Note: Descriptions are shown in the official language in which they were submitted.
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ENZYMATIC HYDROLYSIS OF FUCANS
BACKGROUND
[0001] Fucans (including fucoidan) are sulfated polysaccharides. In general
terms, this means that
they are molecules made up of a number of sugar groups, and also have sulfur
atoms attached to
the sugar groups. The main sugar group is called "fucose", which is sugar that
has 6 carbon atoms
and has the chemical formula C6E11205. "Fucoidan" (or fucoidin) indicates
fucans derived from
brown algae (seaweed). Fucans can exist alone, or in a mixture of other
sugars, for example in a
mixture of sugars such as xylose, galactose, glucose, glucuronic acid and/or
mannose. These other
sugars may be extracted from the seaweed or other source with the fucan.
Although fucans are
currently derived from natural sources such as the brown algae (seaweeds), sea
cucumbers, etc.,
mentioned herein, "fucan" includes polymer molecules having the chemical and
structural motifs
of the fucans as discussed herein regardless of the ultimate source(s) of the
fucans.
[0002] Fucoidan can be obtained from a variety of species of brown algae
including but not limited
to: Adenocystis utricularis, Ascophyllum nodosum, Chorda ilium, Cystoseirabies
marina,
Durvillaea antarctica, Ecklonia kurome, Ecklonia maxima, Eisenia bicyclis,
Fucus evanescens,
Fucus vesiculosis, Hizikia fusiforme, Himanthalia Elongata, Kjellmaniella
crassifolia, Laminaria
brasiliensis, Laminaria cichorioides, Laminaria hyperborea, Laminaria
japonica, Laminaria
saccharina, Lessonia trabeculata, Macrocystis pyrifera, Pelvetia fastigiata,
Pelvetia
Canaliculata, Saccharina japonica, Saccharina latissima, Sargassum
stenophylum, Sargassum
thunbergii, Sargassum confusum, Sargassum fusiforme and Undaria pinnatifida.
These exemplary
species are all from the taxonomic class Phaeophyceae and the majority of
these species fall into
the families of Fucales and Laminariaceae.
[0003] Fucans including fucoidan have been shown to be efficacious in serving
as a barrier device
to prevent, inhibit, and treat the formation of fibrous adhesions. They have
also found use in the
treatment of other related diseases and conditions.
[0004] Thus, there has gone unmet a need for improved methods in the
preparation of fucan
compositions having desired molecular weight distributions. Previous works
discuss the use of a
fucan specific fucanase from a flavobacterian strain 5W5, known as substrate
specific MfFcnA
enzyme (Colin, et al., 2006) in the hydrolysis of some fucans. The present
methods, systems, etc.,
are directed, among other advantages, to providing enzymes capable of
hydrolyzing selected fucan
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compositions and methods for obtaining desired molecular weight fucan
distribution compositions
from a feedstock fucan composition using these enzymes.
SUMMARY
[0005] Methods, systems, compositions etc., are provided for obtaining and
utilizing fucanases
capable of enzymatic hydrolysis of fucans including fucans in feedstock fucan
compositions to
obtain a desired lower molecular weight distribution fucan relative to the
feedstock fucan
composition. The embodiments herein include fucanase-genetic and fucanase-
amino acid
sequences that may be used in the production or synthesis of fucanases and the
production of fucan
compositions having reduced average molecular weight fucans after hydrolysis
by the fucanase(s).
The methods for hydrolyzing the feedstock fucan compositions using the
produced fucanases
provides fucan substrate selectivity and time-based control during the
enzymatic hydrolysis.
[0006] The present methods, systems, etc., include obtaining desired fucan
compositions
comprising a desired molecular weight distribution from a feedstock fucan
composition, as well
as compositions comprising such a desired fucan molecular weight distribution,
and methods of
use of such compositions. In one aspect, the methods comprise:
[0007] The present compositions, systems and methods, etc., provide
compositions that can
comprise a P5AFcnA fucanase and/or a P19DFcnA fucanase in an aqueous solution
and an added
fucan. The added fucan can be a feedstock fucan composition, which composition
can have a pH
of about 6.5-9.0, a temperature of about 15 C-40 C, and be at conditions such
that the fucanase
can hydrolyze glycosidic bonds in a fucan.
[0008] In certain embodiments, the present compositions, systems and methods,
etc., comprise an
enzyme such as a fucanase produced by a prokaryotic or eukaryotic entity, the
fucanase coded by
a gene sequence according to any one of SEQ ID NOS. 1 to 4, or a gene sequence
based on such
SEQ ID NOS. in which at least 0, 1, 2, 3, 4 or 5 codons and fewer than 1%, 2%,
3%, 4% or 5%
of codons are substituted. In further embodiments, the present compositions,
systems and methods,
etc., comprise a fucanase produced by a prokaryotic or eukaryotic entity, the
fucanase can
comprise an amino acid sequence according to any one of SEQ ID NOS. 5 to 9 in
which at least
in which at least 0, 1, 2, 3, 4 or 5 amino acid and fewer than 1%, 2%, 3%, 4%
or 5% of amino
acids are substituted. The prokaryotic entity can be a Psychromonas species or
Escherichia Coil.
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[0009] The methods can comprise enzymatically hydrolyzing a fucan in an
aqueous solution, not
in a Psychromonas species, for example in a manmade solution contained in a
manmade vessel,
using a Psychromonas fucanase or other fucanase as discussed herein. The fucan
can be a fucoidan
and can be obtained from at least one of Adenocystis utricularis, Ascophyllum
nodosum, Chorda
ilium, Cystoseirabies marina, Durvillaea antarctica, Ecklonia kurome, Ecklonia
maxima, Eisenia
bicyclis, Fucus evanescens, Fucus vesiculosis, Hizikia fusiforme, Himanthalia
Elongata,
Kjellmaniella crassifolia, Laminaria brasiliensis, Laminaria cichorioides,
Laminaria hyperborea,
Laminaria japonica, Laminaria saccharina, Lessonia trabeculata, Macrocystis
pyrifer a, Pelvetia
fastigiata, Pelvetia Canaliculata, Saccharina japonica, Saccharina latissima,
Sargassum
stenophylum, Sargassum thunbergii, Sargassum confusum, Sargassum fusiforme and
Undaria
pinnatifida.. In certain embodiments, the fucan or fucoidan is obtained from
at least one of
Saccharina japonica, Laminaria hyperborea, Macrocystis pyifera and Chorda
ilium. The
hydrolzying can result in the average molecular weight of the fucan being
reduced by at least about
5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%. The hydrolzying can
result in at
least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the
glycosidic bonds
in the fucan being hydrolyzed.
[00010] Also provided are methods for obtaining desired lower molecular weight
fucan
compositions relative to a feedstock fucan composition by enzymatic hydrolysis
of fucans in the
feedstock fucan composition. The methods can comprise:
providing the feedstock fucan compositions and a Psychromonas fucanase in an
aqueous
solution;
incubating the feedstock fucan compositions and the Psychromonas fucanase
under
conditions sufficient to hydrolyze fucans in the feedstock fucan compositions
to produce
hydrolyzed fucans and hydrolysis remnant molecules; and
separating the hydrolyzed fucans from the hydrolysis remnant molecules and the
fucanase to obtain the desired molecular weight fucan compositions.
[00011] The fucanase can be coded by a gene sequence and/or comprise an amino
acid sequence
as discussed herein, for example according to one of SEQ ID NOS. 1-8, and the
fucan/fucan
feedstock composition can be sourced as discussed herein. The average
molecular weight of,
and/or glycosidic bonds present in, the fucan/fucan composition can be reduced
by at least 5%,
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%.
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[00012] Also provided are expression vectors comprising an expressible P5AFcnA
fucanase gene,
P19DFcnA fucanase gene, or other sequence as discussed herein, as well as
enzymes and fucanases
as discussed herein and expressed from, and typically collected and purified
from, such expression
vectors.
[00013] These and other aspects, features and embodiments are set forth
within this
application, including the following Detailed Description and attached
drawings. Unless
expressly stated otherwise, all embodiments, aspects, features, etc., can be
mixed and matched,
combined and permuted in any desired manner.
BRIEF DESCRIPTION OF THE DRAWINGS
[00014] FIG.1 depicts a flow chart for an exemplary method of obtaining a
lower molecular
weight fucan composition relative to a feedstock fucan composition by
enzymatic hydrolysis.
[00015] FIG 2A provides an example of a C-PAGE gel showing hydrolysis of a
fucoidan
composition extracted from Saccharina Japonica by P5AFcnA fucanase and
P19DFcnA fucanase
and absence of hydrolysis of a fucoidan composition extracted from Saccharina
Japonica by
MfFcnA fucanase.
[00016] FIG 2B provides an example of a C-PAGE gel showing hydrolysis of a
fucoidan
composition extracted from Laminaria Hyperborea by P5AFcnA fucanase and
P19DFcnA
fucanase and absence of hydrolysis of such fucoidan composition by MfFcnA
fucanase.
[00017] FIG 2C provides an example of a C-PAGE gel showing hydrolysis of a
fucoidan
composition extracted from Macrocystis pyrifera by P5AFcnA fucanase and
P19DFcnA fucanase
and absence of hydrolysis of such fucoidan composition by MfFcnA fucanase.
[00018] The drawings, including the flow chart, present exemplary embodiments
of the present
disclosure. Actual embodiments of the systems, methods, etc., herein may
include further features
or steps not shown in the drawings. The exemplifications set out herein
illustrate embodiments of
the systems, methods, etc., in one or more forms, and such exemplifications
are not to be construed
as limiting the scope of the disclosure in any manner. The embodiments herein
are not exhaustive
and do not limit the disclosure to the precise form disclosed, for example in
the following detailed
description.
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DETAILED DESCRIPTION
[00019] The methods, systems, compositions etc., herein are related to
fucanases capable of
enzymatic hydrolysis of feedstock fucan compositions to obtain a desired lower
molecular weight
distribution fucans relative to a feedstock fucan composition. Thus, the
fucanases discussed herein
are used to selectively, controllably cleave fucans to reduce their size so
that the molecular weight
distribution of the fucans in a feedstock composition is selectively,
controllably reduced to provide
a modified fucan composition comprising a desired, lower molecular weight
distribution of
modified fucans.
[00020] Certain embodiments of the methods etc. are shown as examples in the
Figures using
fucoidan as example of a fucan. The compositions of the coding region fucanase-
genetic sequences
and fucanase-amino acid sequences of the respective native and synthetic
fucanases are discussed.
The lower molecular weight fucan composition obtained as a product of the
enzymatic hydrolysis
of the feedstock fucan composition has a lower average molecular weight
distribution relative to
the feedstock fucan distribution. This shift in molecular weight distribution
may be accompanied
by an alteration in the dispersity and shape of the distribution.
[00021] Turning to the development of certain fucanases discussed herein, the
fucoidanolytic
Psychromonas species SW5A and SW19D were isolated from brown macro algae
collected from
the shores of Cadboro Bay and Willows Beach (Victoria, Vancouver Island,
British Columbia,
Canada). Genomic DNA was extracted from cultured Psychromonas species SW5A and
SW19D
and next-generation sequencing of their genomes was performed on an Illumina
MiSeq platform.
The genomes were annotated to identify putative genes encoding carbohydrate-
active enzymes.
The fucanase-gene sequences encoding the enzymes P5AFcnA and P19DFcnA were
identified in
the genomes of the Psychromonas species Psychromonas sp. SW5A and Psychromonas
sp.
SW19D during this annotation process.
[00022] The
fucanase-gene sequences encoding the enzymes P5AFcnA and P19DFcnA
were obtained from genomes of Psychromonas species Psychromonas sp. SW5A and
Psychromonas sp. SW19D. Synthetic fucanase-genes encoding the enzymes P5AFcnA
and
P19DFcnA that were codon optimized for expression in E. coil and pre-inserted
into the E. coil
expression plasmid pET28a were ordered from Genscript . These constructs were
referred to as
pET28a_p5AFcnA and pET28a P19DFcnA. The sequence fidelity of the constructs
was
confirmed by bi-directional DNA sequencing. Fucanases were expressed in an E.
coil over
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expression system with a six-histidine tag. To purify the recombinant protein,
the cells were
harvested, chemically lysed, and centrifuged to separate the cellular debris
from cell lysate
supernatant. The protein was then purified from the lysate by immobilized
nickel affinity
chromatography. Purified protein was dialyzed into 20 mM Tris pH 8.5 with 0.5
M NaCl buffer
and concentrated to 29 jilVi for further use. The coding region sequences for
the fucanase-genes
of native and synthetic P5AFcnA fucanase are shown in Tables 1A and 1B
respectively, SEQ ID
NOS. 1 and 2, respectively. The coding region sequences for the fucanase-genes
of native and
synthetic P19DFcnA fucanase are shown in Tables 1C and 1D respectively, SEQ ID
NOS. 3 and
4, respectively. The amino acid sequences for the native and synthetic P5AFcnA
fucanase are
shown in Tables 1E and 1F respectively, SEQ ID NOS. 5 and 6, respectively. The
amino acid
sequences for the native and synthetic P19DFcnA fucanase are shown in Tables
1G and 111
respectively, SEQ ID NOS. 7 and 8, respectively. In some embodiments, the
nucleic acid
sequences shown herein are controllably expressed in suitable expression
vectors,
https://en.wikipedia.org/wiki/Expression vector. Such expression vectors can
be optimized for
either prokaryotic or eukaryotic expression and can include plasmids,
expression viruses, cell-
free systems, etc., as desired. The expression vectors can also include
selected primers, origins of
replication, etc., as desired.
[00023] The fucanase-gene sequence shown in Table 1A, SEQ ID NO. 1, will
hereafter be referred
to as the native P5AFcnA gene sequence. The fucanase-gene sequence shown in
Table 1B, SEQ
ID NO. 2, will hereafter be referred to as the synthetic P5AFcnA gene
sequence. The fucanase-
gene sequence shown in Table 1C, SEQ ID NO. 3, will hereafter be referred to
as the native
P19DFcnA gene sequence. The fucanase-gene sequence shown in Table 1D, SEQ ID
NO. 4, will
hereafter be referred to as the synthetic P19DFcnA gene sequence. The
synthetic sequences are
optimized for expression of the enzyme with E. coil. The amino acid sequence
shown in Table
1E, SEQ ID NO. 5, will hereafter be referred to as the native P5AFcnA amino
acid sequence. The
amino acid sequence shown in Table 1F, SEQ ID NO. 6, will hereafter be
referred to as the
synthetic P5AFcnA amino acid sequence. The amino acid sequence shown in Table
1G, SEQ ID
NO. 7, will hereafter be referred to as the native P19DFcnA amino acid
sequence. The amino acid
sequence shown in Table 111, SEQ ID NO. 8, will hereafter be referred to as
the synthetic
P19DFcnA amino acid sequence.
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[00024]
Table 1A: Coding region sequence for the fucanase-gene used to code native
P5AFcnA
fucanase, SEQ ID NO. 1
20 30 40 50 60
ATGTTAATTT CAGTTACTGT TTTAGTAGGC TGTGGAAGCA GTAGTGATGA AGTTGAGTCT
70 80 90 100 110 120
ACAAATACAA CTGACTCATA TAACATTAAT AACACAGAAG CGACTGGTAT TGAATACAAT
130 140 150 160 170 180
GCTAGTTGGA TGGCAGGTAC TTGGGGGATT ACACAACGTG TAGACGGTGG TTATAAATTG
190 200 210 220 230 240
GATAACTCCG CAGACTCCTC TAATTGGCAG GCAGGTGCAG AAGAAATTGT TACTAACATT
250 260 270 280 290 300
CCTGCGGCAG AATACGTCAT AACATCATTT ACCCACCCAG CGCACGGTCA CCTATTCACA
310 320 330 340 350 360
TTACGAACTA ATAATAATGT GGATGTATCA GCAATTCACC CTGATATGGT ACCCACATTA
370 380 390 400 410 420
GAAAATGAAA AAATCATTCT TGATGTTATT AATATATATC GCGCTGCGGG TAAAAAAGTA
430 440 450 460 470 480
ATTCTATATT TAAATTCAGC AGGACCTTCA ATGGCAGAGG AGCGAGGTGA TACTGATATC
490 500 510 520 530 540
CAAGCTGCAT GGGATGAATA CTATATCAAT GAGTGGGATG GAGATGAAGC AGCTGCTTGG
550 560 570 580 590 600
CGTAATTTAG CTCGAGGATA TGTAGAGCGT TTCGATGGTT TAGTAGATGG CTATTGGTTA
610 620 630 640 650 660
GACAACTCCA GAAACTTGCC AGGTGAGGTA TCTGACTTTG TTGCTATGCT GCGTAGCGTT
670 680 690 700 710 720
GATCCCGAAT TAACGATTGC CGTTAATTAT GACCAACACT ACTTTACCGA TGATAACGGA
730 740 750 760 770 780
GAATATTTAT ATGTTGATTC TGACGGTTTA GATGATGAAG ATGAAAGCGA TTATAAAATA
790 800 810 820 830 840
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GTAAAACATG TAGTGACAAA TGAATACATG GATTTTACTA ACGGACATGT CACACCATTA
850 860 870 880 890 900
GGGCGAGGTG CGCCTCCCAA TTCTTGGGCT TATGAGGAAT ATACTATTCC AGATATGATT
910 920 930 940 950 960
GAGGTACCAT GGGAAACCTA TGATGGCAGT AAATATGCAT TAAAACACGG CTGGTTCCCT
970 980 990 1000 1010 1020
ATTAGAAACT CGTGGAGTGG TTCAAAAGCT GAACTTATGT TTGATGTTGA GCAAGCTTAT
1030 1040 1050 1060 1070 1080
CGATTTGTTA GAACAGTTAC TGATGGAGGA GCTGCAATGA CATGGTCAAC GACTCAAGAC
1090 1100 1110 1120 1130 1140
AATGGCTACA TGACTGCCGA TGAAATGAGT ATAATGATTG AAATTAGTAA TAGAATGACA
1150 1160 1170 1180 1190 1200
CAAACCCCTA AACCAGATTA CTCAGTTTAT GAAAGGCCAA AAGGCGCATA TCTAGTGAGT
1210
GAAATAGAAT AA
Table 1B: Coding region sequence for the fucanase-gene used to code synthetic
P5AFcnA
fucanase, SEQ ID NO. 2
20 30 40 50 60
ATGGGCAGCA GCCATCATCA TCATCATCAC AGCAGCGGCC TGGTGCCGCG CGGCAGCCAT
70 80 90 100 110 120
ATGGGTAGCA GCAGCGACGA GGTTGAAAGC ACCAACACCA CCGATAGCTA CAACATTAAC
130 140 150 160 170 180
AACACCGAGG CGACCGGCAT CGAATATAAC GCGAGCTGGA TGGCGGGTAC CTGGGGCATT
190 200 210 220 230 240
ACCCAGCGTG TGGACGGTGG CTACAAGCTG GACAACAGCG CGGATAGCAG CAACTGGCAA
250 260 270 280 290 300
GCGGGTGCGG AGGAAATTGT GACCAACATC CCGGCGGCGG AATACGTTAT TACCAGCTTC
310 320 330 340 350 360
ACCCATCCGG CGCATGGTCA CCTGTTTACC CTGCGTACCA ACAACAACGT GGACGTTAGC
370 380 390 400 410 420
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GCGATCCACC CGGATATGGT TCCGACCCTG GAGAACGAAA AAATCATTCT GGACGTGATC
430 440 450 460 470 480
AACATTTACC GTGCGGCGGG TAAGAAAGTT ATCCTGTATC TGAACAGCGC GGGTCCGAGC
490 500 510 520 530 540
ATGGCGGAGG AACGTGGCGA CACCGATATT CAGGCGGCGT GGGATGAGTA CTATATCAAC
550 560 570 580 590 600
GAGTGGGATG GTGATGAAGC GGCGGCGTGG CGTAACCTGG CGCGTGGCTA CGTGGAGCGT
610 620 630 640 650 660
TTCGACGGTC TGGTTGATGG CTATTGGCTG GACAACAGCC GTAACCTGCC GGGTGAAGTG
670 680 690 700 710 720
AGCGACTTTG TTGCGATGCT GCGTAGCGTG GATCCGGAGC TGACCATTGC GGTTAACTAC
730 740 750 760 770 780
GATCAACACT ATTTCACCGA CGATAACGGT GAATACCTGT ATGTGGACAG CGATGGCCTG
790 800 810 820 830 840
GACGATGAGG ACGAAAGCGA TTACAAGATC GTTAAACACG TGGTTACCAA CGAGTATATG
850 860 870 880 890 900
GACTTTACCA ACGGTCATGT GACCCCGCTG GGTCGTGGCG CGCCGCCGAA CAGCTGGGCG
910 920 930 940 950 960
TACGAGGAAT ATACCATTCC GGACATGATC GAGGTTCCGT GGGAAACCTA CGATGGTAGC
970 980 990 1000 1010 1020
AAGTATGCGC TGAAACACGG CTGGTTCCCG ATTCGTAACA GCTGGAGCGG CAGCAAGGCG
1030 1040 1050 1060 1070 1080
GAACTGATGT TCGACGTGGA GCAGGCGTAC CGTTTTGTGC GTACCGTTAC CGATGGTGGC
1090 1100 1110 1120 1130 1140
GCGGCGATGA CCTGGAGCAC CACCCAAGAC AACGGTTATA TGACCGCGGA TGAAATGAGC
1150 1160 1170 1180 1190 1200
ATCATGATTG AGATCAGCAA CCGTATGACC CAGACCCCGA AGCCGGATTA CAGCGTGTAT
1210 1220 1230 1240
GAACGTCCGA AAGGCGCGTA CCTGGTTAGC GAGATCGAAT AA
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Table 1C: Coding region sequence for the fucanase-gene used to code native
P19DFcnA
fucanase, SEQ ID NO. 3
20 30 40 50 60
ATGTTAATCG GCTGTGGTGG AAGTAGTGCA AATCAAACTC AATCTACGAG CGACCAGGAC
70 80 90 100 110 120
TCATCTGAAG TTAATGAAAC GGTGAGCTTA GACAGCGAAT ATACAGCTAA TTGGATGGCT
130 140 150 160 170 180
GGTGCTTGGG GCATAACTCA ACGCGTAGAC GGCGGTTATA AACTTGATGC TTCTGTAGAA
190 200 210 220 230 240
ACAGGAAAAT ATGATTGGGT CGCTGGTGCA GAAGAAATTG TTGAAAATAT TCCTTCAGCT
250 260 270 280 290 300
GGTTATGTAA TTACATCATT TACTCATCCC GCCCATGGTT TTTTATATAC TTTAAGAGAT
310 320 330 340 350 360
AATGAAAACG TAGATGTTGC TGCTATTCAT CCTGATATGG TGCCTTCTTT AGAAAATGAA
370 380 390 400 410 420
AAAATCATCT TTGATGTTAT TAATGTATAT AAATCTGCCG GTAAGAAAGT ATTATTATAT
430 440 450 460 470 480
TTGAATACGG CAGGTCCTAC TCATGCTGCA GATAGAAACT CCCCCGAAAT TCAAGATGCA
490 500 510 520 530 540
TGGGATGATT ATGTAAATAC GAATTGGAAT GGGGATCATG GTGCAGCTTG GAGGAATCTA
550 560 570 580 590 600
GTTGAAGGTT ACGCTAAAAG ATTCAAAGGT TTAGTTGATG GGTTTTGGTT AGATAATTCA
610 620 630 640 650 660
AAAAACATGG CTGGAGGTCA AAAAGAAATC CCTGAATTTG TTGCAATGCT TAGAGATATT
670 680 690 700 710 720
GATCCGTCAT TTGCAATAGG CGTAAATTAT GAAACTCATT ATTTTGAAGA TGAAGATGGT
730 740 750 760 770 780
AACTACTTAA AAGTAGCATC AGATAGCATA GATGATAACG ATGATCGTGA ATACAAAATA
790 800 810 820 830 840
ATAAAACATG TAGTGACCAA TGAATATATG GACTTTACTA ATGGCCATGT CACTCCAATG
850 860 870 880 890 900
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GGACAAGGTG CCCCACCTAA TTCTTGGGGG TATGAAGAAT ATACAATCCC ACACATGATT
910 920 930 940 950 960
GAAAAACCTT GGGATAGTGT TGATGGTAAT CATTATGCAC TTATGCATGG TTGGTTCCCT
970 980 990 1000 1010 1020
ATCAGATTCT CTTGGAGCGG TTCAGGTGCT GAGCTCATGT TTGAAACTGA ACAAGCTTAT
1030 1040 1050 1060 1070 1080
CGGTTTGTCC GCACGATCAC TGATGGTGGT GCAGCAATGA CATGGTCAAC CACTCAAAAA
1090 1100 1110 1120 1130 1140
AAGGGTTATA TGTCAGCAGA TGAAATGGAT ATAATGATTG AAATTAACAA CAGAATGACA
1150 1160 1170 1180 1190 1200
CAAGCCCCTA AACTAGATTA CGAAGCTTAT GAAAGACCAG AAGGGGCATA TTTGGTTGGT
1210
GAAATAGAAT AA
Table 1D: Coding region sequence for the fucanase-gene used to code synthetic
P 19DFcnA
fucanase, SEQ ID NO. 4
20 30 40 50 60
ATGGGCAGCA GCCATCATCA TCATCATCAC AGCAGCGGCC TGGTGCCGCG CGGCAGCCAT
70 80 90 100 110 120
ATGAACCAGA CCCAAAGCAC CAGCGACCAG GATAGCAGCG AGGTGAACGA AACCGTTAGC
130 140 150 160 170 180
CTGGACAGCG AATATACCGC GAACTGGATG GCGGGTGCGT GGGGCATTAC CCAACGTGTG
190 200 210 220 230 240
GACGGTGGCT ACAAGCTGGA TGCGAGCGTT GAAACCGGTA AATATGATTG GGTGGCGGGC
250 260 270 280 290 300
GCGGAGGAAA TTGTTGAGAA CATCCCGAGC GCGGGTTACG TGATCACCAG CTTCACCCAC
310 320 330 340 350 360
CCGGCGCACG GCTTTCTGTA TACCCTGCGT GACAACGAGA ACGTGGATGT TGCGGCGATT
370 380 390 400 410 420
CACCCGGACA TGGTGCCGAG CCTGGAGAAC GAAAAGATCA TCTTCGATGT GATCAACGTT
430 440 450 460 470 480
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TACAAGAGCG CGGGTAAGAA AGTTCTGCTG TATCTGAACA CCGCGGGTCC GACCCATGCG
490 500 510 520 530 540
GCGGACCGTA ACAGCCCGGA AATTCAGGAT GCGTGGGACG ATTACGTGAA CACCAACTGG
550 560 570 580 590 600
AACGGTGACC ACGGTGCGGC GTGGCGTAAC CTGGTGGAGG GTTATGCGAA GCGTTTCAAA
610 620 630 640 650 660
GGTCTGGTTG ACGGCTTTTG GCTGGATAAC AGCAAGAACA TGGCGGGTGG CCAAAAAGAG
670 680 690 700 710 720
ATTCCGGAAT TCGTGGCGAT GCTGCGTGAC ATTGATCCGA GCTTTGCGAT CGGTGTTAAC
730 740 750 760 770 780
TACGAAACCC ACTATTTCGA GGACGAAGAT GGCAACTACC TGAAGGTTGC GAGCGACAGC
790 800 810 820 830 840
ATCGACGATA ACGACGATCG TGAATACAAG ATCATTAAAC ACGTGGTTAC CAACGAGTAT
850 860 870 880 890 900
ATGGATTTCA CCAACGGTCA TGTGACCCCG ATGGGTCAAG GTGCTCCGCC GAACAGCTGG
910 920 930 940 950 960
GGCTACGAGG AATATACCAT TCCGCACATG ATCGAAAAAC CGTGGGACAG CGTTGATGGT
970 980 990 1000 1010 1020
AACCACTATG CGCTGATGCA CGGCTGGTTC CCGATTCGTT TTAGCTGGAG CGGTAGCGGC
1030 1040 1050 1060 1070 1080
GCGGAGCTGA TGTTCGAAAC CGAACAGGCG TACCGTTTTG TTCGTACCAT TACCGATGGT
1090 1100 1110 1120 1130 1140
GGCGCGGCGA TGACCTGGAG CACCACCCAA AAGAAAGGTT ATATGAGCGC GGACGAAATG
1150 1160 1170 1180 1190 1200
GATATCATGA TTGAGATCAA CAACCGTATG ACCCAAGCGC CGAAGCTGGA TTACGAGGCG
1210 1220 1230 1240
TATGAACGTC CGGAGGGTGC GTACCTGGTT GGCGAGATCG AATAA
Table 1E: Amino acid sequence for the fucanase-gene used to code native
P5AFcnA fucanase,
SEQ ID NO. 5
20 30 40 50 60
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MLISVTVLVG CGSSSDEVES TNTTDSYNIN NTEATGIEYN ASWMAGTWGI TQRVDGGYKL
70 80 90 100 110 120
DNSADSSNWQ AGAEEIVTNI PAAEYVITSF THPAHGHLFT LRTNNNVDVS AIHPDMVPTL
130 140 150 160 170 180
ENEKIILDVI NIYRAAGKKV ILYLNSAGPS MAEERGDTDI QAAWDEYYIN EWDGDEAAAW
190 200 210 220 230 240
RNLARGYVER FDGLVDGYWL DNSRNLPGEV SDFVAMLRSV DPELTIAVNY DQHYFTDDNG
250 260 270 280 290 300
EYLYVDSDGL DDEDESDYKI VKHVVTNEYM DFTNGHVTPL GRGAPPNSWA YEEYTIPDMI
310 320 330 340 350 360
EVPWETYDGS KYALKHGWFP IRNSWSGSKA ELMFDVEQAY RFVRTVTDGG AAMTWSTTQD
370 380 390 400
NGYMTADEMS IMIEISNRMT QTPKPDYSVY ERPKGAYLVS EIE
Table IF: Amino acid sequence for the fucanase-gene used to code synthetic
P5AFcnA
fucanase, SEQ ID NO. 6
20 30 40 50 60
MGSSHHHHHH SSGLVPRGSH MGSSSDEVES TNTTDSYNIN NTEATGIEYN ASWMAGTWGI
70 80 90 100 110 120
TQRVDGGYKL DNSADSSNWQ AGAEEIVTNI PAAEYVITSF THPAHGHLFT LRTNNNVDVS
130 140 150 160 170 180
AIHPDMVPTL ENEKIILDVI
NIYRAAGKKV ILYLNSAGPS MAEERGDTDI QAAWDEYYIN
190 200 210 220 230 240
EWDGDEAAAW RNLARGYVER FDGLVDGYWL DNSRNLPGEV SDFVAMLRSV DPELTIAVNY
250 260 270 280 290 300
DQHYFTDDNG EYLYVDSDGL DDEDESDYKI VKHVVTNEYM DFTNGHVTPL GRGAPPNSWA
310 320 330 340 350 360
YEEYTIPDMI EVPWETYDGS KYALKHGWFP IRNSWSGSKA ELMFDVEQAY RFVRTVTDGG
370 380 390 400 410
AAMTWSTTQD NGYMTADEMS IMIEISNRMT QTPKPDYSVY ERPKGAYLVS EIE
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Table 1G: Amino acid sequence for the fucanase-gene used to code native
P19DFcnA fucanase,
SEQ ID NO. 7
20 30 40 50 60
MLIGCGGSSA NQTQSTSDQD SSEVNETVSL DSEYTANWMA GAWGITQRVD GGYKLDASVE
70 80 90 100 110 120
TGKYDWVAGA EEIVENIPSA GYVITSFTHP AHGFLYTLRD NENVDVAAIH PDMVPSLENE
130 140 150 160 170 180
KIIFDVINVY KSAGKKVLLY
LNTAGPTHAA DRNSPEIQDA WDDYVNTNWN GDHGAAWRNL
190 200 210 220 230 240
VEGYAKRFKG LVDGFWLDNS KNMAGGQKEI PEFVAMLRDI DPSFAIGVNY ETHYFEDEDG
250 260 270 280 290 300
NYLKVASDSI DDNDDREYKI IKHVVTNEYM DFTNGHVTPM GQGAPPNSWG YEEYTIPHMI
310 320 330 340 350 360
EKPWDSVDGN HYALMHGWFP IRFSWSGSGA ELM FETEQAY
RFVRTITDGG AAMTWSTTQK
370 380 390 400
KGYMSADEMD IMIEINNRMT QAPKLDYEAY ERPEGAYLVG EIE
Table 111: Amino acid sequence for the fucanase-gene used to code synthetic
P19DFcnA fucanase,
SEQ ID NO. 8
10 20 30 40 50 60
MGSSHHHHHH SSGLVPRGSH MNQTQSTSDQ DSSEVNETVS LDSEYTANWM AGAWGITQRV
70 80 90 100 110 120
DGGYKLDASV ETGKYDWVAG AEEIVENIPS AGYVITSFTH PAHGFLYTLR DNENVDVAAI
130 140 150 160 170 180
HPDMVPSLEN EKIIFDVINV
YKSAGKKVLL YLNTAGPTHA ADRNSPEIQD AWDDYVNTNW
190 200 210 220 230 240
NGDHGAAWRN LVEGYAKRFK GLVDGFWLDN SKNMAGGQKE
IPEFVAMLRD IDPSFAIGVN
250 260 270 280 290 300
YETHYFEDED GNYLKVASDS IDDNDDREYK IIKHVVTNEY MDFTNGHVTP MGQGAPPNSW
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310 320 330 340 350
360
GYEEYTIPHM IEKPWDSVDG NHYALMHGWF PIRFSWSGSG AELMFETEQA YRFVRTITDG
370 380 390 400 410
GAAMTWSTTQ KKGYMSADEM DIMIEINNRM TQAPKLDYEA YERPEGAYLV GEIE
Enzymatic hydrolysis of fucans
[00025] The flow chart of FIG. 1 depicts an exemplary method [100] for
obtaining a desired lower
molecular weight fucan composition relative to a feedstock fucan composition,
i.e., for reducing
the average molecular weight average of the fucans in the composition, by
enzymatic hydrolysis
of the feedstock fucan composition. In this exemplary, non-limiting method,
the method
comprises: providing [110] the feedstock fucan composition in an aqueous
solution (i.e., a man-
made, typically buffered, solution); incubating [120] the feedstock fucan
composition in solution
with a fucanase to produce a solution comprising the hydrolyzed fucan
composition, the fucanase
and hydrolysis-remnant molecules; and separating [130] the solution comprising
the hydrolyzed
fucan composition from the hydrolysis remnant molecules and the fucanase to
obtain the desired
lower molecular weight fucan composition.
[00026] Providing [110] the feedstock fucan composition may comprise providing
fucans/a
feedstock fucoidan composition extracted from at least one of Adenocystis
utricularis,
Ascophyllum nodosum, Chorda ilium, Cystoseirabies marina, Durvillaea
antarctica, Ecklonia
kurome, Ecklonia maxima, Eisenia bicyclis, Fucus evanescens, Fucus
vesiculosis, Hizikia
fusiforme, Himanthalia Elongata, Kjellmaniella crassifolia, Laminaria
brasiliensis, Laminaria
cichorioides, Laminaria hyperborea, Laminaria japonica, Laminaria saccharina,
Lessonia
trabeculata, Macrocystis pyrifera, Pelvetia fastigiata, Pelvetia Canaliculata,
Saccharina
japonica, Saccharina latissima, Sargassum stenophylum, Sargassum thunbergii,
Sargassum
confusum, Sargassum fusiforme and Undaria pinnatifida. In certain embodiments,
the fucan may
be obtained from at least one of Saccharina japonica, Laminaria hyperborea,
Macrocystis pyifer a
and Chorda Filum.
[00027] Providing [110] the feedstock fucan composition in an aqueous solution
may comprise
providing the feedstock fucan composition at a fucan concentration of between
about 0.01% w/v
and about 30% w/v in solution. Providing [110] the feedstock fucan composition
in an aqueous
solution may comprise providing the feedstock fucan composition at a fucan
concentration of
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between about 0.05% w/v and about 10% w/v in solution. Providing [110] the
feedstock fucan
composition in an aqueous solution may comprise providing the feedstock fucan
composition at a
fucan concentration of between about 0.1% w/v and about 5% w/v in solution.
[00028] Providing [110] the feedstock fucan composition in an aqueous solution
may comprise
providing the feedstock fucan composition in a solution comprising at least
one of monosodium
phosphate, disodium phosphate, trisodium phosphate, phosphate buffer,
phosphate buffered saline,
tricine buffer, borate buffer, trisaminomethane buffer (also known as Tris
buffer), sodium chloride,
TrizmaTm buffer, beta-hydroxy-4-(2-hydroxyethyl)piperazine- 1 -
propanesulphonic acid (also
known as HEPP SO) buffer, piperazine-1,4-bis(2-hydroxypropanesulfonic acid)
dihydrate (also
known as POPSO) buffer, triethanolamine (TEA) buffer, 3-[4-(2-
Hydroxyethyl)piperazin-1-
yl]propane-1-sulfonic acid (EPPS) buffer, glycine buffer, N-(2-
Hydroxyethyl)piperazine-N'-(4-
butanesulfonic acid) (HEPBS) buffer, 2-[4-(2-hydroxyethyl)piperazin-1-
yl]ethanesulfonic acid
(HEPES) buffer, [tris(hydroxymethyl)methylamino]propanesulfonic acid (TAPS)
buffer, 2-
amino-2-methy1-1,3-propanediol (also known as AMPD) buffer, N-
tris(Hydroxymethyl)methy1-
4-amino-butanesulfonic acid (also known as TABS) buffer, N-(1,1-Dimethy1-2-
hydroxyethyl)-3-
amino-2-hydroxy-propanesulfonic acid (AMPSO) buffer, 2-(cyclo-
hexylamino)ethanesulfonic
acid (CHES) buffer, 3-(cyclohexylamino)-2-hydroxy-1-propane-sulfonic acid
(CAPSO) buffer, 2-
amino-2-methyl-1-propanol (also known as AMP) buffer, 3-(cyclohexylamino)-1-
propane-
sulfonic acid (CAPS) buffer, 4-(cyclohexylamino)-1-butanesulfonic acid (CABS)
buffer, citrate
buffer, citrate-phosphate buffer, sodium carbonate-bicarbonate buffer and
ammonium bicarbonate.
The buffered solution can have a pH that is, for example, mildly acidic to
mildly basic, for example
a pH of about 5.5-10.5, 6.5-9.0, 7.5-8.8, or 8.0-8.5.
[00029] Incubating [120] the feedstock fucan composition with a fucanase may
comprise
incubating the feedstock fucan composition with a Psychromonas fucanase.
Incubating [120] the
feedstock fucan composition with a fucanase may comprise incubating the
feedstock fucan
composition with a recombinant fucanase produced by E. Coil. Incubating [120]
the feedstock
fucan composition with a fucanase may comprise incubating the feedstock fucan
composition with
at least one, or both, of P5AFcnA and P 1 9DFcnA fucanases, for example
synthetically produced,
non-natural P5AFcnA and P 1 9DFcnA fucanases. Incubating [120] the feedstock
fucan
composition with a fucanase may comprise incubating the feedstock fucan
composition with a
fucanase translated from its fucanase-gene or modified fucanase-gene by either
of E. Coil or a
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Psychromonas species, or other suitable prokaryotic or eukaryotic
host/production species. The
methods herein may comprise producing an enzyme by expressing a fucanase-gene
sequence of at
least one of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4 in
which fewer than
5% of codons are substituted. Incubating [120] the feedstock fucan composition
with a fucanase
may comprise incubating the feedstock fucan composition with a fucanase coded
by a sequence in
which at least one, three or five, but fewer than 1%, 3%, or 5% of codons or
amino acids of the
native P5AFcnA sequence, SEQ ID NOS. 1 and 5, respectively, are substituted.
Incubating [120]
the feedstock fucan composition with a fucanase may comprise incubating the
feedstock fucan
composition with a fucanase coded by a sequence in which fewer than 1%, 3%, or
5% of codons
or amino acids of the synthetic P5AFcnA sequence, SEQ ID NOS. 2 and 6,
respectively, are
substituted. Incubating [120] the feedstock fucan composition with a fucanase
may comprise
incubating the feedstock fucan composition with a fucanase coded by a sequence
in which at least
one, three or five, but fewer than 1%, 3%, or 5% of codons or amino acids of
the native P19DFcnA
sequence, SEQ ID NOS. 3 and 7, respectively, are substituted. Incubating [120]
the feedstock
fucan composition with a fucanase may comprise incubating the feedstock fucan
composition with
a fucanase coded by a sequence in which fewer than 1%, 3%, or 5% of codons of
the synthetic
P19DFcnA sequence, SEQ ID NOS. 4 and 8, respectively, are substituted.
[00030] Incubating [120] the feedstock fucan composition with a fucanase may
comprise
maintaining the feedstock fucan composition-fucanase mixture at between about
0 C to about 60
C, between about 15 C to 40 C, between about 20 C to 30 C, for example,
about 23 C, about
25 C and about 27 C.
[00031] Incubating [120] the feedstock fucan composition with a fucanase may
comprise at least
one form of agitating the mixture, for example via stirring, shaking, rocking,
or mixing the
feedstock fucan composition-fucanase mixture, for example for at least 1
minute up to throughout
the incubation period.
[00032] Incubating [120] the feedstock fucan composition with a fucanase may
comprise
incubating the feedstock fucan composition-fucanase mixture for between about
30 minutes and
about 300 hours, between about 1 hour and about 100 hours, between about 2
hours and about 50
hours, between about 3 hours and about 24 hours, for example about 5 hours,
about 10 hours, about
15 hours and about 20 hours.
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[00033] Incubating [120] the feedstock fucan composition with a fucanase may
comprise
incubating the feedstock fucan composition-fucanase mixture until a desired
molecular weight in
the fucan composition has been obtained. Incubating [120] the feedstock fucan
composition with
a fucanase may comprise incubating the feedstock fucan composition-fucanase
mixture until at
least about a 5% or 10% reduction in the average molecular weight of the fucan
composition is
obtained, at least about a 20% reduction in the average molecular weight of
the fucan composition
is obtained, for example, until about a 40%, about a 50% or about a 60%
reduction in the average
molecular weight of the fucan composition is obtained. Incubating [120] the
feedstock fucan
composition with a fucanase may comprise incubating the feedstock fucan
composition-fucanase
mixture until at least 5% or 10% of the glycosidic bonds in fucans in the
feedstock fucan
composition are hydrolyzed, at least 20% of the glycosidic bonds in fucans in
the feedstock fucan
composition are hydrolyzed, for example, until about 40%, about 50% and about
60% of the
glycosidic bonds are hydrolyzed.
[00034] Separating [130] the solution comprising the hydrolyzed fucan
composition from the
hydrolysis remnant molecules and the fucanase may comprise quenching the
fucanase in the
solution comprising the hydrolyzed fucan composition with a quenching agent to
terminate the
hydrolysis before the separating. Quenching the fucanase in the solution may
comprise rendering
the solution alkaline. Rendering the solution alkaline may comprise increasing
the pH of the
solution to between about 9-14. Rendering the solution alkaline may comprise
adding at least one
of NaOH, KOH and LiOH to the solution. Quenching the fucanase in the solution
may also or
alternatively comprise heating the solution to between 60-100 C, for example
to at or above about
80 C. Quenching the fucanase in the solution may also or alternatively
comprise precipitating the
fucanase with a precipitant such as at least one of ethanol, isopropanol,
propanol and methanol.
[00035] Separating [130] the hydrolyzed fucan composition from the hydrolysis
remnant
molecules and the fucanase may comprise diafiltering the solution over a
tangential flow filtration
(TFF) filter. The diafiltering may comprise diafiltering the solution
comprising the hydrolyzed
fucan composition with at least one of distilled water, a salt solution and a
buffer solution. The
diafiltering may comprise diafiltering the solution comprising the hydrolyzed
fucan composition
over a TFF filter having a molecular weight cut-off smaller than a desired
molecular weight in the
desired fucan composition. The diafiltering may comprise diafiltrating the
feedstock fucan
composition in solution across a TFF filter with any of a 5kDa, 10kDa, 30kDa,
50kDa, 70kDa or
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100kDa molecular weight cut-off. Separating [130] the hydrolyzed fucan
composition from the
hydrolysis remnant molecules and the fucanase may comprise at least one of
centrifugation,
filtration and sedimentation.
EXAMPLES
[00036] The above method discussed in FIG. 1 was applied to individual
feedstock fucoidan
compositions extracted from Saccharina japonica, Ascophyllum nodosum, Pelvetia
canaliculata,
Fucus vesiculosus, Laminaria hyperborea and Macrocystis pyifera. The fucanases
used were the
previously discussed MfFcnA fucanase (Colin, et al., 2006) and the P5AFcnA and
P19DFcnA
fucanases obtained as discussed above. Feedstock fucoidan compositions were
dissolved at about
between 0.1 and 0.2% w/v in 20mM Tris buffer at pH 8.5 with 0.5M NaCl. 29
jilVi solutions of
MfFcnA, P5AFcnA or P19DFcnA in the same Tris buffer at pH 8.5 with 0.5M NaCl
were
independently added to separate solutions of feedstock fucoidan composition to
a final
concentration of 10 [tM fucanase. The feedstock fucoidan composition-fucanase
mixtures were
all incubated at 25 C for 15 hours on an orbital shaker at 100 rpm.
[00037] After 15 hours, the occurrence of hydrolysis in each individual
feedstock fucoidan
composition-fucanase mixture was analyzed using a carbohydrate-polyacrylamide
gel
electrophoresis (C-PAGE) technique: 10 [IL of the hydrolyzed fucoidan
composition-fucanase
mixture was mixed with 10 [IL of a loading dye (10% v/v glycerol, 0.01% w/v
bromophenol blue
in water). The resulting mixture was loaded onto a 24% w/v C-PAGE gel run on
ice first at a
voltage of 100 Volts for 15 minutes, secondly at a voltage of 150 Volts for 20
minutes and finally
at a voltage of 200 Volts for 20 minutes.
[00038] The results of the C-PAGE analysis are shown in Table 2 below where
the occurrence of
hydrolysis is assessed. Examples of the absence of hydrolysis and the presence
of hydrolysis on
the C-PAGE gel are shown in FIG. 2A, FIG. 2B and FIG. 2C. In the table, a `+'
denotes where
hydrolysis of the fucan composition was observed on the gel, a `-' denotes
where the hydrolysis
of the fucan composition was not observed on the gel and `+/-' denotes where
minimal hydrolysis
is observed.
Source of the feedstock fucoidan composition MfFcnA P5AFcnA P19DFcnA
Saccharina japonica
Ascophyllum nodosum
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Pelvetia canaliculata
Fucus vesiculosus +I-
Laminaria hyperborea
Macrocystis pyifera
Table 2. Enzymatic hydrolysis of fucoidan using three different fucanases
[00039] The method discussed above and in FIG.1 was applied, under
different
conditions, to individual feedstock fucoidan compositions extracted from
different sources of
Macrocystis pyrifera, Chorda ilium, Ascophylum nodosum, Laminaria hyperborea,
Saccharina
japonica and Fucus vesiculosus. The feedstock fucoidan compositions were
dissolved at 0.25%
w/v and incubated with 111M of the same fucanases at room temperature for 40
hours. The same
Tris buffer composition was used adjusted to a pH of 8Ø
[00040] Analysis was conducted on C-PAGE, the results are shown in Table 3
below
where the occurrence of hydrolysis is assessed. In the table, a `+' denotes
where hydrolysis of the
fucan composition was observed on the gel, a `-' denotes where the hydrolysis
of the fucan
composition was not observed on the gel and `+/-' denotes where minimal
hydrolysis is
observed.
Source of the feedstock fucoidan composition MfFcnA P5AFcnA Pl9DFcnA
Macrocystis pyrifera
Chorda filum +I- +I-
Ascophylum nodosum +I-
Laminaria hyperborea
Saccharina japonica +I- +I- +I-
Fucus vesiculosus
Table 3. Enzymatic hydrolysis of fucoidan using three different fucanases
[00041] Table 2 and Table 3 together with FIG. 2A, FIG. 2B and FIG. 2C
indicate that the
P5AFcnA fucanase and P19DFcnA fucanase show a different enzymatic activity
compared with
the previously discussed MfFcnA fucanase. The P5AFcnA fucanase and P19DFcnA
fucanase are
able to enzymatically degrade fucoidans extracted from Laminaria hyperborea
and Macrocystis
pyifera and can degrade Saccharina japonica to a greater extent, where the
MfFcnA fucanase is
unable to accomplish this. The variability in the results between the two
separate experiments run
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are illustrative of the fact that fucoidans extracted from different sources
even of the same species
may show slight differences in backbone structures which may be dependent on
geographical or
seasonal variation.
[00042] The present application is further directed to fucan compositions made
according to the
various elements of the methods, systems etc., discussed herein as well as to
methods of using the
compositions and to systems and devices, etc., configured to perform the
methods herein and
obtain the desired fucan compositions.
Parts List:
100 Method for obtaining a desired lower molecular weight fucan composition
relative to a
feedstock fucan composition by enzymatic hydrolysis of the feedstock fucan
composition.
110 Providing the feedstock fucan composition in an aqueous solution.
120 Incubating the feedstock fucan composition in solution with a fucanase to
produce a
solution comprising the hydrolyzed fucan composition, the fucanase and
hydrolysis
remnant molecules.
130 Separating the solution comprising the hydrolyzed fucan composition
from the hydrolysis
remnant molecules and the fucanase to obtain the desired molecular weight
fucan
composition.
[00043] All terms used herein are used in accordance with their ordinary
meanings unless the
context or definition clearly indicates otherwise. Also, unless expressly
indicated otherwise, in
the specification the use of "or" includes "and" and vice-versa. Non-limiting
terms are not to be
construed as limiting unless expressly stated, or the context clearly
indicates, otherwise (for
example, "including," "having," and "comprising" typically indicate "including
without
limitation"). Singular forms, including in the claims, such as "a," "an," and
"the" include the
plural reference unless expressly stated, or the context clearly indicates,
otherwise.
[00044] Unless otherwise stated, adjectives herein such as "substantially" and
"about" that modify
a condition or relationship characteristic of a feature or features of an
embodiment, indicate that
the condition or characteristic is defined to within tolerances that are
acceptable for operation of
the embodiment for an application for which it is intended.
[00045] The scope of the present methods, compositions, systems, etc.,
includes both means plus
function and step plus function concepts. However, the claims are not to be
interpreted as
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indicating a "means plus function" relationship unless the word "means" is
specifically recited in
a claim, and are to be interpreted as indicating a "means plus function"
relationship where the word
"means" is specifically recited in a claim. Similarly, the claims are not to
be interpreted as
indicating a "step plus function" relationship unless the word "step" is
specifically recited in a
claim, and are to be interpreted as indicating a "step plus function"
relationship where the word
"step" is specifically recited in a claim.
[00046] From the foregoing, it will be appreciated that, although specific
embodiments have been
discussed herein for purposes of illustration, various modifications may be
made without deviating
from the spirit and scope of the discussion herein. Accordingly, the systems
and methods, etc.,
include such modifications as well as all permutations and combinations of the
subject matter set
forth herein and are not limited except as by the appended claims or other
claim having adequate
support in the discussion and figures herein.
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