WO2020024043A1

WO2020024043A1 - Enzymatic hydrolysis of fucans

Info

Publication number: WO2020024043A1
Application number: PCT/CA2019/051025
Authority: WO
Inventors: Alisdair BORASTON; Chelsea Joy VICKERS; Orly Ester SALAMA-ALBER; Kento Takehito ABE
Original assignee: Arc Medical Devices Inc.
Priority date: 2018-07-31
Filing date: 2019-07-24
Publication date: 2020-02-06
Also published as: CA3106453A1; KR20210038539A; EP3830272A1; BR112021000629A2; MX2021000859A; CN112654709A; AU2019314769A1; IL280167A; JP2021531791A; SG11202100386XA

Abstract

Fucanases from Psychromonas species, including P5AFcnA fucanase and P19DFcnA fucanase. Also, methods, systems, compositions, etc., related thereto including fucan compositions having reduced average molecular weight after hydrolysis by such fucanases.

Description

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 C6H12O5. "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 utricular is, Ascophyllum nodosum, Chorda filum, Cystoseirabies marina, Durvillaea antarctica, Ecklonia kurome, Ecklonia maxima, Eisenia bicyclis, Fucus evanescens, Fucus vesiculosis, Hizikia fiisiforme, 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 SW5, known as substrate specific MfFcnA enzyme (Colin, et ak, 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 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 Pl9DFcnA 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 l5°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 Coli. [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 & 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 filum , Cystoseirabies marina , Durvillaea antarctica , Ecklonia kurome , Ecklonia maxima , Eisenia hi eye I is., Fucus evanescens, Fucus vesiculosis , Hizikia fusiforme , Himanthalia Elongata, Kjellmaniella crassifolia , Laminaria hrasiliensis , Laminaria cichorioides , Laminaria hyperhorea , Laminaria japonica , Laminaria saccharina , Lessonia traheculata , Macrocystis pyrifera , Pelvetia fastigiata , Pelvetia Canaliculata , Saccharina japonica , Saccharina latissima, Sargassum stenophylum , Sargassum thunhergii , Sargassum confusum , Sargassum fusiforme and Undaria pinnatifida.. In certain embodiments, the fucan or fucoidan is obtained from at least one of Saccharina japonica, Laminaria hyperhorea, Macrocystis pyifera and Chorda filum. 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%. [00012] Also provided are expression vectors comprising an expressible P5AFcnA fucanase gene, Pl9DFcnA 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.l 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 Pl9DFcnA 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 Hyperhorea by P5AFcnA fucanase and Pl9DFcnA 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 Pl9DFcnA 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. 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 Pl9DFcnA 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 Pl9DFcnA were obtained from genomes of Psychromonas species Psychromonas sp. SW5A and

Psychromonas sp. SW19D. Synthetic fucanase-genes encoding the enzymes P5AFcnA and Pl9DFcnA that were codon optimized for expression in E. coli and pre-inserted into the E. coli expression plasmid pET28a were ordered from Genscript®. These constructs were referred to as pET28a_p5AFcnA and pET28a_Pl9DFcnA. The sequence fidelity of the constructs was confirmed by bi-directional DNA sequencing. Fucanases were expressed in an E. coli over 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 mM for further use. The coding region sequences for the fucanase-genes of native and synthetic P5AFcnA fucanase are shown in Tables 1A and IB respectively, SEQ ID NOS. 1 and 2, respectively. The coding region sequences for the fucanase-genes of native and synthetic Pl9DFcnA fucanase are shown in Tables 1C and ID respectively, SEQ ID NOS. 3 and 4, respectively. The amino acid sequences for the native and synthetic P5AFcnA fucanase are shown in Tables IE and IF respectively, SEQ ID NOS. 5 and 6, respectively. The amino acid sequences for the native and synthetic Pl9DFcnA fucanase are shown in Tables 1G and 1H 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 IB, 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 Pl9DFcnA gene sequence. The fucanase-gene sequence shown in Table ID, SEQ ID NO. 4, will hereafter be referred to as the synthetic Pl9DFcnA gene sequence. The synthetic sequences are optimized for expression of the enzyme with E. coli. The amino acid sequence shown in Table IE, SEQ ID NO. 5, will hereafter be referred to as the native P5AFcnA amino acid sequence. The amino acid sequence shown in Table IF, 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 Pl9DFcnA amino acid sequence. The amino acid sequence shown in Table 1H, SEQ ID NO. 8, will hereafter be referred to as the synthetic Pl9DFcnA amino acid sequence. [00024]

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 filum, 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 pyifera 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 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, di sodium phosphate, tri sodium phosphate, phosphate buffer, phosphate buffered saline, tricine buffer, borate buffer, trisaminomethane buffer (also known as Tris buffer), sodium chloride, Trizma™ buffer, beta-hydroxy-4-(2-hydroxyethyl)piperazine-l-propanesulphonic acid (also known as HEPPSO) buffer, piperazine- l,4-bis(2-hydroxypropanesulfonic acid) dihydrate (also known as POPSO) buffer, triethanolamine (TEA) buffer, 3 -[4-(2 -Hydroxy ethyl)piperazin-l - yl]propane-l -sulfonic acid (EPPS) buffer, glycine buffer, N-(2-Hydroxyethyl)piperazine-N'-(4- butanesulfonic acid) (HEPBS) buffer, 2-[4-(2-hydroxyethyl)piperazin-l-yl]ethanesulfonic acid (HEPES) buffer, [tris(hydroxymethyl)methylamino]propanesulfonic acid (TAPS) buffer, 2- amino-2-methyl- 1,3 -propanediol (also known as AMPD) buffer, N-tris(Hydroxymethyl)methyl- 4-amino-butanesulfonic acid (also known as TABS) buffer, N-(l, l-Dimethyl-2-hydroxyethyl)-3- amino-2-hydroxy-propanesulfonic acid (AMPSO) buffer, 2-(cyclo-hexylamino)ethanesulfonic acid (CHES) buffer, 3 -(cyclohexylamino)-2-hydroxy-l -propane-sulfonic acid (CAPSO) buffer, 2- amino-2-methyl-l -propanol (also known as AMP) buffer, 3 -(cy cl ohexylamino)-l -propane- sulfonic acid (CAPS) buffer, 4-(cyclohexylamino)-l-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. Coli. 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 Pl9DFcnA fucanases, for example synthetically produced, non-natural P5AFcnA and Pl9DFcnA 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. Coli or a 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 Pl9DFcn A 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 Pl9DFcnA 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. [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, lOkDa, 30kDa, 50kDa, 70kDa or lOOkDa 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 Pl9DFcnA 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 mM solutions of MfFcnA, P5AFcnA or Pl9DFcnA 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 mM 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 pL of the hydrolyzed fucoidan composition-fucanase mixture was mixed with 10 pL 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‘+1-’ denotes where minimal hydrolysis is observed.

Table 2. Enzymatic hydrolysis of fucoidan using three different fucanases

[00039] The method discussed above and in FIG.l was applied, under different conditions, to individual feedstock fucoidan compositions extracted from different sources of Macrocystis pyrifera, Chorda filum, Ascophylum nodosum, Laminaria hyperborea, Saccharina japonica and Fucus vesiculosus. The feedstock fucoidan compositions were dissolved at 0.25% w/v and incubated with 1 mM of the same fucanases at room temperature for 40 hours. The same Tris buffer composition was used adjusted to a pH of 8.0.

[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.

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 Pl9DFcnA fucanase show a different enzymatic activity compared with the previously discussed MfFcnA fucanase. The P5AFcnA fucanase and Pl9DFcnA 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 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 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.

Claims

What is claimed is:

1. A composition comprising a P5 AFcnA fucanase in an aqueous solution and an added fucan.

2. A composition comprising a Pl9DFcnA fucanase in an aqueous solution and an added fucan.

3. The composition of claim 1 or 2 wherein the added fucan is a feedstock fucan

composition.

4. The composition of any of claims 1 to 3 wherein the composition has a pH of about 6.5- 9.0.

5. The composition of any of claims 1 to 4 wherein the composition has a temperature of about l5°C-40°C.

6. The composition of any of claims 1 to 5 wherein the composition is at conditions such that the fucanase can hydrolyze glycosidic bonds in a fucan.

7. A fucanase produced by a eukaryotic entity, the fucanase coded by a gene sequence according to any one of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 or SEQ ID NO. 4 in which fewer than 5% of codons are substituted.

8. A fucanase produced by a Psychromonas species, the fucanase coded by a gene sequence according to any one of SEQ ID NO. 1 or SEQ ID NO. 3 in which at least 1 codon and fewer than 5% of codons are substituted.

9. A fucanase produced by a Psychromonas species, the fucanase coded by a gene sequence according to any one of SEQ ID NO. 2 or SEQ ID NO. 4 in which fewer than 5% of codons are substituted.

10. A fucanase produced by a prokaryotic entity other than a Psychromonas species, the fucanase coded by a gene sequence according to any one of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 or SEQ ID NO. 4 in which fewer than 5% of codons are substituted.

11. A fucanase produced by a eukaryotic entity, the fucanase comprising an amino acid

sequence according to any one of SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, or SEQ ID NO. 8 in which fewer than 5% of amino acids are substituted.

12. A fucanase produced by a Psychromonas species, the fucanase comprising an amino acid sequence according to any one of SEQ ID NO. 5 or SEQ ID NO. 7 in which at least 1 amino acid and fewer than 5% of amino acids are substituted.

13. A fucanase produced by a Psychromonas species, the fucanase comprising an amino acid sequence according to any one of SEQ ID NO. 6 or SEQ ID NO. 8 in which fewer than 5% of amino acids are substituted.

14. A fucanase produced by a prokaryotic entity other than a Psychromonas species, the fucanase comprising an amino acid sequence according to any one of SEQ ID NO. 5,

SEQ ID NO. 6, SEQ ID NO. 7, or SEQ ID NO. 8 in which fewer than 5% of amino acids are substituted.

15. The fucanase of claim 10 or 14 wherein the prokaryotic entity is E. Coli.

16. A method comprising selectively enzymatically hydrolyzing a fucan in a manmade

aqueous solution using a Psychromonas fucanase.

17. The method of claim 16 wherein the Psychromonas fucanase is coded by a gene sequence according to any 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.

18. The method of claim 16 wherein the Psychromonas fucanase is coded by a gene sequence according to any one of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO.

4 in which fewer than 3% of codons are substituted.

19. The method of claim 16 wherein the Psychromonas fucanase is coded by a gene sequence according to any one of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO.

4 in which fewer than 1% of codons are substituted.

20. The method of claim 16 wherein the Psychromonas fucanase comprises an amino acid sequence according to any one of SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8 in which fewer than 5% of amino acids are substituted.

21. The method of claim 16 wherein the Psychromonas fucanase comprises an amino acid sequence according to any one of SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8 in which fewer than 3% of amino acids are substituted.

22. The method of claim 16 wherein the Psychromonas fucanase comprises an amino acid sequence according to any one of SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8 in which fewer than 1% of amino acids are substituted.

23. The method of claim 16 wherein the fucan is a fucoidan is obtained from at least one of Adenocystis utricularis , Ascophyllum nodosum , Chorda filum , Cystoseirabies marina , Durvillaea antarctica , Ecklonia kurome, Ecklonia maxima , Eisenia hicyclis , Fucus evanescens, Fucus vesicu/osis, Hizikia fusiforme , Himanthalia Elongata, Kjellmaniella crassifolia , Laminaria hrasi/iensis. Laminaria cichorioides. Laminaria hyperhorea , Laminaria japonica , Laminaria saccharina , Lessonia traheculata , Macrocystis pyrifera , Pelvetia fastigiata , Pelvetia Canaliculata , Saccharina japonica , Saccharina latissima, Sargassum stenophylum , Sargassum thunhergii , Sargassum confusum , Sargassum fusiforme and Undaria pinnatifida.

24. The method of claim 16 wherein the fucan is a fucoidan is obtained from at least one of Saccharina japonica, Laminaria hyperhorea, Macrocystis pyifera and Chorda filum.

25. The method of any one of claims 16-24 wherein the average molecular weight of the fucan is reduced by at least 5%.

26. The method of any one of claims 16-24 wherein the average molecular weight of the fucan is reduced by about 10%.

27. The method of any one of claims 16-24 wherein the average molecular weight of the fucan is reduced by about 20%.

28. The method of any one of claims 16-24 wherein the average molecular weight of the fucan is reduced by about 30%.

29. The method of any one of claims 16-24 wherein the average molecular weight of the fucan is reduced by about 40%.

30. The method of any one of claims 16-24 wherein the average molecular weight of the fucan is reduced by about 50%.

31. The method of any one of claims 16-24 wherein the average molecular weight of the fucan is reduced by about 60%.

32. The method of any one of claims 16-24 wherein the average molecular weight of the fucan is reduced by about 70%.

33. The method of any one of claims 16-24 wherein the average molecular weight of the fucan is reduced by about 80%.

34. The method of any one of claims 16-24 wherein the average molecular weight of the fucan is reduced by about 90%.

35. The method of any one of claims 16-24 wherein the average molecular weight of the fucan is reduced by about 95%.

36. The method of any one of claims 16-24 wherein at least 5% of the glycosidic bonds in the fucan are hydrolyzed.

37. The method of any one of claims 16-24 wherein at least 10% of the glycosidic bonds in the fucan are hydrolyzed.

38. The method of any one of claims 16-24 wherein at least 20% of the glycosidic bonds in the fucan are hydrolyzed.

39. The method of any one of claims 16-24 wherein at least 30% of the glycosidic bonds in the fucan are hydrolyzed.

40. The method of any one of claims 16-24 wherein at least 40% of the glycosidic bonds in the fucan are hydrolyzed.

41. The method of any one of claims 16-24 wherein at least 50% of the glycosidic bonds in the fucan are hydrolyzed.

42. The method of any one of claims 16-24 wherein at least 60% of the glycosidic bonds in the fucan are hydrolyzed.

43. The method of any one of claims 16-24 wherein at least 70% of the glycosidic bonds in the fucan are hydrolyzed.

44. The method of any one of claims 16-24 wherein at least 80% of the glycosidic bonds in the fucan are hydrolyzed.

45. The method of any one of claims 16-24 wherein at least 90% of the glycosidic bonds in the fucan are hydrolyzed.

46. The method of any one of claims 16-24 wherein at least 95% of the glycosidic bonds in the fucan are hydrolyzed.

47. A method for obtaining a desired lower molecular weight fucan composition relative to a feedstock fucan composition by enzymatic hydrolysis of fucans in the feedstock fucan composition, the method comprising:

providing the feedstock fucan composition and a Psychromonas fucanase in an aqueous solution; incubating the feedstock fucan composition and the Psychromonas fucanase under conditions sufficient to hydrolyze fucans in the feedstock fucan composition 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 composition.

48. The method of claim 47, wherein the fucanase is coded by a gene sequence according to one of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4.

49. The method of claim 47, wherein the fucanase is comprises an amino acid sequence according to one of SEQ ID NO. 5, SEQ ID NO. 6 SEQ ID NO. 7 and SEQ ID NO. 8.

50. The method of claim 47, wherein the fucan is obtained from at least one of Adenocystis utricularis , Ascophyllum nodosum , Chorda filum , Cystoseirabies marina , Durvillaea antarctica , Ecklonia kurome , Ecklonia maxima , Eisenia hicyclis , Fucus evanescens, Fucus vesiculosis , Hizikia fusiforme , Himanthalia Elongata, Kjellmaniella crassifolia , Laminaria hrasi/iensis. Laminaria cichorioides , Laminaria hyperhorea , Laminaria japonica , Laminaria saccharina , Lessonia traheculata , Macrocystis pyrifera , Pelvetia fastigiata , Pelvetia Canaliculata , Saccharina japonica , Saccharina latissima, Sargassum stenophylum , Sargassum thunhergii , Sargassum confusum , Sargassum fusiforme and Undaria pinnatifida.

51. The method of claim 47, wherein the fucan is obtained from at least one of Saccharina japonica, Laminaria hyperhorea, Macrocystis pyifera and Chorda Filum.

52. The method of any one of claims 47-51 wherein the average molecular weight of the fucan is reduced by at least 5%.

53. The method of any one of claims 47-51 wherein the average molecular weight of the fucan is reduced by about 10%.

54. The method of any one of claims 47-51 wherein the average molecular weight of the fucan is reduced by about 20%.

55. The method of any one of claims 47-51 wherein the average molecular weight of the fucan is reduced by about 30%.

56. The method of any one of claims 47-51 wherein the average molecular weight of the fucan is reduced by about 40%.

57. The method of any one of claims 47-51 wherein the average molecular weight of the fucan is reduced by about 50%.

58. The method of any one of claims 47-51 wherein the average molecular weight of the fucan is reduced by about 60%.

59. The method of any one of claims 47-51 wherein the average molecular weight of the fucan is reduced by about 70%.

60. The method of any one of claims 47-51 wherein the average molecular weight of the fucan is reduced by about 80%.

61. The method of any one of claims 47-51 wherein the average molecular weight of the fucan is reduced by about 90%.

62. The method of any one of claims 47-51 wherein the average molecular weight of the fucan is reduced by about 95%.

63. The method of any one of claims 47-51 wherein at least 5% of the glycosidic bonds in the fucan are hydrolyzed.

64. The method of any one of claims 47-51 wherein at least 10% of the glycosidic bonds in the fucan are hydrolyzed.

65. The method of any one of claims 47-51 wherein at least 20% of the glycosidic bonds in the fucan are hydrolyzed.

66 The method of any one of claims 47-51 wherein at least 30% of the glycosidic bonds in the fucan are hydrolyzed.

67. The method of any one of claims 47-51 wherein at least 40% of the glycosidic bonds in the fucan are hydrolyzed.

68 The method of any one of claims 47-51 wherein at least 50% of the glycosidic bonds in the fucan are hydrolyzed.

69. The method of any one of claims 47-51 wherein at least 60% of the glycosidic bonds in the fucan are hydrolyzed.

70. The method of any one of claims 47-51 wherein at least 70% of the glycosidic bonds in the fucan are hydrolyzed.

71. The method of any one of claims 47-51 wherein at least 80% of the glycosidic bonds in the fucan are hydrolyzed.

72. The method of any one of claims 47-51 wherein at least 90% of the glycosidic bonds in the fucan are hydrolyzed.

73. The method of any one of claims 47-51 wherein at least 95% of the glycosidic bonds in the fucan are hydrolyzed.

74. An expression vector comprising an expressible P5AFcnA fucanase gene.

75. An expression vector comprising an expressible Pl9DFcnA fucanase gene.

76. An expression vector comprising an expressible Psychromonas fucanase gene.

77. An expression vector comprising an expressible gene sequence according to any one of SEQ ID NO. 1, or SEQ ID NO. 3 in which fewer than 5% of codons are substituted.

78. An expression vector comprising an expressible gene sequence according to any one of SEQ ID NO. 2 or SEQ ID NO. 4 in which fewer than 5% of codons are substituted.

79. The expression vector of claim 74-78 wherein the expression vector is a plasmid.

80. The expression vector of claim 74-78 wherein the expression vector is a prokaryotic- expression plasmid.

81. The expression vector of claim 80 wherein the prokaryotic-expression plasmid is

configured for expression in E. coli.

82. The expression vector of claim 74-78 wherein the expression vector is a eukaryotic- expression plasmid.

83. A method of making an enzyme comprising expressing the gene according to any one of claims 74 to 82 and collecting the enzyme expressed by the gene.

84. A method of making an enzyme comprising expressing the gene sequence according to claim 71 to 82 and collecting the enzyme expressed from such gene.

85. The method of claim 83 or 84 wherein the enzyme is a fucanase.

86. A method of using an expressed fucanase comprising providing a fucanase obtained from an expression vector according to any one of claims 74 to 82 and combining the expressed fucanase with a fucan under conditions selected for the fucanase to hydrolyze the fucan.

87. The method of claim 86 wherein the fucan is provided in a feedstock fucan composition.

88. The method of claim 86 or 87 wherein the expressed fucanase hydrolyzes glycosidic bonds within the fucan.