Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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BIOCHEMICAL PROCESS END COMPOSITION
This invention relates to a novel process
for the biodegradation of pentosans, to an enzyme
useful therefore and a process for obtaining a composition
containing it.
Pentosans are a class of polymers of one
or more naturally-occurring pentoses. They occur
in a variety of natural sources, such as cereals,
e.g. wheat and oats, plants erg. tea and coffee,
seaweed, fruits, vegetables and sorghum, and their
presence is undesirable in a number of applications
to which the natural source may be put. Thus,
for example, pentosans occur in cereal flour, in
which they bind water, and they contribute to stiffening
or staling of bread after baking. reduction
in the pentosan content of the flour reduces the
liability to stiffening in this situation. In
another example, pentosans occur in coffee and
occur in soluble gums co-extracted with the coffee
in the manufacture of instant coffee granules or
powder. Reducing the pentosan content of such
materials enables higher solid levels to be achieved
in the extraction with consequent economies in
the manufacturing process.
In our British Patent Specification No. 1421127,
we describe a method for the production of enzymes
which will degrade ~-glucans derived from barley
and related B-1,4/~-1,3 glucans. Roy principal
enzyme obtained is a 4/3~1,3-glucanase, of
particular value in the treatment of barley, although
other ~-glucanase, laminarinase, hydroxyethyl cellulose,
cellulose and aimless activities are said also
to be able to be present. Microorganisms of the
species Penicillium emersonii are employed as the
enzyme source, and in particular the strain which
was deposited with the Commonwealth Mycological
Institute, Knew, England under the number IMP 116815.
I! I I
We have now been able to ferment TalaromYces
emersonil (formerly known as Penicillium emersonii)
in order to produce another enzyme, a pentosanase,
which will catalyze the degradation of pentosans,
particularly pentosans derived from wheat The
class of such enzymes is generally known and various
individual enzymes have been used in, inter alias
baking, starch manufacture, farming, brewing, the
extraction of vegetable tissue and the manufacture
lo of vegetable coloring materials. However, in
many of these applications, high temperatures are
routinely employed and there is a need for a thermos table
pentosanase which will retain good activity over
a wide range of elevated temperatures.
Thus in one aspect of the present invention
we provide an enzyme obtainable by fermentation
from a strain of the species Talaromyces emersonii,
in particular from TalaromYces emersonii Stork,
sync Penicillium emersonii Stork IMP 116815, or
a mutant thereof, which is capable of catalyzing
the degradation of pentosans~ The new enzyme has
been found lo, be produced in good yield and at
a concentration highly satisfactory for commercial
use.
In a particular aspect of the invention we
provide an enzyme obtainable by fermentation from
a strain of the species T. emersonii, in particular
T. emersonii IMP ]16815, or a mutant thereof, which
is capable of catalyzing the degradation of Dylan.
Dylan is a polymer of the Penrose Zulus.
The new enzyme has been found to be thermos table,
i.e. it retains good activity at elevated temperatures,
and hence is of particular value in the high temperature
degradation of pentosans. For example, in stability
measurements, using enzyme prepared according to
the procedure of Example l described hereinafter,
the enzyme still retained 50% of its initial activity
Al ~2~i~35
after heating at 95~C for six minutes at pi 5.0
in the absence of substrate. The enzyme also exhibits
an advantageous temperature-activity relationship,
with optimum activity using oat hull Dylan as substrate
at 87 + 2C and retention of 45% of optimum activity
at 95C. This is unusual in an enzyme of finagle
origin, and contrasts for example, with the ~-glucanase
described above which has a poorly defined optimum
at 60-70C and only retains 40~ of this peak activity0 at 80C~
The enzyme according to the invention has
the following characteristics, measured using a
crude enzyme preparation obtained by the procedure
of Example 1 described hereinafter:5 (a) the pi for the optimum activity of the new
enzyme is 5.0, measured at 50C and using
oat hull Dylan as the pentosan substrate;
(b) the temperature for the optimum activity
of the new enzyme is 87+2C, measured at
pi 5.0 using oat hull Dylan as the pentosan
substrate;
(c) the presence of previously added Zulus,
glucose or maltose has no effect on enzyme
activity when subsequently assayed at pi
5.0 and 50C using oat hull Dylan substrate.
The enzyme according to the invention may
generally be prepared by fermenting an inoculum
of T. emersonii, for example T. Emerson IMP 116815
or a mutant thereof in a nutrient medium therefore.
However, we have further found that the amount
of pentosanase naturally produced may be increased
relative to the amount of 3-glucanase produced
by suitable modification of the fermentation medium.
An enzyme preparation obtained directly by the
fermentation of Talaromyces emersonii, desirably
T. emersonii IMP 116815 in which the level of pentosanase
activity relative to any ~-1,4/~-1,3-glucanase
activity is higher as a result of such modification
than that which may conventionally be obtained
is a preferred feature of the invention, as is
a process for its preparation.
The fermentation may be carried out by methods
well-known in the fermentation industry. Thus
the strain of T. emersonii may be cultured under
aerobic conditions, preferably in submerged culture,
with agitation or stirring with air or oxygen.
The fermentation medium employed should contain
an assimilable source of carbon, a digestible source
of nitrogen and, if desired, growth-promoting substances
as well as inorganic salts.
Suitable carbon sources include materials
rich in pentosans, for example, cereals such as
barley and wheat, distillers solubles or other
malt-or grain-distillation by-products, cellulose
e.g. Polka Floe, or Dylan.
Suitable nitrogen sources include, for example,
barley, distillers solubles or other malt- or grain-
distillation by-products, soya meal, nitrates or
ammonium salts such as ammonium dihydrogen phosphate.
Where it is desired to increase the level
of pentosanase activity relative to any ~-glucanase
activity, a carbon or nitrogen source selected
from Scotagran (a mix of solubles with dried barley
grains), ammonium nitrate, pot ale syrup, maize
pellets (dried maize grains mixed with solubles),
Curve syrup and corn steep liquor will desirably
be used.
Inorganic salts which may be used in the
fermentation medium may be, for example, sulfites
or chlorides of potassium, magnesium or sodium.
Growth promoting substances which may be
used include trace elements such as manganese,
iron, zinc, copper or phosphorus.
Advantageously, the fermentation medium contains
barley in a concentration in the range 0.2 to 3.5
w/v, distillers solubles or other malt or grain
distillation by-products in a concentration in
the range 0.4 to 5.5% w/v, and 0.2 to 3.0% w/v
cellulose.
Culturing conditions such as temperature,
pi and fermentation time are selected such that
the strain employed may accumulate a maximum amount
of the desired enzyme For example, the fermentation
is advantageously carried out at a temperature
ranging from 35-60C, preferably 50-54C, at a
pi from 3.5-4.5 and for from 1-20 days, preferably
7-10 days.
The crude culture liquid can be used directly
for its enzymatic action, or if desired the whole
culture broth may be dried and the resulting powder
used. If desired, some purification of the pentosanase
may be carried out, e.g. by chromatographic techniques,
to provide an enzyme preparation having increased
pentosanase activity relative to any ~-1,4/1,3-
glucanase activity and a method for effecting such
purification and the product obtained comprise
further aspects of the invention.
Alternatively, the enzyme, which is exocellular,
may be extracted from the fermentation product
by, for example, conventional methods. Thus for
example a first stage is normally to filter off
the Muslim formed, preferably by means of precut
filtration, i.e. using a filter which has been
coated with a filter aid. The resulting filtrate
may be used directly, or, conveniently, may be
concentrated, preferably in vacua to yield the
enzyme in a liquid concentrate form. The liquid
concentrate may itself be employed as the enzyme
source. Materials such as sodium chloride, sodium
bonniest or sodium metabisulphite which confer
enzyme storage stability, enzyme thermostability
I 35
-- 6
and/or bacteriological stability may if desired
be added to such liquid concentrates. Alternatively,
the liquid concentrate may be absorbed in a suitable
solid, for example ground wheat or barley, optionally
in the presence of a carrier such as sodium carboxymethyl
cellulose, and the resulting damp mass dried to
yield an active enzyme product. If desired, the
liquid concentrate itself may be dried, e.g. by
spray drying, freeze drying or roller drying to
yield a dry enzyme composition.
Unless the extraction is rigorous, which
for commercial purposes is usually avoided for
economic reasons and is not necessary given the
range of uses to which a cruder composition can
be put, the composition will usually contain other
enzyme activities.
Where a solid enzyme product is desired,
this may be obtained from the filtrate or a liquid
concentrate thereof by conventional methods, such
as precipitation by addition for example of an
excess of a water-miscible organic solvent such
as an alcohol e.g. ethanol or a kitten e.g. acetone.
Either direct precipitation or precipitation onto
a carrier may be used typical carriers including
for example starch methyl cellulose or sodium
carboxymethyl cellulose.
The inoculum ox T. emersonii used in the
. .
fermentation may be obtained by, for example, scaling
up from a surface culture of the organism. Scaling
up to productive fermentation may conveniently
be effected by carrying out a laboratory stage
of vegetative growth, followed by one or more seed
stages in stirred fermentation vessels.
Thus, in a typical inoculum preparation,
the organism is streaked onto a solid nutrient
medium, e.g. an ajar medium containing petunia
(e.g. 0.5~ w/v), sodium chloride (e.g. 0.4% wove),
;226~35
glycerol (e.g. 0.75~ w/v), molasses twig 0.8~
w/v), potassium dihydrogen phosphate (e.g. 0.006%
w/v) and magnesium sulfite (e.g. 0.005% McCoy
w/v) which has previously been sterilized, e.g.
by autoclaving at about 120C for 15 minutes, and
allowed to cool. Incubation is preferably carried
out at about 37C for about 10 days, after which
the spores produced are used to inoculate a liquid
medium for vegetative growth, sterilized by e.g.
autoclaving, at about 120C for 15 minutes, and
containing malt extract (e.g. 3.3~ w/v), yeast
extract (e.g. 2.0~ w/v) and ammonium dihydrogen
phosphate (e.g. 0.6~ w/v). The culture is preferably
effected until good mycelial growth is present,
for example, when using about 500ml of medium,
for about 72 hours at 45C.
The resulting vegetative culture is then
used to inoculate a first stirred seed stage.
The medium for this stage preferably contains similar
components to those described previously for the
main fermentation medium, and is preferably prepared
by sterilizing all the ingredients together in
the seed stage vessel e.g. by steam injection.
A typical medium for the seed stage is 'Medium
A' shown hereinafter in Table 1. Using about 40
liters of medium, culturing is preferably effected
at about 50C for about 76 hours with stirring,
e.g. at 420 rum and aeration, e.g. 50 litres/minute
of air.
The seed culture thus obtained may then be
used to inoculate a producing stage as described
above or, if more seed is required, may be used
in further seed stages, identical in nature to
be first.
Mutants of T. emersonii for use in the above
fermentation processes may be obtained by conventional
methods of strain improvements, e.g. by the use
I
of ionizing radiation (for example X and y-rays;
us light; or us light in the presence of a photo-
sensitizing agent such as 8-methoxypsoralen), chemicals
(e.g. nitrous oxide; hydroxylamine; pyrimidine
base analogies such as 5-bromouracil; assuredness;
alkylating agents such as ethyl methanesulphonate
or mustard gas; hydrogen peroxide; phenols or formal-
Dodd), heat or genetic techniques (ego recombination,
transduction, transformation, lysogenisation, lysogenic
conversion and selective techniques for spontaneous
mutants).
The desired enzyme activity may be determined
in the culture liquid or at any point in an isolation
procedure by a simple test designed to determine
the reducing sugars released by the action of the
enzyme on a pentosan substrate. Thus, in one test,
a sample of the enzyme preparation to be measured
is suitably diluted and is then incubated for 10
minutes with an excess of the pentosan Dylan, at
50C in pi 5.0 acetate buffer. The sugar released
is determined as a maltose equivalent by adding
alkaline 3,5-dinitrosalicylic acid reagent, heating
for 5 minutes on a boiling water bath, quenching
in ice water, measuring the absorption of the solution
at 540nm and converting this to a maltose equivalent
by reference to a standard calibration graph obtained
by preparing standard dilutions of maltose of known
moisture content and reacting these with the donator-
salicylic reagent under the conditions lust described.
As used herein, one unit of enzyme releases 1 my
of maltose equivalent per minute under the test
conditions at 50C and pi 5Ø
The enzyme according to the invention is
useful for a variety of purposes for which other
pentosanases are used. Thus, for example, it may
be used in baking, to reduce the natural pentosan
content of wheat flour and so improve the keeping
qualities of bread. It may be used in starch manufacture
and hydrolysis, to lower the pentosan content of
cereals and reduce the viscosity of starch slurries
which otherwise adversely affect the efficiency
and cost of starch recovery and subsequent hydrolysis
It may be used in farming, to upgrade the quality
of mixed silage and animal feeds. It may be used
in brewing, for the improved production and extraction
of fermentable sugars when the mash includes e.g.
wheat or sorghum, and for the prevention or treatment
of certain types of haze. It may be used for the
extraction of vegetable tissue, for example in
the extraction of tea, coffee and fruits; and to
extract alginates from seaweed; and in the manufacture
of natural vegetable coloring materials. In some
of these applications, the thermostability of the
enzyme is particularly advantageous in view of
the temperatures at which the processes are conducted.
In a further aspect of the invention, therefore,
we provide a method of reducing the pentosan content
of a pentosan-containing material which comprises
contacting the said material with an enzyme of
the invention.
Typical pentosans which may be degraded with
the enzyme of the invention include those found
in cereals such as wheat and oats; plants such as
tea and coffee and in extracts thereof; fruits;
vegetables; seaweed and sorghum. In one example,
the pentosan may be Dylan, for example an oat hull
Dylan. In a preferred embodiment, the pentosan-
containing material which is to be degraded is
derived from wheat.
In a further embodiment of the invention
we provide a method of reducing the Dylan content
of a xylan-containing material wherein the material
is contacted with an enzyme according to the invention.
The enzyme or composition according to the
33~
- 10 -
invention may be used employing methods customarily
found in enzyme technology. Thus for example the
enzyme may be contacted with an aqueous medium
containing the substrate either in suspension,
admix or in a solution. The reaction temperature
will vary according to the exact nature of the
reaction but will in general be in the range 35-
95C, advantageously at the higher end of the range,
in some applications at 90C and above The pi
of the reaction mixture may be for example in the
range 4.0-6.5, preferably 4.5-5.5 and in particular
at pi 5Ø If desired, the reaction mixture may
contain other added enzymes, for example aimless.
The strain of Toolers emersonii which
we have used is a sub-culture of that which was
deposited at the Commonwealth Mycological Institute,
Knew, England under Number IMP 116815 in 1972 before
the Budapest Treaty on the International Recognition
of the Deposit of Microorganisms for the purposes
of Patent Procedure (Budapest 1977) was signed.
We have recently redeposited this strain (on
1984) under slumber IMP 290604 at the same depository
but under the conditions of the Treaty of Budapest
and it is our belief that the two strains are identical
and that either strain may be used.
The following Examples illustrate the invention.
All temperatures are in C. All percentages are
in w/v.
I
E _ pie 1
Talaromyces emersonii IMP 116815 was cultured
at 37 for 10 days on an ajar medium sterilized
in an autoclave and containing 0.5% petunia, 0.4~
Nail, 0.75% glycerol, 0.8% molasses, 0.006% KH2PO4
and 0.005~ McCoy).
The spores produced were used to inoculate
flasks of a liquid medium sterilized by autoclaving
and containing 3.3% malt extract, 2.0% yeast extract
and 0.6% ammonium dihydrogen phosphate. The flasks
were placed on a rotary shaker (210 rev/min; 4.9 cm
throw) for 3 days at 45.
The resulting vegetative culture (400 ml)
was used to inoculate 40 liters of Medium A) Table 1
sterilized by steam injection in a 50 lithe capacity
stainless steel fermenter. The culture was stirred
at 420 rev/min and aerated at 50 litres/min for
75 hours at 50.
The resulting culture (4.5 L) was used to
inoculate 40 liters of Medium A (Table 1) sterilized
by steam injection in a 50 lithe capacity stainless
steel fermenter. The culture was stirred at 420
rev/min and aerated at 50 litres/min for 75 hours
at 50.
The resulting culture (4.5 L) was used to
inoculate 40 liters of Medium A sterilized by steam
injection in a 50 lithe capacity stainless steel
fermenter. The culture was stirred at 420 rev/min
and aerated at 50 litres/min for 36 hours at 50.
The resulting culture (16 L) was used to
inoculate 32 liters of Medium B (Table 1) sterilized
by steam injection in a 50 lithe capacity stainless
steel fermenter.
The culture was aerated at 50 litres/min.
35 and stirred intermittently at 420 rev/min. at 50
for 10 days. Sterile water was added at an average
rate of 1.5 liters per day.
- 12 -
Assay of the exocellular fluid yielded a
concentration of the enzyme according to the invention
Of 36 u/
Table 1
Ingredient Medium A (~) Medium B (~)
Ground Barley 1.0 3.38
Distillers Solubles 2.25 5.06
Polka Floe 2025 2.53
( H4)2 H2PO4 0.34 0 77
K2SO4 0.12 0.27
M9S04'7H20 0.01 0.11
Nail 0.11 0.11
MnSO4-4H2o .S 0.001
15 Phase 0.004 0.008
ZnSO4.7H2O 0.004 0.008
QUIZ 0 0005 0.001
POW 0.5 1.02
Water to 100
Example 2
Demonstration of variation in pentosanase: glucanase
ratio on fermentation
a) Spores were produced as in Example 1 and
used to inoculate a seed stage in 250 ml
flasks containing 40 ml of Medium A sterilized
by autoclaving. The flasks were incubated
at 45 for 72 hours, shaking at 200 rev/min.
The resulting culture (4 ml) was used to
inoculate 40 ml of Medium B sterilized by
autoclaving in 250 ml flasks. after incubating
at 45 for 12 days, shaking at 200 rev/min,
the exocellular culture fluid was assayed
or pentosanase (P) and barley ~-glucanase
(G) activities and the P : G ratio was calculated.
b) Using essentially the same system as in a)
above.
I
Omitting the Polka Floe and including oat
spells Dylan (2.2%) gave a P : G ratio 71%
higher than that obtained in a).
Example 3
In order to demonstrate pentosanase activity
in the enzyme produced in Example 1, a 1% pentosan
solution was incubated at 50 in buffer at pi 5.0
in the presence of added enzyme (test system) or
with the addition of an equivalent amount of water
(control system). The pentosan substrate used
was Dylan, obtained from oat hulls.
Samples of the test system were withdrawn
after 10 minutes, 30 minutes and 22.5 hours, quenched
to prevent any further reaction and then spotted
on to 20 cm square thin layer chromatography plates
covered with 0.25 mm kieselgel. Spots of selected
standard sugar solutions and of the control system
were also applied.
The mobile phase was n-butanol:pyridine:ethanol:
water (20:15:25:10). Detection was by spraying
the dried plates with 5% ammonium molybdate in
5% sulfuric acid followed by heating at 105 for
15 miss.
All the test samples showed inter aria a
spot of identical Of value to that given by the
standard sugar solution Zulus.
Since the control sample gave only a single
spot on the origin, this test spot can not have
derived either from Zulus contamination of the
substrate or from its chemical hydrolysis: it must
have arisen by enzymic degradation of the pentosan.
Example 4
The following comparative test was designed
to illustrate the potential utility of the enzyme
according to the invention in an enzymatic wheat
;26~35
- 14 -
starch digestion process:
Enzyme (2 ml) prepared according to Example 1,
low grade wheat flour (155 g), sodium chlorite
(2 9), calcium chloride (0.17 g), 'Termamyl' bacterial
alpha aimless (Nova Industry A/S) (1 ml) and water
(250 ml) were shaken thoroughly in a glass flask.
The pi was adjusted to 6.5 using caustic soda.
The neck of the container was covered to minimize
evaporation and the flask was held for 5 hours
at a temperature of 90. Starch hydrolysis occurred
(negative iodine test result) and the divest viscosity
was 43 cups.
In a control experiment omitting the enzyme
of the invention the final viscosity of the digest
was 127 cups.
The following Examples illustrate experiments
performed to characterize the enzyme according
to the invention In each Example, the enzyme
used was prepared according to the method of Example
1 except that small amounts of sodium bonniest
and sodium met:abisulphite were added to the exocellular
fluid as bacteriological stabilizers. Enzyme activity
was determined in each Example using an excess
of 1% aqueous oat hull. Dylan as the pentosan substrate
and measuring the increase in reducing activity
after a 10 mix incubation, against a Zulus calibration
curve. The pi and temperature of each incubation
are stated in the Examples.
-
Example 5
The pH-activity relationship was obtained
at a temperature of 50
pi 4.0 4.5 5.0 5.5 6.0 6.5
.
% Maximum Activity 62 85 100 84 70 47
_ _ __ I_._ _ _ _ _ _ _ ._ _ _ _ ._ _ _ , _ _. _ _ _ _ _ ___ _ __ __ _
ago Pry'
I
- 15 -
Significant hydrolysis occurs throughout
the range illustrated.
Example 6
Thermal stability was studied by holding
samples at temperatures of 60, 70, 80, 90 and 95
in the absence of substrate and withdrawing amounts
at timed intervals to obtained a family of residual
activity-time curves. The activity remaining was
measured at pi 5.0 and 50:
- at 60, there was no detectable loss in activity
after 80 miss,
- at 70, 70~ of the initial activity was still
present after 1 h,
15 - at 80, 70% of the initial activity was still
present after 30 miss,
- at 90, 50~ of the initial activity was still
present after 8 miss,
- at 95, 50% of the initial activity was still
present after 6 miss.
Example 7
The temperature - activity relationship was
obtained at a pi of 5Ø Inspection of the activity-
temperature curve showed that peak activity occurred at 87+2. Activities at the individual temperatures
studied, expressed as percentages of this maximum,
were as follows:
% Maximum Activity 13 24 41 57 86 [100] 90 45
Temperature 40 50 60 70 80 [87+2] 90 95
The results confirm the inherent thermal
stability of the new enzyme, as shown in example
5. The enzyme can clearly be used at temperatures
in excess of 90.
~6~33~
-- 16 -
This contrasts with a poorly defined optimum
of 60-70 for the barley ~-glucanase of British
Specification 1,421,127. At 80, only 40% of peak
activity is recorded.
Example 8
In a preliminary study to determine whether
activity of the enzymes was affected by products
of the reaction and/or other sugars present in
a typical digest in an industrial process, three
simple sugars (Zulus, glucose and maltose) were
separately added to the enzyme, which was then
assayed at 50 and pi 5Ø Each sugar was added
in an amount equivalent to the amount of Zulus
liberated from the substrate under the assay conditions.
There was no inhibition or promotion of enzyme
activity, and thus, on this evidence, there would
be no interference with enzyme activity from the
accumulation of simple sugars that will occur in
many industrial applications.
