenestrated
chloroplast and large dark lipid droplets, and (d) the relative thickness of
20 the capsule.
Furthermore, the scanning electron micrographs of desiccated
samples of encapsulated Chlamydomonas segnis Ettl cells in Figure 9
reveal the heavily coated cells with mantles or thick chlamyses embedded
in the dried thick matrix of extracellular polysaccharides. Figure 9 shows
25 scanning electron micrographs of desiccated cells of Chlamydomonas
segnis Ettl at mid encapsulation phase, (a) coated with thick mantles of
mucopolysaccharides, (b) in a fractured layer of dried extracellular
mucopolysaccharide material released in the culture medium, and (c and
d) embedded in the thick matrix of this material.
Visualization of the polysaccharide(s) content of the capsule
in the immobilized cells of the said microalga shown in Figure 10 is
achieved by autofluorescence due to the ability of the capsule material to
CA 02249103 1998-09-30
- 42 -
dissipate ultraviolet A, and by the use of metachromatic dyes as toluidine
blue (1% in borax) or solutions of alcian blue in water.
Figure 10 shows photomicrographs,with a scale bar of 60 ,um,
of: (a) immobilized spaced cells of Chlamydomonas segnis Ettl at mid
encapsulation phase, (b) the autofluorescence of the capsule material using
excitation filters UV 330-380 nm and a Nikon epifluorescence microscope,
and (c) encapsulated cells stained with alcian blue, or (d) toluidine blue to
visualize the capsule.
Figure 11 depicts the retarding effect of the presence of one
10 ~M boric acid (+ Boron) in the low salt boron-deficient nutrient medium
(- Boron) given in TABLE 1, column A, on cell encapsulation as indicated
by the downturn of the cell number in shallow cultures of
Chlamydomonas segnis Ettl, incubated at 25~C, bubbled with air for 72
hours at 25 Wm-2 to 30 Wm-2, and thereafter with 5% CO2 in air (volume
15 by volume) at 60 Wm-2 (high light). The example presented in Figure 11
shows that in shallow cultures the encapsulation phase, which is indicated
by the downturn in cell number after 96 hours incubation at 25 Wm-2 to 30
Wm-2 followed by 60 Wm-2 at the 72nd hour, is delayed for 96 hours when
boric acid or sodium borate is included in the low salt boron-deficient
20 medium, given in TABLE 1, column A, at a final concentration of one
micromolar. Probably, in the boron repleted medium mechanisms other
than cell encapsulation are operating for photoprotection under the
stressful cultural conditions caused by nutrient depletion after 96 hours
incubation at 60 Wm-2.
Figure 12 depicts the inhibitory effect of aerating
Chlamydomonas segnis Ettl cells with 5% CO2 in N2 instead of 5% CO2 in
air (volume by volume) at the end of the exponential phase of growth, on
the encapsulation phase in shallow cultures provided with the low salt
boron-deficient nutrient medium given in TABLE 1, column A, incubated
30 at 25~C, bubbled with air for 72 hours at 25 Wm-2 to 30 Wm-2, followed by
60 Wm-2 (high light) and aeration with 5% CO2 in air, which is replaced by
5% CO2 in N2 at the 96 hour to remove ambient oxygen. The graph in
CA 02249103 1998-09-30
-43-
Figure 12 provides strong evidence that cell encapsulation and capsule
enlargement are linked to the presence of air levels of oxygen. This is
because bubbling shallow cultures of Chlamydomonas segnis Ettl by the
end of the exponential phase of growth with 5% CO2 in nitrogen gas
5 instead of 5% CO2 in air (volume by volume) at 60 Wm-2 restores celI
division as indicated by the almost linear increase in cell number.
Another observation not shown in Figure 12 is that switching
off light and maintaining the culture in darkness for 48 to 72 hours instead
of bubbling the culture with 5% CO2 in nitrogen gas also restores cell
10 division. This suggests that air levels of oxygen promote the microalgal
cell encapsulation in continuously illuminated nutrient depleted cultures
of Chlamydomonas segnis Ettl, i.e. under conditions of excessive light.
Thus, cell encapsulation in cultures of the microalga is a
means for protection against oxygen radicals which are generated through
15 the p~rtial reduction of oxygen ll.olecule~ during ph~to~yl.'hetic elec~on
transport as explained in Plant Physiology Textbooks, (e.g. Mohr and
Schopfer, Plant Physiology, Springer-Verlag, pgs 173-175 (1995)). Such
electrons are not used for biosynthetic activity towards cell growth and
division in nutrient depleted media. The high activities of photosystems I
20 and II as well as catalase at the early phase of cell encapsulation, shown inTABLE 2, support this conclusion and point to the potential use of CENC,
harvested at early to mid encapsulation phases, as an effective ingredient
for protection against oxygen radicals.
EXAMPLE 7
25 l~xtraction of CENC from the Algal Medium
For the production of encapsulated algal biomass of
Chlamydomonas segnis Ettl with large capsules, deep cultures were grown
in the modified Woods Hole MBL nutrient medium pH 7.2 as given in
TABLE 1, column B, using the aforedescribed method of culturing the said
30 microalga. When the greenish microalgal biomass becomes viscous due
to the formation of large metachromatic capsules, i.e. at mid encapsulation
phase, the microalgal suspension was withdrawn from the culture vessels
CA 02249103 1998-09-30
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into glass or plexiglass standing settling containers, using a vacuum device
as an aspirator pump. The encapsulated microalgal biomass was then left
for 48 hours to 96 hours, depending on the cell density, at 4-6~C to separate
from the liquid medium and settle at the bottom of the container forming
5 the microalgal slurry. The latter can also be obtained by centrifugation, if,
for example, a high speed refrigerated centrifuge with large rotor and
plastic centrifuge bottles of 500 ml capacity is available. A vacuum device,
however, may be required for the removal by suction of the supernatant
and thereafter the microalgal slurry for further processing.
Thus, to one volume of the microalgal slurry four volumes
of 95% ethanol were added to give a final ethanol concentration of 70% to
80%, depending on the thickness of the said slurry.
The mixture was then left either at room temperature or at
4-6~C to settle for about 4 to 6 days, to allow the extraction of chlorophylls,
15 accessory pigments and ethanol soluble cellular compounds, as well as the
dehydration of the mucopolysaccharide(s) of the microalgal capsule. As a
result, the supernatant of the ethanol-microalgal slurry mixture turns
greenish or yellowish green in colour, and the encapsulated microalgal
slurry forms a white to yellowish coloured pellet at the bottom of the
20 settling container. After removing the supernatant by suction, the solid
pellet of the achlorophyllous dehydrated encapsulated microalgal biomass
was resuspended in about half- to one-litre 95% ethanol to wash out
residual pigments and ethanol-soluble cellular compounds. The
dehydrated, relatively clear and moist encapsulated microalgal biomass
25 was collected after the removal of the supernatant by suction,
centrifugation, or by filtration using glass fiber, teflon, or nylon filters with
pore size between one and three micrometer, depending on the thickness
of the said biomass. The microalgal crust obtained by filtration, and the
compact moist biomass secured by centrifugation or scooped out after
30 removing the supernatant, is the caked encapsulated Chlamydomonas
segnis Ettl, i.e. CENC, which is the raw source for algal hyaluronic acid-like
substance.
CA 02249l03 l998-09-30
-45-
EXAMPLE 8
Charact~ri~tion of CENC
Greenish suspensions of Chlamydomonas segnis Ettl
encapsulated cells, harvested from cultures exposed to excessive light (60
Wm-2 to 150 Wm-2) for 24 to 72 hours, i.e. at early to mid-encapsulation
phase, can readily settle in 75% to 80% ethanol within 48 to 72 hours at 4~C
to 6~C or at room temperature. Yellowish suspensions of the said
microalga, obtained after prolonged exposure to excessive light, i.e. at late
encapsulation phase, require more time to settle in ethanol due to the
accumulation of lipid droplets in chloroplasts. The ethanol dehydrated
encapsulated cells of Chlamydomonas segnis Ettl yield a moist cake,
which has at a concentration of 1.0% in water (dry weight per volume)
higher viscosity than the commercial gums xanthan and guar, and
exhibits a degree of shear thinning similar to the pseudoplastic xanthan
gum solution.
Figure 13 shows photomicrographs of: (a) a sample of CENC
homogenate, (b) a two-gram slightly moist pellet of CENC,(c) the size
increase of the moist pellet of CENC during water imbibition, and (d) three
samples of CENC during hydration exhibiting colour shades.
As shown in Figure 13 (a), CENC is highly hydrophilic and
forms a stable colloidal homogenate when blended with water. The
example photographed in Figure 13 (b) and (c) shows that a two-gram
pellet of a slightly moist preparation of CENC imbibes about 200 ml of
distilled water within 24 to 48 hours at room temperature. The colour and
texture of CENC may vary according to the age of the microalgal biomass
harvested during the encapsulation phase. The orange colour of the
CENC sample number 3 in Figure 13 (d) may be due to the presence of
extraplastidic carotenoids, as reported by Burczyk et al., Planta, 151, pgs
247-250 (1981), which may accumulate at late encapsulation phase.
Prolonged desiccation of the moist CENC, e.g. for 96 hours using
anhydrous calcium sulfate of the Hammond Dreirite Company, will
abolish its ability to imbibe water and rehydrate.
CA 02249l03 l998-09-30
-46-
Figure 14 depicts the apparent viscosity of 1% CENC
homogenate in water, compared to 1% solution of either xanthan or guar
in water, as a function of shear rate, determined by a Bohlin Viscometer
(Bohlin Reologi, Lund, Sweden).
As seen in Figure 14, viscosity measurements for CENC
homogenate in distilled water point clearly to its high viscosity with shear
thinning similar to the pseudoplastic xanthan gum solution. CENC
homogenate is a gel, which does not show thixotropy and has higher
viscosity than the commercial gums xanthan and guar. Furthermore,
10 CENC homogenates prepared in open air and stored for two weeks at
room temperature or for over a year at 4-6~C have not been the subject of
fungal or bacterial contamination, as judged from the absence of microbial
growth. This observation suggests that CENC must be at least bacterio-
and/or fungi-static.
15 EXAMPLE 9
Extraction of Chlamyhyaluronic acid from CENC
By use of the process of the present invention, removal of
protein from the precipitated material is not necessarily required because
of the relatively low absorption of its aqueous solutions at 280 nm.
20 Furthermore, the protein content of the said material does not exceed 0.5%
of its weight as determined by the method of Bradford, Analytical
Biochemistry, 72, pgs 248-254 (1976), using Coomassie Brilliant blue G 250.
Furthermore, electrophoresed aqueous solutions of 2-5 mg ml-l of the
precipitated capsular extract on a 6% polyacrylamide gel (as described by
25 Badour and Kim, Canadian J. of Botany, 66, pgs 1750-1754 (1988)), and
stained with 0.5 % alcian blue dissolved in water, migrated as
homogeneous streaks along two thirds the length of the gel matrix with
blue coloured portion at the top, and faint blue towards the bottom. This
suggests that the CENC precipitated extracts consist of the same acid
30 mucopolysaccharide but with different molecular weights, the largest at
the top and the smallest at the bottom of the streak. Similar observations
have been reported by DeAngelis et al., J of Biological Chemistry, 268, pgs
CA 02249l03 l998-09-30
-47-
14568-14571 (1993) for purified hyaluronic acid extracts from transformed
bacteria.
Accordingly, the CENC precipitated extract appears to be at
least partially pure, and further purification would be mainly focused on
the removal of the low molecular weight population and/or inhibiting
the degradation of the high molecular weight population during
extraction.
Thus, for the extraction of the metachromatic material, which
is clearly visualized within the capsules of the CENC preparation by
10 toluidine blue as shown in Figure 15 (a), a batch of eight to twelve grams
of the moist CENC, which contains about 50% of its weight as water
(TABLE 3), is used. This batch is usually obtained from a four-litre
Chlamydomonas seg~is Ettl deep culture after four to six days growth and
encapsulation period, depending on the density of the microalgal
suspension used for inoculation.
Figure 15 shows photomicrographs of samples from CENC
homogenate stained with toluidine blue, (a) before extraction, exhibiting
the metachromatic capsular content, (b) after extraction with the buffered
saline solution (0.8 M NaCI dissolved in 0.05 M phosphate buffer of p H 7),
exhibiting intact cells inside the ghost capsule with strata indicated by
arrows, and (c) exhibiting the acapsular appearance of CENC cells after
extraction.
Eight to twelve grams of the aforedescribed ethanol
dehydrated moist CENC were homogenized in 200 ml of 0.05 M phosphate
buffer, p H7 to 7.5 by a blender or through continuous stirring for one to
two hours in a closed container at room temperature. The resulting
homogenate was extracted for about two hours by adding either sodium
chloride, sodium acetate or potassium chloride under continuous stirring
to a final concentration of 0.8 M to 1.0 M. One volume of the solubilized
30 fraction, which is clarified either by sieve filtration or by centrifugation, is
precipitated by the addition of four volumes of 95% ethanol. After 24
hours to 48 hours, the clear supernatant is removed through suction using
CA 02249l03 l998-09-30
-48-
a vacuum device, and the precipitated mucopolysaccharide salt is collected
directly as pellets by centrifugation or as fairly thick suspensoids in
ethanol, and gently aerated with nitrogen gas to remove traces of ethanol
before storage at -16~C. One litre of the encapsulated cells of
ChZamydomonas segnis Ettl from deep cultures produced according to the
culturing method described herein, yields between 0.38 gram to 0.75 gram
of the mucopolysaccaride salt. Although sodium chloride has been
routinely used for the extraction of the capsular material as shown in
TABLE 3, it may be replaced by sodium acetate for better extraction.
10 Acetate salts were the preferred agents for the extraction of hyaluronic acidfrom vitreous humor, as reported by Karl Meyer in Physiological Reviews,
27, pgs 335-359(1947).
As seen from Figure 15 (b), efficient extraction of the capsular
material results in the negative metachromasy of the capsule which
otherwise stains with toluidine blue due to the presence of the acid
mucopolysaccharide within. Only intact vegetative cells of
Chlamydomonas segnis Ettl can be visualized in the extracted samples of
CENC, as shown in Figure 15 (C). If capsular metachromasy persists, CENC
homogenate is extracted repeatedly with 0.8 M to 1.0 M of the said salts.
Precipitation of the extracted material is achieved by adding to one
volume of the clear salt extracts four volumes of 95% ethanol. The
mixture is then left either at room temperature or at 4~C to 6~C for 24 to 48
hours for the complete precipitation of the white mucopolysaccharide salt
at the bottom of the settling container.
EXAMPLE 10
Partial Purification of Chlamyhyaluronic acid Extract
After removing the bulk of ethanol by suction, thick
suspensoids of the precipitate in ethanol are transferred into centrifuge
tubes for further removal of ethanol by suction after sedimentation, or
directly by centrifugation. The wet pellet of the precipitate in the
centrifuge tubes, or the wet residue obtained if narrow beakers are used
instead of tubes, is placed in a desiccator or a closed container equipped
CA 02249l03 l998-09-30
-49-
with manifolds designed to allow gentle aeration of the wet precipitates
with nitrogen gas at room or slightly higher temperature. The dried
mucopolysaccharide salts are then scooped out, sealed and stored at -16~C.
As seen from the example in TABLE 3, one litre of Chlamydomonas
segn is Ettl cultures, using the culturing method described in this
invention and harvested at about mid encapsulation phase, yields on the
average about 2.45 grams of CENC, or 1.22 grams dry biomass that provides
0.57 gram of the mucopolysaccharide salt.
EXAMPLE 11
10 ~haracterizatiQn Qf Chlamyhyaluronic acid obtained from Algae
Conventionally, the methods used to identify hyaluronic acid
or its salts are based on the changes in the properties of this acid
mucopolysaccharide in solutions when depolymerized by hyaluronate
lyase (EC 4.2.2.1), or by hyaluronidase (EC 3.2.1.35). According to Rapport et
al., J. Biological Chemistry, 186, pgs 615-623 (1950), changes in the
properties of hyaluronic acid can be measured through: (1) the decrease in
viscosity, (2) the loss of ability to form stable colloidal suspension, i.e.
turbidity, with acidified protein solutions at pH 4.2, and (3) the increase in
reducing groups, i.e. reducing sugar content of the hyaluronic acid
solution.
Because the absolute rate of viscosity decrease varies with
different hyaluronic acid preparations and other unknown factors, this
method was used herein only to determine the intrinsic viscosity of
chlamyhyaluronic acid and that of hyaluronic acid from human umbilical
cord for comparison.
The turbidimetric or the turbidity reducing method described
by Dorfman, A., Methods in Enzymology, I, pgs 166-173 (1955) and
Arvidson, S. O., in: Staphylococci and Staphylococcal Infections, 2, pgs
749-750, edited by Easmon, C. S. F. and Adlam, C., Academic Press (1983)
was used. TABLE 4 shows that by increasing the concentration of
chlamyhyaluronic acid or the human umbilical cord hyaluronic acid, the
turbidity increases almost linearly within the 0.4 mg to 2.0 mg ml-l of
CA 02249103 1998-09-30
- 50 -
chlamyhyaluronic acid and between 0.4 mg to 1.6 mg ml-l of the said
hyaluronic acid. The results also show that the fraction in
chlamyhyaluronic acid which binds to the protein and causes the turbidity
is about 36.7% of that in the umbilical cord hyaluronic acid, as indicated by
5 the difference in absorbance between the two solutions.
Also, samples of chlamyhyaluronic acid solutions of about 2.0
mg ml-l in 0.02 M acetate buffer, pH 5.0, treated with about 70 units of
hyaluronate lyase from Streptomyces hyalurolyticus (Sigma, No. H-1136),
or with about 980 units of hyaluronidase from bovine testes (Sigma, type
10 rv-s, No.3884) for 6-12 hours at 37~C indicate 40-60% decreases in turbidity. Such decreases are associated with increases in the reducing sugars, as
determined by the arsenomolybdate method of Nelson, as described by
Ashwell, G., Methods in Enzymology, III, pgs 73-87 (1957). In other
examples shown in Figure 16 and Figure 17, the minute amounts of
15 reducing compounds produced within 10 to 45 minutes through
chlamyhyaluronic acid depolymerization by the said enzymes can be
measured spectrophotometrically by the increase in absorption at 230 nm,
as described by Greiling, H., Hoppe-Seylers Zeitschrift fur physiologische
Chemie, 309, pgs 239-242 (1957), when the enzymically treated and
20 untreated samples are compared.
Figure 16 depicts the increase in absorption around 230 nm of
chlamyhyaluronic acid (0.2 mg ml-l of 0.02 M acetate buffer, pH5.0) after
incubation with about 70 units of hyaluronate lyase at 55~C for 45 minutes
(upper graph), compared to a control sample without the said enzyme
25 (lower graph), using Hewlett Packard Diode Array Spectrophotometer
Model 8452A.
Figure 17 depicts the increase in absorption around 230 nm of
chlamyhyaluronic acid (0.8 mg ml-l of 0.02 M acetate buffer, pH 5.0) after
incubation with about 980 units of hyaluronidase at 37~C for 10 minutes
30 (upper graph), compared to a control sample without the said enzyme
(lower graph), using Hewlett Packard Diode Array Spectrophotometer
Model 8452A.
CA 02249l03 l998-09-30
-51-
Figure 18 shows a print-out of a cellulose thin layer
chromatogram, developed with butanol, acetic acid, and water (50: 15: 35),
after exposure to ultraviolet radiation in a Gel Doc 1000 Video Gel
Documentation System (BIO-RAD) to visualize the products formed by
the degradation of chlamyhyaluronic acid (c and d), and hyaluronic acid
from human umbilical cord (e and f) with hyaluronate lyase at 37~C for six
hours, and the absence of such products (a and h) or their reduced level (b
and g) in samples without enzyme or with heated enzyme, respectively.
Figure 19 shows increases in the degradation products indicated in Figure
10 18, as a result of extending the incubation period to twelve hours at 37~C.
Figure 20 shows a print-out of a chromatogram produced as described in
Figure 18, except that hyaluronidase is the enzyme used.
As seen from the thin layer chromatograms in Figures 18-20,
ultraviolet absorbing spots from the enzymically treated
15 chlamyhyaluronic acid are almost mirror images of those formed in the
enzymically treated hyaluronic acid from human umbilical cord.
However, from Figure 21, it appears from the relatively low intrinsic
viscosity of chlamyhyaluronic acid, as compared to that of the human
umbilical cord hyaluronic acid in Figure 22, that the majority of the
20 polymer population in the former is of low molecular weight. Improving
the method of chlamyhyaluronic acid extraction, e.g. by inhibiting the
endogeneous enzymes that could cause its depolymerization during the
preparation of CENC, is expected to increase the proportion of high
molecular weight fraction in the extract.
Figure 21 depicts the reduced viscosity of chlamyhyaluronic
acid (potassium salt) as a function of concentration, using a Ubbelohde
viscometer, and the intrinsic viscosity is determined by extrapolating to
zero concentration with an asymptotic model.
Figure 22 depicts the reduced viscosity of hyaluronic acid
30 (sodium salt) from human umbilical cord, as a function of concentration,
using a Ubbelohde viscometer, and the intrinsic viscosity is determined by
extrapolating to zero concentration with a linear model.
CA 02249103 1998-09-30
-52 -
Thus chlamyhyaluronic acid (the partially purified sodium
and potassium salts of the mucopolysaccharide extracted from CENC after
being homogenized in 0.05M phosphate buffer at pH 7-7.5), was found to
contain less than 0.5% protein, to be degraded by Streptomyces
5 hyalurolyticus hyaluronate-lyase (EC 4.2.2.1) and to form stable colloidal
suspensions with acidic serum albumin, i.e. develops turbidity in a
manner similar to animal hyaluronic acid.
Chlamyhyaluronic acid does not appear to be identical to
hyaluronic acid obtained from umbilical cord or rooster comb.
Differences between chalmyhyaluronic acid and umbilical
cord hyaluronic acid were shown by comparing the 500 MH~ NMR
(nuclear magnetic resonance) spectra of the algal and human umbilical
cord sodium salts of hyaluronic acid solutions in D2O (deuterium oxide) at
300K. The peak of the algal hyaluronic acid at 1.7599 ppm was off the peak
15 of acetyl group ~hown at 1.8980 pprlP~ by the huma~. umbilical cord
hyaluronic acid. Also, the peaks associated with carbohydrates were 3.2349
ppm to 4.0879 ppm for the human umbilical cord hyaluronic acid, and in
the range of 3.4907 ppm to 3.8873 ppm for chalmyhyaluronic acid. More
specifically, the carbohydrate region shows five peaks at different ppm,
20 namely at 3.8873 ppm, 3.8125 ppm, 3.7052 ppm, 3.6008 ppm and 3.4907
ppm. The highest peaks are at 3.7052 ppm and 3.6008 ppm, which are
different from the human umbilical cord hyaluronic acid carbohydrate
peak at 3.7116 ppm.
Differences were also observed between chalmyhyaluronic
25 acid and rooster cori,b hyal-uronic acid. Sol-u.ions of hyaluronic acid
prepared from rooster comb form a complex with bivalent copper
according to Figueroa et al., Biochemical and Biophysical Research
Communications, 74:460-465 (1977), show an absorption band at 238 nm.
Chlamyhyaluronic acid solutions also form a copper-complex, but differ in
30 showing an absorption band at 232 nm.
However, chlamyhyaluronic acid is similar to the hyaluronic
acid known in the art. It is degraded by hyaluronate lyase and it reacts
CA 02249l03 l998-09-30
-53-
with proteins to form either a granular precipitate or a stable "mucin" clot,
i.e. it forms stable colloidal suspensions with acidic serum. This property,
as well as its antibacterial activity, point to its potential for efficacy as anagent for wound healing.
EXAMPLE 12
(~ommercial Production
Using the novel process described in this application,
Chlamydomonas segnis Ettl can be induced in cultures to produce
encapsulated cells with extracellular chlamyhyaluronic acid in its capsules.
10 Such cultures may be utilized commercially by known algal biomass
production systems. Such systems provide culture containers or vessels
designed to allow light absorbance, aeration and harvesting the algal
biomass on a large scale. Existing systems which are used in algal
biotechnology, and described in Hydrobiologia, 204-205, pgs 401-408 (1990),
Biotechnology Techniques~ 4, pgS 321-324 ~1990)~ Bioresource Technology,
38, pgs 233-235 (1991), and in Biotechnology Advances, 8, pgs 709-728(1990),
can be modified to produce mass cultures of encapsulated
Chlamydomonas segnis Ettl. The economic feasibility parallels that of
mass cultivation of microalgae such as Spirulina and Dunaliella for
human nutrition as health food, which has achieved economic success.
Preparation of CENC by a two-step procedure consisting of
dehydrating and settling the algal biomass, will provide a relatively clean,
inexpensive source of chlamyhyaluronic acid. Partial purification of the
dehydrated raw algal preparation would yield on the average about 0.6 g
chlamyhyaluronic acid per one litre algal suspension or per 0.98-1.67 g
algal dry matter. In contrast, about 1250 grams rooster combs are required
for the production of one gram hyaluronic acid by Fermentech UK of
Woking, Surrey, as reported in Chemistry and Indus~ry, 11: 374 (1986), and
76 g of dried human umbilical cord would yield 1.5 g of hyaluronic acid as
reported in the early study of Meyer and Palmer, Journal of Biological
Chemistry, 114, pgs 689-703(1936).
CA 02249103 1998-09-30
Having illustrated and described the principles of the
invention in a preferred embodiment, it should be appreciated to those
skilled in the art that the invention can be modified in arrangement and
detail without departure from such principles. We claim all modifications
5 coming within the scope of the following claims.
All publications, patents and patent applications referred to
herein are incorporated by reference in their entirety to the same extent as
if each individual publication, patent or patent application was specifically
and individually indicated to be incorporated by reference in its entirety.
CA 02249103 1998-09-30
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TABLE 1
Composition of the low salt Woods Hole MBL pH7.2 medium before (A) and after
mol1ifi~ Afi-ln (B), compared to the high salt nutrient medium (C) routinely used for growing
Chlamydomonas segnis Ettl at pH7+0.1.
A (~ i.. B ~ n~n~rAtir~n C C~-)n~l~n~rA~i~n
Salt (mM) Salt (mM) Salt (mM)
CaCl2-2H2O 0.25 CaCl2-2H2O 0.25 Cacl2 0.10
MgSO4 7H2o 0.15 MgSO4 7H2O 0.15 MgSO4 7H2O 1.00
NaHCO3 0.15 NaHCO3~ 0.00 NaH2PO4.H2O 3.70
K2HPO4 0.05 K2HPO4 0.05 K2HPO4.2H20 4.50
NaNO3 1.00 NaNO3 1.00 KNO3 10.00
Na2SiO3-9H20 0.10 Na2SiO3 9H20~ 0.00
Na2EDTA 0.012 Na2EDTA 0.012 Na2EDTA 0.025
Fecl3 6H2O Q012 Fecl3 GH2O 0.012 FeSO4 7H2O 0.025
(llM) (llM) (llM)
CuSO4.5H2O 0.04 CuSO4.5H2O 0.04 CuS04 0.038
ZnsO4 7H2o 0.076 znSO4.7H2O 0.076 ZnSO4.7H2O 0.350
CoCI2.6H2O 0.04 CoCk.6H2O 0.04 CoCI2.6H2O 0.034
MnCI2 4H2O 0.91 Mncl2 4H2O 0.91 MnSO4 4H2O 0.120
Na2MoO4 2H2O 0.025 Na2MoO4.2H2O 0.025 (NH4)6Mo7O24.4H2O 0.0049
Tris-HCI Buffer, Tris-HCI Buffer H3BO3 1.5
pH7.2 4000 pH7.2 500
Vitamins (llg) 101 Vitamins (~lg) 000
Total with ~ Total without L2~ Total (mM) l9.;~iZ
Vitamins (mM) Vitamins & salts
marked~ (mM)
CA 02249103 1998-09-30
-56-
TABLE 2
Rates of photosynthetic oxygen evolution (Ps), activities of photosystem I (PSI),
photosystem II (PSII), and catalase (Cat) from low salt boron-deficient shallow
cultures of Chlamydomonas segnis Ettl at early (24 h) and late (72 h) encapsulation
phases, using the methods adopted by Badour and Irvine, Botanica Acta, 103, pgs
149-154 (1990).
,u mol ~2 mg Chlorophyll-l h-l (n=4)
24h 72h
Ps 1016.5 + 99.7 406.4 + 33.6
PSII 1401.0 i 139.4 455.1 ~ 41.9
PSI 1371.9 99.2 431.8 i 62.0
Cat 2591.1 + 149.0 1092.4 +118.4
CA 02249103 1998-09-30
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TABLE 3
Yields of the phosphate buffered (pH 7.0)-sodium chloride (0.8 M) extracts of CENC,
after being precipitated in about 80% ethanol and dried by gentle aeration with
nitrogen gas; values in brackets are the water contents of CENC samples given inpercentage of their wet weights.
Grams per litre of Chlamydomonas segnis Ettl encapsulated culture
Example Moist Desiccated Dried Ethanol
No. CENC CENC Precipitate
2.07 (52.7) 0.98 0.38
2 2.69 (48.3) 1.39 0.66
3 1.97 (58.4) 0.82 0.47
4 3.08 (45.8) 1.67 0.75
CA 02249103 1998-09-30
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TABLE 4
Increased turbidity, measured at 500 nm with Ultroscopic 2000 (Pl~rm~ Biotech)
Spectrophotometer, due to the increases in the concentrations of Chlamyhyaluronic
acid (ChlamyHA) and human umbilical cord hyaluronic acid (U cord HA), using one
ml of hyaluronic acid solutions in 0.02 M acetate buffer, pH5.0, to which ten mls of the
acid albumin solution as describedby Dorfman, Methods in Enzymology, I, pgs.l66-173
(1957), are added.
ChlamyHA U cord HA
mg ml-l Absorbance ~hsorb~n' ~ Absorbance Ahsorb~rlce
[HA] [HA~
0.4 0.102 0.255 0.261 0.653
0.8 0.211 0.264 0.544 0.680
1.2 0.294 0.245 0.802 0.668
1.6 0.389 0.243 1.060 0.663
2.0 0.526 0.263 1.209 0.605
CA 02249103 1998-09-30
- 59 -
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