CN101103047A - Modified alginates, methods of production and use - Google Patents

Abstract

Process for preparing a modified alginate polymers are disclosed. The processes comprise the steps of covalently attaching a modifying moiety to one or more unmodified monomeric subunits of an alginate polymer; and changing one or more unmodified mannuronic (M) monomeric subunits of the alginate polymer to one or more unmodified guluronic (G) monomeric subunits by an enzymatic epimerization reaction performed in any order. Processes for preparing alginate gels, fiber, and compositions are also disclosed. Modified alginates in which only M monomeric subunits are modified, and alginate gels, fibers and compositions comprising the same, are disclosed.

Description

Alginate of modification and its production and application

Invention field

The present invention relates to the alginate of the modification of the enzyme method of modifying preparation by alginate polymer as herein described, and its production and application.

Background of invention

The application requires the U.S. Provisional Application submitted on November 12nd, 2004 No. 60/627057, the right of priority of the U.S. Provisional Application the 60/630867th that No. the 60/627247th, the U.S. Provisional Application that on November 12nd, 2004 submitted to and on November 24th, 2004 submit to, these three parts of applications are included in this by reference.

On chemical sense, alginate are 1 → 4 beta-D-mannuronic acid (M) that connects arranged with block form along molecular chain and the linear copolymer of α-1-guluronic acid (G), are scattered with the zone of alternating structure (MG-block) in the homopolymerization zone of M (M-block) and G (G-block) residue.In nature, alginate are at first with the form preparation of the polymannuronic acid of homopolymerization, be converted into by the epimerization reaction after the polymerization and comprise M and the monomeric heteropolymer of G, the C-5 that the epimerization reaction after the described polymerization relates on the polymannuronic acid M residue transforms.This reaction is by the enzyme catalysis of polymannuronic acid C-5 epimerization.

Recently, find seven kinds of different polymannuronic acid C-5-epimerization enzyme genes of genome encoding of the bacterium azotobacter vinelandii of generation alginate.These genes sort in intestinal bacteria, clone and express; Consequent enzyme is called as AlgE1-AlgE7.Because all natural alginate all are to transform from the polymannuronic acid of homopolymerization to the identical basic C-5 of G by M to form, so the significant difference of composition of finding in the polysaccharide and sequence only is because the different catalytic properties of different epimerization enzymes cause.For example, AlgE4 mainly forms the alginate of MG-block, and AlgE6 then introduces polymkeric substance with long G-block.The availability of these alginate modification enzymes and application thereof make it possible to prepare the structure with customization and the alginate of physical properties.

Alginate form cross linked gel in the presence of divalent cation, the G monomer subunits and the ionic crosslinking of polymkeric substance.Alginate form gel fast and depend on the content of G residue and the sequence pattern of G and M residue in the presence of millimolar concentration calcium.

In past 10 years, people are more and more interesting to be applied to the more and more final use of high request, biological example technology, biomedicine and medicinal application with alginate.Using alginate is an example of this trend as the immobilization material of cell and biological catalyst.These systems may use in industry, medical science and agricultural is very extensive, from being prepared ethanol by yeast and preparing monoclonal antibody by hybridoma, to coming the scale operation synthetic seed by capturing plant embryo.

Alginate jelly also can be used as the cell epimatrix material (ECM) of fixed cell, transplanting and organizational project.Yet, though the Protanal TXF 200 hydrogel has interesting physics and mass transfer character; But because any biological inert (for example, cell adhesion and signal conduction) is restricted its application.Though it is the fixing technology as mild as a dove of viable cell that alginate are captured, special interaction need take place to realize its propagation and growth with matrix in many cells.The behavior of this dependence fixed is common in most of mammalian cells; Yet there is not interaction the alginate network in itself.

Because known alginate are non-bioadhesive materials,, thereby participate in the recognition process of cell-cell and cell-ECM directly so introducing cell-specific aglucon or extracellular signal transduction molecule are necessary as peptide or oligose.Like this, reported third generation biomaterial, can significantly improve the interaction with cell, disclosed the new chance and the future development in polymer engineering and tissue regeneration field based on the alginate of this modification.Yet except basic biological property, the platform design of fully simulating ECM also depends on physical properties, example gel formation, physical strength and stability.

The ion-transfer gelationization character of alginate makes it become attracting candidate target in biotechnology and the medical use, especially seals the field at cell and tissue.For example; contain the alginate-poly-L-Lysine capsule (capsule) of Lang Gehanshi pancreas islet but be presented at reverting diabetes in the macrofauna; wherein, stable and the permeable barrier protection transplanted cells of selectivity that capsule showed avoids contacting host's immunity system.

Various aglucons are coupled to alginate polymer to improve cell/matrix phase mutual effect, have equaled the US 6,642,362 that announced on November 4th, 2003 as Mooney.The subject matter of alginate being carried out chemical modification is that this modification usually is not a chemo-selective.In other words, modification can occur on the two kinds of sugar monomers (guluronic acid (G) and mannuronic acid (M)) that constitute alginate.Also known gel formation (especially gel-strength) is the character relevant with the quantity of unmodified G.The chemical modification of alginate described in the prior art has been described the replacement that is not limited only to M residue (M unit) and takes place in the G of G-block residue (G unit), thereby reduce the amount that can get the G residue, thereby weaken the collaborative combination of divalent cation and reduce gel formation speed, gel formation speed reduces and causes in the salt solution gel formation relatively poor and swelling is uncontrolled.As described herein, " residue " refers to single M or G unit, and " block " refers to a plurality of M, G or MG unit.

Reported by the 1-amino-1-deoxidation-beta galactose residue on the uronic acid group of introducing the polysaccharide chain and synthesized the alginate and the sign thereof of semi-lactosi-replacement.To the identification of beta galactose part and consider results reported, the alginate of recommending to use modification are hepatocellularly sealed and are adhered to improve as the biomaterial of suitable formation gel based on the asialoglycoprotein receptor that exists on the hepatocellular cell surface (ASGP-R).Yet the feature of the alginate of modification on molecular level shows that introducing side-chain radical on the alginate chain mainly influences the G residue, thereby weakened that calcium-in conjunction with character, the result forms the lower hydrogel of stability.Protanal TXF 200 hydrogel rigidity and the stability of having reported modification significantly reduce.Therefore, introducing the cell-specific aglucon on the polysaccharide chain can cause the mechanical property of hydrogel to descend.

In this respect, the alginate that have the selectivity modification of side chain molecule on the preparation mannuronic acid residue have been represented significant improvement with the calcium of restriction hydrogel in conjunction with loss and stability lost.Similarly, need to improve the character of the alginate of modification with side chain molecule, with the positively charged ion of restriction hydrogel in conjunction with loss and stability lost.Can after replacing, control by the present invention the machinery and the swelling property of alginate jelly with various aglucons as described below.

Summary of the invention

The present invention relates to prepare the method for the alginate polymer of modification.This method may further comprise the steps: make modification partly be covalently bonded in the one or more unmodified monomer subunits of alginate polymer and the epimerization reaction by enzyme makes one or more unmodified mannuronic acid (M) monomer subunits of alginate polymer be converted into one or more unmodified guluronic acids (G) monomer subunits.Reactions steps can random order be carried out and can any sequence be repeated repeatedly.

The invention still further relates to the method for preparing alginate jelly and fiber.This method may further comprise the steps: the alginate polymer and the divalence gelling ion that mix multiple modification in solvent.In some embodiments, viable cell is encapsulated in the alginate jelly.

The present invention relates to the alginate polymer of modification, wherein, only the M monomer subunits is modification, and described modification is not an acetylization reaction.

The present invention relates to comprise the alginate jelly and the fiber of the alginate polymer of modification, wherein, only the M monomer subunits is modification, and described modification is not an acetylization reaction.

The invention still further relates to preparation method that be shaped or unfashioned solid non-crosslinked alginate compositions.

Brief Description Of Drawings

Fig. 1 has shown that the two-step approach selectivity replaces the example of the ManA residue in the alginate.The first step is to replace polymannuronic acid with GalN.The C-5 epimerization enzyme that second step was to use reorganization to produce carries out the C-5 epimerization reaction.Embodiment 1 expression process shown in Figure 1.

Fig. 2: by the swelling of the calcium alginate gel pearl of following composition preparation: square: utmost point North Sea band; Circle: through the polymannuronic acid (12% semi-lactosi) of modification and epimerization; Trilateral: the utmost point North Sea band (14% semi-lactosi) of modification and the replacing number of times of salt brine solution (NaCl 0.9%).

Fig. 3: the physical strength that following composition is measured with Young's modulus: 1: unmodified utmost point North Sea band; 2: the utmost point North Sea band (14% semi-lactosi) of modification; 3: through the polymannuronic acid (12% semi-lactosi) of modification and epimerization.

Fig. 4: the selectivity modification on the M residue is to the calcium alginate gel pearl swollen influence by the preparation of following composition: from the alginate (zero) of the modification of utmost point North Sea band (Laminaria hyperborean), through the polymannuronic acid (mouth) of modification and epimerization and the replacing number of times of utmost point North Sea band alginate (△) and salt brine solution (NaCl 0.9%).

Photocrosslinking is to the influence (A) of the stability among the 50mM EDTA with to the influence of the swollen in the 0.9%NaCl solution (B), uncrosslinked sample (mouth), photocrosslinking sample (zero) on the alginate capsule that Fig. 5: M-replaces.

The 300MHz of Fig. 6: MGal, MGalE4 and MGalE4E6 ¹H-NMR spectrogram (dystopy zone).H1-G represents the dystopy signal of the guluronic acid residue introduced, the H5 signal of the guluronic acid residue that H5-G (G) expression is adjacent with another guluronic acid part.

Fig. 7: a) compared epimerization enzyme AlgE4, b) compared epimerization enzyme AlgE6 polymerization is replaced MG to polymannuronic acid with to introducing the efficient (%) of the MGal sample of single G residue in the polymer chain ²⁰(FG=0.47) with to the efficient (%) of the MGalE4 sample of introducing single G residue (light gray) and GG dyad (Dark grey) in the polymer chain.

Fig. 8: a) use pNH ₂The polymannuronic acid of Ph β Gal (d.s.=0.18) modification, and use b) the AlgE4 epimerization (form: F by final polymkeric substance _G=0.26; F _GG=0), use c then) the AlgE6 epimerization (form: F by final polymkeric substance _G=0.36; F _GG=0.17) 300MHz ¹The H-NMR spectrogram.

Fig. 9: a) MGal, b) MGalE4 and c) MGalE4E6 add calcium (for all samples, [Ca ²⁺]/[polymkeric substance]=0.26) (-) and the circular dichroism spectrogram of (--) before afterwards.

Figure 10: for by sample MGalE4E6 (trilateral), the gel that LhypGal (circle) and alginate (from utmost point North Sea band) (square) obtain, a) G ' and the b) variation of δ in first 1000 seconds, for MGalE4E6 (-), LhypGal (--) and from the alginate of utmost point North Sea band (...), c) G ' is in the variation of the setting up period of calcium gel.In the gel that obtains by 1.5% polymers soln, add 20mM CaCO ₃And 40mMGDL.

Figure 11: the storage modulus G ' (solid mark) and the out-of-phase modulus G of the hydrogel that obtains by utmost point North Sea band alginate (square), LhypGal (circle) and MGalE4E6 (trilateral) " (hollow mark).In the gel that obtains by 1.5% polymers soln, add 20mM CaCO ₃With 40mM GDL.

Figure 12: a) the cylindrical Young's modulus of gel (E) that obtains from utmost point North Sea band, LhypGal and MGalE4E6.In all three kinds of samples, [Ca ²⁺]/[the G residue] mol ratio be 0.59.Value representation is mean value s.d. (n=8).B) in the gel right cylinder that is obtained by utmost point North Sea band (square), LhypGal (trilateral) and MGalE4E6 (circle), dehydrating condensation is to [Ca ²⁺]/[polymkeric substance] dependency.Value representation is mean value ± s.d. (n=8).

Figure 13: for alginate, increase salt brine solution replacing number of times and can improve the stability of calcium pearl, be expressed as absolute diameter (d from utmost point North Sea band (square), LhypGal (trilateral) and MGalE4E6 (circle) ₀The initial diameter of=pearl) increase.Value representation is mean value ± s.d..

Figure 14: the enzyme method of the alginate of selectivity modification on the preparation M residue.S=1-amino-1-deoxidation-β-D-semi-lactosi or pNH ₂Ph-β-D-galactopyranoside.

Detailed description of the preferred embodiment

Alginate are the general names with the linear copolymer family of the D-mannuronic acid of various ratios and series arrangement and L-guluronic acid.The ratio and the length of adjacent G residue block are closely related in the ability of alginate polymer and divalent cation such as calcium formation gel and the character of gained gel and the polymer chain.

The invention provides the method for alginate being carried out modification, this method needed for two steps at least: the first step, make modification partly be covalently bonded in the one or more unmodified monomer subunits of alginate polymer, in second step, the epimerization reaction by enzyme makes one or more unmodified mannuronic acid (M) monomer subunits of alginate polymer be converted into one or more unmodified guluronic acids (G) monomer subunits.According to the inventive method, these steps can be carried out in random order.And, can make modification partly be covalently bonded in a plurality of steps of one or more unmodified monomer subunits of alginate polymer, and, can carry out making one or more unmodified mannuronic acid (M) monomer subunits of alginate polymer be converted into a plurality of steps of one or more unmodified guluronic acids (G) monomer subunits by the epimerization reaction of enzyme.Can random order carry out above-mentioned a plurality of step.Can on carboxyl or hydroxyl, carry out modification to monomer subunits.

According to the chemical property and the size of component, the replacement of functional group will reduce the ability of polymer formation gel in the alginate.By increasing G block content this effect is minimized.Some preferred embodiment in, the replacement of functional group is limited to the alginate that use only contains M and replaces the M residue as the parent material that carries out modification.After the modification, unmodified M is converted into G by epimerization reaction.

Prepared the alginate polymer that only the M monomer subunits has been carried out the modification of modification.The alginate polymer of modification can comprise unmodified M and unmodified G.Modification is not acetylize, though acetylize can take place some M.In other words, some M monomer subunits of these polymkeric substance can be carried out modification by the modified-reaction except that acetylization reaction, and no matter whether other M monomer subunits of these polymkeric substance is acetylation.Some preferred embodiment in, by adding the modification part as semi-lactosi or its oligomer, seminose or its oligomer, sLe ^x(NeuAc α 2-3Gal β 1-[4Fuc α 1-3] GlcNAc), GlcNAc, HA-oligomer (transparent attachment proteins (hyaladhesins); Hyaluronic acid binding protein), RDG peptide, YIGSR peptide, REDV peptide, IKVAV peptide, KHIFSDDSSE peptide and KRSR peptide, the alginate polymer of the only modification of modification M monomer subunits is carried out modification.The alginate polymer that only contains the modification of M monomer subunits can be used for preparing alginate jelly and fiber.

Initial alginate can have the M and the G of different content, organize into groups in the various structural arrangement modes of MM, GG and/or MG block.Chemical reaction step will cause that substitution reaction (the M residue of modification and the G residue of modification) takes place applicable alginate on M and G residue.The enzyme step is converted into the G residue by the M residue that makes desired number, thereby changes the content of M and G in the alginate.For example, be converted into MG or GG block by making the MM block, or make the MG block be converted into the GG block, can increase the content of G.

In some embodiments, the alginate of high M content are 50%, 60%, 70%, 80%, 90%, 95% or 95+% useful, that for example M content is M and G gross weight at least.An embodiment of the invention adopted the homopolymer of mannuronic acid such as the initial alginate that the M residue is rich in the polymannuronic acid conduct before chemical reaction.These homopolymer for example can prepare by the AIgG negative mutant of the Pseudomonas aeruginosa (Pseudomonasaeruginosa) described in the disclosed WO04011628 on February 5th, 2004 (its content is included in this by reference), pseudomonas syringae (Pseudomonas syringae) or Pseudomonas fluorescens (P.fluorescens).Other examples of high M alginate have been disclosed among the WO 03046199A2 (its content is included in this by reference).

According to the present invention, the modification part can be any chemical structure, but is preferably selected from: monose, oligosaccharides, mononucleotide, oligonucleotide, amino acid, peptide and protein.In some embodiments, modification partly is selected from United States Patent (USP) 6,642, listed structure in 362.In some embodiments, modification partly comprises carbon-to-carbon double bond or the triple bond that Raolical polymerizable can take place.Monose can be, for example, lactose, semi-lactosi, sucrose, fructose, seminose and Mierocrystalline cellulose.Oligosaccharides can be homopolymer or the heteropolymer that is made of monose such as lactose, semi-lactosi, sucrose, fructose, seminose and Mierocrystalline cellulose.Oligosaccharides preferably has 2-10 monomer, more preferably 2-3 monomer.Mononucleotide can be, for example, and VITAMIN B4, guanine, cytosine(Cyt), thymidine or uridylic.Oligonucleotide can be homopolymer or the heteropolymer that is made of mononucleotide such as VITAMIN B4, guanine, cytosine(Cyt), thymidine or uridylic.Oligonucleotide preferably has 2-150 monomer, more preferably 2-50 monomer, more preferably 5-35 monomer, more preferably 10-20 monomer.Amino acid can be any and any synthetic amino-acid residue in 26 kinds of natural origin amino acid.Peptide can be a homopolymer as poly--Methionin, or heteropolymer.Peptide preferably has 2-25 monomer, more preferably 2-20 monomer, more preferably 2-15 monomer, more preferably 2-10 monomer, more preferably 2-5 monomer, more preferably 2,3 or 4 monomers.Protein can be any proteinaceous molecule, for example cell attaching or adhesion molecule, receptor protein or aglucon.Protein preferably has the amino acid more than 25, in some embodiments, can be 25-200 amino acid or longer.

In some embodiments, modification partly is the galactosyl oligosaccharides, for example in conjunction with the oligosaccharides of ASGPR asialoglycoprotein receptor or gala lectin.ASPGR is the liver cell adhesion receptor.The gala lectin is the cell adhesion acceptor.In some embodiments, modification partly is sLe ^x(NeuAc α 2-3Gal β 1-[4Fuc α 1-3] GlcNAc) be sectine, cell-cell recognition molecule.In some embodiments, modification partly is GlcNAc, and promptly ASGP also is useful in liver cell adheres to.In some embodiments, modification partly is HA-oligomer (hyaladhesins useful in the endothelial cell proliferation; Hyaluronic acid binding protein).In some embodiments, modification partly is the mannose group oligosaccharides, for example in conjunction with the oligosaccharides of mannose binding lectin (Mannose binding lectine) or Langerin.Mannose binding lectin is relevant with Keratinocytic propagation.Langerin is the acceptor of Langerhans cell.

In some embodiments, the modification part can be the RDG peptide, for example derives from the peptide of fibronectin or vitronectin.The RDG peptide can be used as cell adhesion and sarcoplast adhesin polypeptide.In some embodiments, the modification part can be the YIGSR peptide, for example derives from the peptide of ln B1.The YIGSR peptide can be used as cellular adhesion peptide.In some embodiments, the modification part can be the REDV peptide, for example derives from the peptide of fibronectin.The REDV peptide can be used as endotheliocyte and adheres to peptide.In some embodiments, the modification part can be the IKVAV peptide, for example derives from the peptide of ln.The IKVAV peptide can be used as the spinous process extension peptide.In some embodiments, the modification part can be the KHIFSDDSSE peptide, for example derives from the peptide of nerve cell adhesion molecule.The KHIFSDDSSE peptide and have 2,3,4 or more a plurality of amino acid whose fragment can be used as the astroglia cell adhesin polypeptide.In some embodiments, the modification part can be the KRSR peptide, for example derives from the peptide of heparin binding domain.The KRSR peptide can serve as the osteocyte adhesin polypeptide.

Alginate polymer can be crosslinked by the key between the modification part.These keys can be covalent linkage, ionic linkage, and can comprise the connection intermediate.The alginate polymer that like this, for example can prepare predetermined shape by non-gelling linking agent.

The alginate sample of modification has following general formula:

A-X

Wherein, A is the alginate polysaccharides, and X is the modification part.A and X are covalently bound by the connecting key that is selected from ester, ether, thioether, disulfide linkage, acid amides, imines, secondary amine, direct carbon-to-carbon (C-C) connecting key, sulfuric ester, sulphonate, phosphoric acid ester, carbamate, carbonic ether etc.

Or

In other words, one or more monomers of alginate can directly or pass through the covalently bound modification part of spacer.Therefore, the alginate sample of modification also can have following general formula:

A-Y-X

Wherein, A and X as mentioned above, Y is the spacer that comprises the alkyl or aryl chain, for example alkyl, thiazolinyl, alkynyl or aromatic yl group.In some embodiments, alkyl group is C ₁-C ₁₅, preferred C ₁-C ₁₀, preferred C ₁-C ₅, preferred C ₁-C ₃Alkyl, thiazolinyl, alkynyl or aromatic yl group.A and Y, and Y and X link to each other by aforesaid connecting key.

Connecting key or connection portion can randomly have or not have spacer with the monomer subunits of connection modification part with alginate polymer.The example of connection portion includes but not limited to: ester, ether, thioether, disulfide linkage, acid amides, imines, secondary amine, directly carbon-to-carbon (C-C) connecting key, sulfuric ester, sulphonate, phosphoric acid ester, carbamate and carbonic ether, with or not with spacer such as alkyl, thiazolinyl, alkynyl, aryl combination.

The ester connecting key refers to following structure:

Or

The ether connecting key refers to-the O-structure that the thioether connecting key refers to-the S-structure that disulfide linkage refers to-the S-S-structure that the acid amides connecting key refers to

Or Structure

The imines connecting key refers to

Structure

Secondary amine or tertiary amine connecting key refer to:

Or

Directly the carbon-to-carbon connecting key refers to-the C-C-structure; Sulphonate and sulfuric ester connecting key refer to respectively:

Or

The phosphoric acid ester connecting key refers to:

The carbamate connecting key refers to:

Or

The carbonic ether connecting key refers to:

The inventive method comprises one or more steps, and wherein, the epimerization reaction by enzyme is converted into the G residue with the one or more unmodified M residue of alginate.The epimerization enzyme is well-known.Example derives from azotobacter vinelandii, and as United States Patent (USP) 5,939,289 is described, and its content is included in this by reference.Other source comprises pseudomonas syringae (J.Biol:chem. such as Bjerkan, the 279th volume, the 28920-28929 page or leaf, its content is included in this by reference) and on August 5th, 2004 disclosed international application the palmate sea-tangle (Laminaria digitata) of disclosing in WO2004065594 number (its content is included in this by reference) is disclosed.

Polymannuronic acid C-5 epimerization enzyme (AlgE enzyme) comprises that a class is by the bacterium such as the coded module albumen of azotobacter vinelandii that produce alginate.United States Patent (USP) 5,939,289 have described the sequence of encoding such enzymes, prepare these enzymes method and preparation have definite G/M than and the alginate of block structure in application.The activity of these isozymes and the pattern difference of inducing epimerization.AlgE-1 and 6 can effectively produce long-chain G-block, and AlgE4 only introduces the MGM sequence.The former is strong effectively gel former, and the latter's enzyme produces flexible chain (reference).For example referring to Table I.

Seven kinds of AlgE epimerization enzymes from azotobacter vinelandii

A-385 amino acid, R-155 amino acid

Table I

Use separately or use different C-5 epimerases with form of mixtures, one goes on foot or one after the other handles, the chemistry and the enzyme step that comprise different order, make initial alginate generation epimerization before for example replacing, and then carry out epimerization, can make all alginate and polymannuronic acid generation epimerization.By changing the amount and the time of substitution value and epimerization, can obtain the alginate molecule of multiple choices replacement.By the amount and the combination thereof of controlled temperature, reaction times, reaction reagent, may command epimerization reaction.For example, in some embodiments, add acid, be heated to 90 ℃ or add 50mM EDTA and shelter the required calcium ion of enzyme reaction, can stop epimerization reaction.By control reaction, can control the unmodified amount that is converted into G, thereby control the amount of G in the alginate of final modification.

The character of initial substance is the character of may command the finished product also.Adopt polymannuronic acid as initial substance, modified-reaction is prior to epimerization reaction, produces the final product that M only is modified.That is,, find that these materials can on carboxyl or hydroxy functional group modification take place, and make it epimerization with C-5 epimerization enzyme then from the polysaccharide (polymannuronic acid) that comprises the mannuronic acid residue.This epimerization reaction takes place on the residue of non-modification, causes the alginate molecule that the selectivity modification takes place on mannuronic acid.

If use polymannuronic acid as initial substance and residue modification prior to any enzymatic conversion from M to G, modified-reaction will cause mannuronic acid to have along the substituting group of polymer chain stochastic distribution.The residue of amount by controlling reaction time, temperature, reactant and the modification of combination may command thereof is with respect to the amount of unmodified residue, the polymannuronic acid that has required M modification degree with generation.In second step of this paper, the polymannuronic acid that replaces with polymannuronic acid-C-5 epimerization enzyme (that is, make the D-M residue be converted into the L-guluronic acid and do not destroy the enzyme of polymer chain) treating part.Because C-5 epimerization enzyme can not transform the M-residue of replacement, final product will be the polymkeric substance that comprises complete G block, and to be connected with forming in conjunction with calcium, substituting group only is arranged on the M residue that remains on soluble fractions.In soluble fractions, substituting group can be freely mutually chemically crosslinked or with exogenous acceptor interaction.

In some embodiments, initial alginate comprise M and G.In this case, chemical replacement can occur on M and the G residue.The alginate that replace with the enzyme treating part can transform a part of unsubstituted M and G residue.An embodiment is the alginate that comprise poly MG block, at first replace at M and/or G group top, but the G that uses specificity to transform residue MG alternating polymerization section then forms enzyme (being AlgE-1) by C-epimerization generation enzyme reaction.

By controlled step order and speed of reaction, the alginate polymer of gained modification can have different modification degree, different M and G modification levels, different unmodified M content and different unmodified G content.

In some embodiments, only M is modified.In some embodiments, M and G are modified.

In some embodiments, being less than 10% residue is modified.In some embodiments, being less than 20% residue is modified.In some embodiments, be modified more than 20% residue.In some embodiments, the residue of 10-80% is modified.In some embodiments, the residue of 20-60% is modified.In some embodiments, the residue of 30-50% is modified.In some embodiments, about 40% residue is modified.

In some embodiments, being less than 20% residue is unmodified G.In some embodiments, be unmodified G more than 20% residue.In some embodiments, the residue of 20-80% is unmodified G.In some embodiments, the residue of 30-60% is unmodified G.In some embodiments, the residue of 40-50% is unmodified G.In some embodiments, about 45% residue is unmodified G.

By mixed and modified alginate and divalence gelling ion such as Ca ⁺⁺, Sr ⁺⁺, Ba ⁺⁺, Zn ⁺⁺, Fe ⁺⁺, Mn ⁺⁺, Cu ⁺⁺, Pb, Co, Ni or its combination, can use the alginate of modification to prepare alginate jelly or fiber.

In some embodiments, use alginate jelly to seal viable cell such as proliferative cell or non-proliferative cell.Cell can be from clone or patient/donor.The example of cell comprises: islet cells, liver cell, neurocyte, renal cortical cell, vascular endothelial cell, Tiroidina and parathyroid gland cell, adrenal cells, thymocyte, gonad cell, chondrocyte, myocyte, heart cell, stem cell, inoblast, keratinocyte or derive from the cell of established cell line, for example 293, MDCK and C2C12 clone.In some embodiments, the cell of sealing comprises the expression vector of one or more cells of coding expressed protein when keeping.In some embodiments, described protein is his spit of fland [Endostatin] of his spit of fland [angiostatin] of cytokine, somatomedin, Regular Insulin or angiogenesis inhibitor such as blood vessel or endothelium, other treatment albumen or other treatment molecule such as medicine.Because the porousness of gel network, be especially preferred material standed for less than the low molecular weight protein of about 60-70kD.In some embodiments, cell exists with many cells aggregation or organizational form.

In some embodiments, prepare the alginate fiber by following method, this method comprises: the alginate polymer of multiple modification is mixed with divalence gelling ion, and extruding comprises the fiber of crosslinked alginate polymer.In some embodiments, by form, molded, cast or the alginate polymer of the multiple modification that is shaped preparation solid non-crosslinked alginate compositions or paste.

In some embodiments, already present alginate jelly or fiber are carried out modification procedure and/or epimerization enzyme step.

Embodiment

Embodiment 1: prepare the polymannuronic acid of modification with 1-amino-1-deoxidation-semi-lactosi

The na form (1.5g) that 1-amino-1-deoxidation-β-D-semi-lactosi (270mg) is joined polymannuronic acid contain N-hydroxy-succinamide (NHS) (1.3g) and 1-ethyl-3-[3-(dimethylamino)-propyl group] carbodiimide hydrochloride (EDC) 0.2M 2-[N-morpholino (2.17g)] in the stirring of ethyl sulfonic acid (MES) buffered soln (pH4.5,400mL) in.Stirred this solution 30 minutes under the room temperature.Make the product dialysis dialysis membrane by weight shutoff 12000-14000 5 days with respect to deionized water.With the product freeze-drying of dialysis, obtain the galactose derivate of pure polymannuronic acid sodium.Output: 1.45g.By ¹H-NMR calculates, and finds that substitution value is 12%.This method forms carboxyl (uronic acid) group of the mannuronic acid that exists in the acid amides target polymer.It will be apparent to one skilled in the art that the substitution value of product can be different by use the different ratios of polymannuronic acid and 1-amino-1-deoxidation-semi-lactosi in above-mentioned reaction.Identical method is applicable to amino acid, peptide, different monose or oligosaccharides, Nucleotide and the photocrosslinkable group that has or do not have the amido-containing group of alkyl or aryl spacer between molecule and amido functional group.

Embodiment 2: the methacrylic ester of synthetic polymannuronic acid

Polymannuronic acid sodium (3g) is dissolved in is cooled to 4 ℃ in the 300mL deionized water and in ice bath.Under the constant agitation, methacrylic anhydride (23g) dropwise is added drop-wise in the cold polymannuronic acid solution, makes the pH value maintain 9.0 by adding an amount of 5M NaOH.4 ℃ are continued down to stir 24 hours.Precipitin reaction product in 96% ethanol, centrifugal and with washing with alcohol 3 times.Then product is dissolved in the water, makes the dialysis membrane of its dialysis by weight shutoff 12000-14000 3 days with respect to deionized water.With the product freeze-drying of dialysis, obtain the methacrylate derivative of pure polymannuronic acid sodium.Output: 2.6g.By ¹H-NMR calculates, and substitution value is 8%.The secondary hydroxyl that exists in the ester target monomeric unit that this method forms.It will be apparent to one skilled in the art that the substitution value of product can be different by use the different ratios of polymannuronic acid and acid anhydrides in above-mentioned reaction.Identical method can be applicable to the amino acid, peptide of suitable modification, different monose and oligosaccharides, Nucleotide and photocrosslinkable groups.

Embodiment 3: use the polymkeric substance of AlgE4 epimerization through modification

To be dissolved in as the polymannuronic acid sample (1g) through modification of acquisition as described in embodiment 1 and 2 and contain CaCl ₂(2.5mM) and in the 50mM MOPS damping fluid (pH6.9) of NaCl (10mM), concentration is 2.5g/L.Add C-5 epimerization enzyme AlgE4 (enzyme/polymer weight ratio=1/200) then, 37 ℃ of following stirred solutions 24 hours.So that the pH value is 1-2, make the epimerization reaction quencher by concentrated hydrochloric acid being joined in the cold polymers soln.Add NaCl (ultimate density 1.5%) in mixture, 4 ℃ keep down spending the night.Centrifugal product of separating out is also used dilute hydrochloric acid (0.05M) washing three times.Product is dissolved in the deionized water, makes the pH value keep a little higher than 7.In this solution, add NaCl (ultimate density 0.2%), and use 96% ethanol sedimentation.Product is filtered,, and make the dialysis membrane of its dialysis by weight shutoff 12000-14000 3 days with respect to deionized water with washing with alcohol 3 times.With the product freeze-drying of dialysis, obtain the epimerization fluidized polymer of pure polymannuronic acid through modification.Output: 0.85g.It will be apparent to one skilled in the art that the epimerization degree can be different by using different reaction times.

Embodiment 4: use the polymkeric substance of AlgE6 epimerization through modification

To be dissolved in as the polymannuronic acid sample (1g) through modification of acquisition as described in embodiment 1 and 2 and contain CaCl ₂(2.5mM) and in the 50mM MOPS damping fluid of NaCl (75mM) (pH6.9), concentration is 2.37g/L.Add C-5 epimerization enzyme AlgE6 (enzyme/polymer weight ratio=1/20) then, 37 ℃ of following stirred solutions 48 hours.So that the pH value is 1-2, make the epimerization reaction quencher by concentrated hydrochloric acid being joined in the cold polymers soln.Add NaCl (ultimate density 1.5%) in mixture, 4 ℃ keep down spending the night.Centrifugal product of separating out is also used dilute hydrochloric acid (0.05M) washing three times.Product is dissolved in the deionized water, makes the pH value keep a little higher than 7.In this solution, add NaCl (ultimate density 0.2%), and use 96% ethanol sedimentation.Product is filtered,, and make the dialysis membrane of its dialysis by weight shutoff 12000-14000 3 days with respect to deionized water with washing with alcohol 3 times.With the product freeze-drying of dialysis, obtain the epimerization fluidized polymer of pure polymannuronic acid through modification.Output: 0.90g.It will be apparent to one skilled in the art that the epimerization degree can be different by using different reaction times.

This method produces two kinds of polymkeric substance:

1. with semi-lactosi the uronic acid group is carried out modification, epimerization then

d.s.＝12％：FG＝0.45；FM＝0.55；FGG＝0.16。

With

2) with photocrosslinkable substituting group hydroxyl is carried out modification, epimerization then

Initial substance: as the polymannuronic acid that has carried out modification as described in the embodiment 2: d.s.=8%, FM=1

The material of epimerization: d.s.=8%; FG=0.54; FM=0.46; FGG=0.37.

Fig. 2 and 3 shown with the alginate from the unmodified and modification (14% semi-lactosi) of utmost point North Sea band and compared, to the influence of the gelating property of galactosylation and polymannuronic acid epimerization.

Embodiment 5: use the combination of AlgE4 and AlgE6, to the epimerization of the polymkeric substance of chemical modification

To be dissolved in as the polymannuronic acid sample (1g) through modification of acquisition as described in embodiment 1 and 2 and contain CaCl ₂(2.5mM) and in the 50mM MOPS damping fluid (pH6.9) of NaCl (10mM), concentration is 2.5g/L.Then, add C-5 epimerization enzyme AlgE4 (enzyme/polymer weight ratio=1/100), 37 ℃ of following stirred solutions 24 hours.Add C-5 epimerization enzyme AlgE6 (enzyme/polymer weight ratio=1/20) then, 37 ℃ of following stirred solutions 24 hours.So that the pH value is 1-2, make the epimerization reaction quencher by concentrated hydrochloric acid being joined in the cold polymers soln.Add NaCl (ultimate density 1.5%) in mixture, 4 ℃ keep down spending the night.Centrifugal product of separating out is also used dilute hydrochloric acid (0.05M) washing three times.Product is dissolved in the deionized water, makes the pH value keep a little higher than 7.In this solution, add NaCl (ultimate density 0.2%), and use 96% ethanol sedimentation.Product is filtered,, and make the dialysis membrane of its dialysis by weight shutoff 12000-14000 3 days with respect to deionized water with washing with alcohol 3 times.With the product freeze-drying of dialysis, obtain the epimerization fluidized polymer of pure polymannuronic acid through modification.Output: 0.90g.It will be appreciated by one of skill in the art that, by using different reaction times, the degree of epimerization can be different, produce G block and polymerization and replace all polymkeric substance between the M residue that replaces of block, are to lack the MM sequence with the difference of the polymkeric substance of AlgE 6 epimerizations.The flexibility that this has just increased polymkeric substance causes higher dehydrating condensation (syneresis) and lower swelling.

Embodiment 6: the polymkeric substance of preparation G-block between the polymerization MG sequence that replaces

To be dissolved in as the polymannuronic acid sample (1g) of acquisition as described in embodiment 1 and 2 and contain CaCl ₂(2.5mM) and in the 50mM MOPS damping fluid (pH6.9) of NaCl (10mM), concentration is 2.5g/L.Add C-5 epimerization enzyme AlgE4 (enzyme/polymer weight ratio=1/100) then, 37 ℃ of following stirred solutions 24 hours.So that the pH value is 1-2, make the epimerization reaction quencher by concentrated hydrochloric acid being joined in the cold polymers soln.Add NaCl (ultimate density 1.5%) in mixture, 4 ℃ keep down spending the night.Centrifugal product of separating out is also used dilute hydrochloric acid (0.05M) washing three times.Product is dissolved in the deionized water, makes the pH value keep a little higher than 7.In this solution, add NaCl (ultimate density 0.2%), and use 96% ethanol sedimentation.Product is filtered,, and make the dialysis membrane of its dialysis by weight shutoff 12000-14000 3 days with respect to deionized water with washing with alcohol 3 times.With the product freeze-drying of dialysis, obtain the epimerization fluidized polymer of pure polymannuronic acid through modification.Output: 0.85g.Form: the molar fraction of G=0.47, the molar fraction of GG=0.It will be apparent to one skilled in the art that the degree of epimerization can be different by using different reaction times.

The polymerization MG for preparing modification with 1-amino-1-deoxidation-semi-lactosi

The buffered soln of the na form (1.5g) that 1-amino-1-deoxidation-β-D-semi-lactosi (270mg) is joined the polymannuronic acid of modification in the stirring of the 0.2M MES that contains NHS (1.3g) and EDC (2.17g) (pH4.5,400mL) in.Stirred this solution 30 minutes under the room temperature.Make the product dialysis dialysis membrane by weight shutoff 12000-14000 5 days with respect to deionized water.With the product freeze-drying of dialysis, obtain the galactose derivate of pure polymannuronic acid sodium.Output: 1.45g.By ¹H-NMR calculates, and finds that substitution value is 12%.This method forms carboxyl (uronic acid) group of the mannuronic acid that exists in the acid amides target polymer.It will be apparent to one skilled in the art that the substitution value of product can be different by use the different ratios of polymerization MG and 1-amino-1-deoxidation-semi-lactosi in above-mentioned reaction.Identical method is applicable to amino acid, peptide, different monose and oligosaccharides, Nucleotide and the photocrosslinkable group that has or do not have the amido-containing group of alkyl or aryl spacer between molecule and amido functional group.

Use the polymerization MG of AlgE1 epimerization through modification

To be dissolved in as the polymannuronic acid sample (1g) through modification of acquisition as described in embodiment 1 and 2 and contain CaCl ₂(2.5mM) and in the 50mM MOPS damping fluid (pH6.9) of NaCl (75mM), concentration is 2.37g/L.Add C-5 epimerization enzyme AlgE1 (enzyme/polymer weight ratio=1/20) then, 37 ℃ of following stirred solutions 48 hours.So that the pH value is 1-2, make the epimerization reaction quencher by concentrated hydrochloric acid being joined in the cold polymers soln.Add NaCl (ultimate density 1.5%) in mixture, 4 ℃ keep down spending the night.Centrifugal product of separating out is also used dilute hydrochloric acid (0.05M) washing three times.Product is dissolved in the deionized water, makes the pH value keep a little higher than 7.In this solution, add NaCl (ultimate density 0.2%), and use 96% ethanol sedimentation.Product is filtered,, and make the dialysis membrane of its dialysis by weight shutoff 12000-14000 3 days with respect to deionized water with washing with alcohol 3 times.With the product freeze-drying of dialysis, obtains the epimerization fluidized polymer of pure polymannuronic acid through modification, it is characterized in that long G block is between by the polymerization MG sequence of M or G replacement.Output: 0.90g.It will be apparent to one skilled in the art that the degree of epimerization can be different by using different reaction times.

Embodiment 7

Adopt the combination of chemistry and enzyme method to obtain only on the M residue, to have beta galactose alginate sample polymkeric substance partly.Use EDC (1-ethyl-3-[3-(dimethylamino)-propyl group] carbodiimide hydrochloride) and NHS (N-hydroxy-succinamide) as coupling agent, 1-amino-1-deoxidation-semi-lactosi is introduced polymannuronic acid by amido linkage.Use two kinds of different C-5 epimerization enzymes that this polymkeric substance is carried out epimerization, with alternately or the G sequence form of homopolymerization introduce the guluronic acid residue.With ¹H-NMR and HPSEC-RI-MALLS are characterized on the M residue the optionally grafting alginate of modification, detect limiting viscosity and calcium-binding capacity thereof by the circular dichroism spectrometry.Compare with the alginate sample of on the G residue, introducing identical sugar moieties, improve through the machinery and the gel formation character of the material of modification.At last, on the M residue, carry out the selectivity modification and cause having advantages of higher stability by the calcium pearl of grafting alginate preparation.

Materials and methods

Provided from the commercially available sample of the isolating sodiun alginate of utmost point North Sea band petiole by FMC Biopolymers (Norway), LF 10/60, (F _G=0.69; F _GG=0.56).High molecular polymannuronic acid (F _G＜0.001) epimerization enzyme-negative mutant (Alg`) of separation autofluorescence pseudomonas.According to Ertesv  g, H.; Skj  k-Braek, G., Methods in Biotechnology, 1999, Carbohydrate Biotechnology Protocols; Bucke, C. compiles; Humana Press Inc., Totowa, NJ., (its content is included in this by reference) carries out purifying and deacetylated described in 10,71.By using Hartmann, M.; Duun, A.S.; Markussen, S.; Grasdalen, H.; Valla, S.; Skj  k-Braek, G.Biochim.Biophys.Acta, the AlGE4 epimerization enzyme described in 2002,1570,104 (its content is included in this by reference) prepares polymerization alternative MG (F by polymannuronic acid _G=0.47; F _GG=0).1-ethyl-3-[3-(dimethylamino)-propyl group] carbodiimide hydrochloride (EDC) and sodium-chlor available from Aldrich Chemical Co. (Milwaukee, WI).N-hydroxy-succinamide (NHS), 2-[N-morpholino] ethyl sulfonic acid (MES) and maltonic acid-delta-lactone (GDL) available from Sigma Chemical Co. (St.Louis, MO).Lime carbonate (median size 4 μ m) available from Merck (Darmstadt, Germany).

Reorganization polymannuronic acid C-5 epimerization enzyme

Prepare polymannuronic acid C-5 epimerization enzyme by the following recombinant escherichia coli strain that ferments: AlgE4 is in JM 105, and AlgE6 is in SURE.By Q-Sepharose FF (Pharmacia, Uppsala, Sweden) these enzymes of hydrophobic interaction chromatograph partial purification on the ion-exchange chromatography on and the phenyl Sepharose FF (Pharmacia).Cultivate with enzyme ³During the polymannuronic acid of H-5-mark, come the enzyme analysis activity by measuring the release of tritium in water.

The polymannuronic acid of semi-lactosi-replacement (MGal)

The na form (1.5g) that 1-amino-1-deoxidation-β-D-semi-lactosi (galactosyl amine) (270mg, 0.2 equivalent) is joined polymannuronic acid is containing NHS and EDC ([EDC]/[polymkeric substance]=1.5; [NHS]/[EDC]=1, [polymkeric substance] are the volumetric molar concentrations of glycopyranoside polymer repeat unit) the stirring of 0.2M MES in buffered soln (pH4.5,400mL) in.Stirred this solution 30 minutes under the room temperature, with respect to NaHCO ₃0.05M dialyzed polymer (being about 12000 by molecular weight of film) 1 day is dialysed with respect to deionized water then, is lower than 2 μ S up to 4 ℃ of following specific conductivity.The pH value is adjusted to 7, and by 0.45 μ m Millipore filter, the freeze-drying acquisition has the polymannuronic acid sample of the modification of 12% semi-lactosi (being introduced into as side-chain radical) with polymer filtration, as ¹H-NMR analyzes (substitution value (d.s.) that is calculated with respect to the dystopy proton intensity of M residue in the polymer chain by the H-1 strength of signal of galactosyl amine) and potentiometric titration is disclosed.

With AlgE4 epimerization (MGalE4)

Polymkeric substance MGal is dissolved in contains CaCl ₂(2.5mM) and in the 50mMMOPS damping fluid (pH6.9) of NaCl (10mM), concentration is 2.5g/L.Add C-5 epimerization enzyme AlgE4 (enzyme/polymer weight ratio=1/100), 37 ℃ were stirred this solution 24 hours down.

With AlgE6 epimerization (MGalE4E6)

Polymkeric substance MGalE4 is dissolved in contains CaCl ₂(2.5mM) and in the 50mMMOPS damping fluid (pH6.9) of NaCl (75mM), concentration is 2.37g/L.Add C-5 epimerization enzyme AlgE6 (enzyme/polymer weight ratio=1/20), 37 ℃ were stirred this solution 48 hours down.

The purifying of epimerization fluidized polymer

By 5M NaCl solution (ultimate density 1.5%) and hydrochloric acid (3M) being joined in the cold polymers soln, make the epimerization reaction quencher so that the pH value is about 1-2.Under 4 ℃ with this mixture store overnight to help precipitation.Throw out is centrifugal and with dilute hydrochloric acid (0.05M) washing three times.Then, throw out is dissolved in the deionized water, makes the pH value keep a little higher than 7 by adding dilute sodium hydroxide.This solution is mixed (ultimate density 0.2%) with 5M NaCl solution, and use ethanol sedimentation.The dissolution precipitation thing, with respect to deionized water dialyse (film by molecular weight be about 12000) be lower than 2 μ S up to 4 ℃ of following specific conductivity, pH value is adjusted to 7, filter and pass through 0.45 μ m Millipore filter and freeze-drying.

Alginate (LhypGal) from the semi-lactosi-replacement of utmost point North Sea band

Past has been reported with the alginate sample of 1-amino-1-deoxidation-semi-lactosi processing from utmost point North Sea band.The alginate of the modification that contains 14% galactose moiety are introduced in acquisition on the G residue, as ¹The H-NMR analysis discloses.

¹The H-NMR spectrography

According to described preparation samples such as Grasdalen.With Bruker WM 300 at 90 ℃, D ₂Record among the O ¹The H-NMR collection of illustrative plates.Chemical shift is expressed as p.p.m., and is downward from the signal of 3-(trimethyl silyl) propanesulfonic acid ester.

Potentiometry

Carry out the equivalent weight of potentiometric determination MGal and MGalE4E6 sample.Use is equipped with the radiometer pHM240 pH meter of glass electrode.Spend the night the H of preparation polymkeric substance by HCl dialysis 3g/L solution with respect to 0.1M ⁺Form.By thoroughly dialysing, remove excessive HCl with respect to deionized water.The freeze-drying collected polymer.With 0.1M NaOH standardized solution (Tritisol, Merck) specific concentrations of the titration known polymer aqueous solution.Find the H of MGal and MGalE4E6 ⁺The repeating unit molar mass of form is respectively 198 ± 4g/mol and 200 ± 3g/mol, realizes well coupling with the theoretical value of calculating based on NMR gained substitution value (195.3g/mol).

The circular dichroism spectrometry

Be recorded in (c～2 * 10 in the deionized water respectively with Jasco J-700 spectropolarimeter ^-3Mol/L) the circular dichroism spectrogram of polymkeric substance MGal, MGalE4 and MGalE4E6 na form (seeing Table 2).Use the quartz cell of 1 centimetre of path length, keep following and set: bandwidth 1nm; Time constant 2s; Scanning speed 20nm/min.For each sample, get mean value with respect to four collection of illustrative plates of background correction.Adding Ca (ClO ₄) ₂Solution makes [Ca ²⁺))/collection of illustrative plates of each sample with afterwards, is write down before in [polymkeric substance]=0.26.

The formation of pearl

By 2% (w/V) polymers soln is added drop-wise to 50mM CaCl ₂In the solution, prepare the calcium pearl from utmost point North Sea band, LhypGal and MGalE4E6 respectively.Use high pressure static electricity pearl producer (7kV, 10mL/h, 0.4mm external diameter draw point, pin is 1.7-cm to the distance of gelling soln) control drop size.Before the use, gained alginate jelly pearl was stirred in gelling soln 30 minutes.

Stability in the salt brine solution

The 0.5mL gel beads is joined in the 3mL salt brine solution (0.9%), measure respectively the dimensional stability of the Protanal TXF 200 pearl that obtains from utmost point North Sea band, LhypGal and MGalE4E6 with being inverted opticmicroscope (Zeiss).Sample was stirred 1 hour.Change several times salt brine solution and before each the replacing, determine capsule diameter (n=25).Use the deionized water rinsing capsule before measuring.

Gelling kinetics and rheological charactristics

(REOLOGICA instruments AB, 22363 Lund Sweden) measure gelling kinetics and dynamic viscoelastic feature to adopt the Stress-Tech rheotron.In brief, respectively in 1.5% solution of utmost point North Sea band, LhypGal and MGalE4E6 (referring to table 2) add CaCO ₃(20mM) and GDL (40mM), this mixture was stirred 30 seconds, measure then.T=20 ℃, during crack=1.00mm, carry out these experiments with serrated plate-plate (d=40mm) measuring apparatus.Timed interval replication G ' and G with 3 minutes " (ω=6.28rad*s ^-1) continue about 18 hours, determine gelling kinetics.By measuring the frequency dependence of storage modulus (G ') and out-of-phase modulus (G "), after inducing gelling, carried out the dynamic viscoelastic sign in 24 hours.Under range of frequency 0.01-50Hz, carry out frequency sweep with constant strain (0.001).With low density silicone oil sealed sample to avoid the side effect relevant in whole gelling experiment with solvent evaporation.

Preparation gel right cylinder and synersis

By Ca with polymers soln and deactivation form ²⁺(CaCO ₃) mix, add the maltonic acid-delta-lactone (GDL) of slow hydrolysis then, keep mol ratio GDL/Ca ²⁺=2, prepare homogeneous calcium gel from utmost point North Sea band, LhypGal and MGalE4E6 respectively.The ultimate density of polymkeric substance is 1% (w/V) in all cases.

Suppose that density value is 1 to calculate, to determine the synersis of calcium alginate gel with respect to the minimizing of the gel weight of initial weight.The aliquots containig of calcium polymer gel solution of preparation is as mentioned above solidified in 24 hole tissue culturing plates, culture plate diameter 16mm, high 18mm (costar, Cambridge, MA).From the hole, take out gel after 24 hours, measure its weight.With (1-W/W ₀Synersis, wherein W and W are calculated in) * 100 ₀Refer to cylindrical final weight of gel and initial weight respectively.

Use Stable Micro Systems TA-XT2 contextual analysis of organization instrument to measure down, calculate the Young's modulus (E) of gained gel by the initial slope of power/deformation curve at 20 ℃.For all gels that shows synerisiss, determine final polymer concentration and according to E ∝ c ²Proofread and correct E.

Viscosity measurement

Adopt Schott-Ger  te AVS/G auto-plant and Ubbelohde type viscometer, under 25 ℃ in 0.1MNaCl the capillary viscosity (seeing Table 2) that reduces of working sample MGal, MGalE4 and MGalE4E6 respectively.Adopt Huggins (equation 1) and Kraemer (equation 2) equation respectively, by analyzing specific viscosity (η _Sp/ c) reduce and relative viscosity logarithm (ln η _Rel/ the concentration dependent that c) reduces is determined inherent viscosity.

η _Sp/ c=[η]+k ' [η] ² C equation 1

(ln η _Rel)/c=[η]-k " [η] ²C equation 2

Wherein, k ' and k " be Huggins and Kraemer constant.

Efficient size exclusion chromatography is in conjunction with multiple angle laser light scattering (HP SEC-RI-MALLS)

The HPSEC-RI-MALLS system is made up of online degasifier (Shimadzu DGU-4A), pump (ShimadzuLC-10AD) and 3 placed in-line posts (TSK GEL G6000/5000/4000 PWXL).Elutriant is the 0.05M Na that contains 0.01M EDTA ₂SO ₄(pH6), speed 0.5mL/min.Detector is refractive index (RI), UV monitor (Pharmacia LKB UV-M II, Amersham Pharmacia Biotech.Uppsala, Sweden), multiple angle laser light scattering (the MALLS-Dawn DSP that has He-Ne laser apparatus 632.8nm, Wyatt Technology Corp., Santa Barbara, CA, USA).Sample is dissolved in the 0.05M Na that contains 0.01M EDTA with the about 1mg/mL of concentration ₂SO ₄In the solution, pH=6 filtered by 0.22 μ m filter before injecting 100 μ L.Employing ASTRA software (edition 4 .70.07, Wyatt Technology Corp., Santa Barbara, CA USA) analyzes the data that are used for determining molecular weight.Used index increment (dn/dc) is 0.15. ³¹The angle match is based on the Debye method, according to the 1 grade polynomial curve fitting of logM (M=molecular weight) with elution volume, obtains weight-average molecular weight Mw and number-average molecular weight Mn.

Result and discussion

Synthesize and sign

Past reports that introducing 1-amino-1-deoxidation-beta galactose on the alginate chain mainly influences the G residue, influence the conformation of gel ability and stability and polymer chain.That optionally introduces side-chain radical on mannuronic acid (M) residue may represent attracting improvement.Consider that these groups do not participate in gel formation, the calcium combination and the gelating property of the alginate of these selectivity modifications are unaffected.Yet because the similarity of uronic acid functional group, in author's ken, the scheme based on blocking group is not to be fit to this purpose.In order to overcome this problem, consider polymannuronic acid is carried out in succession chemical modification, then by two kinds of epimerizations of C-5 epimerization enzyme induction (Figure 14).

The first step by the N-glycosidic link, is introduced 1-amino-1-deoxidation-semi-lactosi (galactosyl amine) on the uronic acid group of polymannuronic acid M residue.In the presence of NHS, employing enriching agent EDC carries out the linked reaction between alginate and the galactosyl amine, has proved that this process is successful.The sugar-substituted polymannuronic acid MGal's of gala ¹The H-NMR collection of illustrative plates as shown in Figure 6.As mentioned above, in case on the M residue, introduce the galactosyl amine moiety, near the 4.75ppm place, can detect the peak from the dystopy proton of the sugar that exists with the side chain form of new formation.Substitution value (12%) that obtains by peak area and polymkeric substance H ⁺The potentiometric titration income value (14%) of form is well relevant.

From the sugar-substituted polymannuronic acid of gala, adopt enzyme AlgE4 and AlgE6, by two epimerization reactions in succession, the guluronic acid residue is introduced polymeric chain.Be different from work in the past,, use two kinds of enzymes independently according to reaching the discrepancy in elevation to the required different sodium chloride concentration of isomerization efficient.In first time epimerization reaction, handled sample MGal 24 hours with AlgE4, thereby the G residue is introduced the alternately long sequence (Figure 14) of MGM, as the binding mode of epimerase expectedly is provided.In fact, be MGalE4's at the epimerization material ¹In the H-NMR collection of illustrative plates (Fig. 6), near the peak of the dystopy proton of the G residue that can clearly detecting the 5.07ppm place makes a fresh start introduces.Estimate by latter's peak area, find the total content (F of G residue _G) be 0.33 (table 2).And, it should be noted that owing to belong to the existence of the H-5 signal of G residue in the alternate sequence, the collection of illustrative plates complicacy in 4.8 to 4.65ppm scanning area increases.

Owing to can distinguish the galactosyl amine that connects on the M residue in homopolymerization or the alternate sequence, to find on the M residue of modification and the obstruction of epimerization reaction on the adjacent group, this can easily detect.In fact, do not detecting the dystopy proton signal that belongs to the galactosyl amine of on the M of contiguous G residue, introducing near the 4.9ppm place, proving that epimerization can not take place the M residue of the M part of contiguous modification.Consider that based on this total epimerization that MGalE4 can be reached and the polymannuronic acid gained result of AlgE4-processing compare.Fig. 7 a has shown the enzyme efficient of representing with the ratio between experiment and theoretical G residue content (%), and the complete epimerization of all obtainable M residues of the calculation assumption of theoretical content produces strict alternative sequence.For the polymannuronic acid that AlgE4-handles, find that the final G content in the strict alternate sequence is 0.47.The theoretical maximum of considering this substrate is 0.50, and calculating enzyme efficient is 94%.For sample MGalE4, epimerization reaction can not take place for the residue of semi-lactosi modification and adjacent M group: this fact causes the theoretical maximum (F of the G residue introduced _G) equal 0.38.Therefore, can think that when enzymic activity was reduced to 86% under latter event, the galactose residue that exists as side chain only caused very little influence to epimerization reaction.

Adopt epimerization enzyme AlgE6 to carry out second time epimerization reaction to introduce homopolymerization G sequence, generation sample MGalE4E6 (Figure 14).Fig. 6 has shown sample MGalE4E6's ¹The dystopy zone of H-NMR collection of illustrative plates.The new signal from the H-5 proton of G residue in the homopolymerization sequence that forms proves near the 4.45ppm place, only has on the M residue in the MGalE4E6 sample of 12% galactose moiety alternately and the existence of homopolymerization G sequence.The content of sample MGalE4E6 monad and dyad is as shown in table 2.Should emphasize the nearly existence of 16%GG dyad, this is the essential feature that forms the calcium gel.

Some signals of polymer chain and signal overlap among the sample MGalE4E6 as the galactose moiety of side chain; This is independently method inspection substitution value of prompting just.Therefore, to the H of polymkeric substance ⁺Form is carried out potentiometric titration.Later a kind of mode is calculated substitution value (15%) and is confirmed, the degraded of N-glycosidic link does not all take place during the purifying of epimerization or final product.

In order to estimate the efficient of epimerization enzyme AlgE6, under identical reaction conditions, handle strict alternative MG polymkeric substance (Fig. 7 b) to sample MGalE4.Under above-mentioned identical hypothesis, can think that the existence of galactosyl amine can significantly not hinder the extra G residue of introducing in polymkeric substance in the polymeric chain.In fact, shown in Fig. 7 b, the observations (67%) that replaces the MG sample with polymerization is compared, and finds that AlgE6 reduces (59%) a little to the efficient of the polymkeric substance of semi-lactosi-modification.But side chain is more remarkable to the effect of introducing the GG dyad, with same enzyme polymerization is replaced shown 41% the comparing of MG sample, and enzyme equals 21% to the efficient of MGalE4.

Table 2 has been summed up the composition of three kinds of samples of monad and dyad form.Exist simultaneously alternately and the G sequence of homopolymerization, sample MGalE4E6 can be described as the alginate sample molecule that optionally has 12% galactosyl amine moiety on the M residue.

For desk study is introduced the influence of spacer to epimerization between polymkeric substance and sugar moieties, with right-aminophenyl-β-D-galactopyranoside (pNH ₂Ph β Gal) is connected on the polymannuronic acid polymer chain.Adopt EDC/NHS chemical method (referring to materials and methods), with 0.3 equivalent pNH ₂PhPGal obtains the polymkeric substance of modification, with the described identical reaction conditions of MGal under epimerization takes place, the gained sample passes through ¹H-NMR analyzes.By collection of illustrative plates shown in Fig. 8 (a-c) as can be known, though substitution value higher relatively (d.s.=18%) has been realized significant epimerization.In fact, among Fig. 8 ¹The quantitative analysis of H-NMR collection of illustrative plates discloses, and considers at pNH ₂Introduce the G residue on the substrate of Ph β Gal-modification, efficient is respectively 82% and 56% for AlgE4 and AlgE6, thereby the C-5 counter-rotating that the rigid spacer group of proof phenyl moiety representative can the unmodified M residue of remarkably influenced.In addition, should be understood that with AlgE4 and AlgE6 handle not can polymer pyrolysis and side-chain radical between amido linkage, as the epimerization sample ¹The resonance that can detect easily that belongs to aromatic ring in the H-NMR collection of illustrative plates proves (referring to Fig. 8 b and c).

Molecule details situation (table 2) by limiting viscosity and SEC-MALLS measuring result preliminary assessment sample MGal, MGalE4 and MGalE4E6.Should emphasize that above-mentioned two kinds of technology all disclose,, be likely because due to the slight lytic activity of enzyme because epimerization causes molar mass to reduce.Though degrade, the polymkeric substance that chemistry of the present invention-enzyme method produces is that MGalE4E6 still has higher relatively molecular weight (about 183000).

Employing is by Flory and Fox ³⁷From the equation 3 that equivalence model (" non-draining " theory) is derived, the persistence length q of assess sample MGal, MGalE4 and MGalE4E6, ³⁶Suppose that alginate are the molecule (worm sample chain) of relative rigidity, estimate persistence length from particular viscosity and molecular mass:

$q=\frac{1}{2}*[\left(\frac{1}{\mathrm{DP}*l}\right)*{\left(\frac{\left[\mathrm{\η}\right]M_w}{\mathrm{\Φ}}\right)}^{\frac{2}{3}}]$ Equation 3

Wherein, DP represents the polymerization degree, the 1st, and actual key length, φ are the functions of chain molecular cell spatial distribution ³⁸3 pairs of mono-disperse systems of equation are effectively strict; And, suppose that φ is a universal constant, in given group of the different polymkeric substance of being considered, be constant at least perhaps.Under these hypothesis ²¹(for MGal, MGalE4 and MGalE4E6 is respectively 12.2 ± 1.2nm to obtain closely similar q value, 13.5 ± 0.6nm and 14.4 ± 0.3nm), prompting is in qualitative level, and the epimerization of M residue can significantly not change the rigidity of the polymkeric substance of these semi-lactosi modifications.

Optics chirality character (Fig. 9 a-c) by circular dichroism spectrometry difference study sample MGal, MGalE4 and MGalE4E6.It should be noted that three kinds of samples show the different molar ellipticities and the curve of function of wavelength owing to introduce the G residue in polymeric chain.In fact, two kinds of sugar components of well-known alginate have different optics chirality behaviors, and the total CD spectrogram of polymkeric substance depends on the relative quantity and the sequence pattern of M and G part.Specifically, the CD spectrogram of GG, MM and MG sequence shows different positions, signal and peak intensity.Circular dichroism spectrum also provides about by above-mentioned three kinds of polymkeric substance (being MGal, MGalE4 and MGalE4E6) though in conjunction with just qualitatively information divalent cation such as calcium, useful.Polymeric sugars aldehydic acid part causes the conformational change of calcium binding sequence to the strong coordination of divalent cation.The latter causes the change of carboxylate groups electronic environment again, detects the variation of the total CD collection of illustrative plates of polymer samples.Before adding known and equivalent calcium and write down the CD collection of illustrative plates of MGal, MGalE4 and MGalE4E6 afterwards respectively, the result is shown in Fig. 9 (a-c).Specifically, the spectrogram that it should be noted that sample MGal do not show take place when adding calcium relevant the change (Fig. 9 a), therefore got rid of homopolymerization M sequence calcium ion is produced the specificity coordinate may.On the contrary, by handling sample MGalE4E6 with equivalent calcium, detect the noticeable change (Fig. 9 c) of collection of illustrative plates, this noticeable change may be interpreted as and only form the orderly homopolymerization G sequence (so-called " egg box " structure) of conformation in MGalE4E6.This result proves that the material of selectivity modification and epimerization has the ability of coordination in conjunction with calcium.Merit attention, shown in Fig. 9 b, though fundamentally lack the GG dyad, polymkeric substance MGalE4 also shows perceptible change when adding calcium.Though need further to analyze, Morris and colleague thereof think that calcium exists the formation that interchain connects between the alternately long sequence of inductive rule can explain observed behavior.

Gel formation and character

For the alginate MGalE4E6 that proposes the selectivity modification as suitable biological activity biomaterial, measured the physical properties of calcium gel, i.e. gelling kinetics, visco-elasticity behavior and Young's modulus.Specifically, compared by sample MGalE4E6 and the calcium-hydrogel that obtains by synthetic sample LhypGal as mentioned above.Should emphasize that though the former only has 1-amino-1-deoxidation-semi-lactosi of 12% on the M residue, the latter is characterised in that, has the identical residue of similar content (14%) on the G part.In this comparison, use unmodified alginate to form material as standard gel from utmost point North Sea band.

By with inactivation form (CaCO ₃) calcium ion join in the polymers soln slow hydrolysis of lactone GDL then, study sample MGalE4E6, LhypGal and respectively from the gel formation kinetics of the alginate of utmost point North Sea band.In all three kinds of samples, the calcium of adding and the mol ratio of polymer repeat unit are 0.26, with the dehydrating condensation (referring to Figure 12 b) of restriction gel.

In this " inner gelling " process, (slowly) hydrolysis of GDL discharges proton, with insoluble CaCO ₃Be converted into HCO ₃ ^-Thereby, provide gel formation required free calcium ions.Sluggishness between lactone mixing and the gel formation makes it possible to the formation and the curing (Figure 10 a-c) of research hydrogel in rheometer.

Figure 10 a has reported the variation of utmost point North Sea band, LhypGal and MGalE4E6 storage modulus (G ') in first 1000 seconds of gel formation process respectively.Data show that introducing galactose moiety on alginate G residue influences gel formation kinetics strongly.In fact, by LhypGal relatively and unmodified alginate sample, can though think that the former did not show the noticeable change of G ' value in first 1000 seconds, 16 times of the latter's storage modulus increases from utmost point North Sea band.On the contrary, galactose content is similar to LhypGal but the sample MGalE4E6 that optionally introduces on the M residue is presented at that storage modulus significantly increases in identical observing time, show with alginate and compare faster formation gel from the semi-lactosi-modification of utmost point North Sea band.The remarkable increase of storage modulus can be traced back to the high-content in the long alternate sequence polymkeric substance among the sample MGalE4E6, may cause faster and more effective being connected to form.

These Considerations are confirmed by Figure 10 b, have reported the variation of the phase angle (δ) that utmost point North Sea band, LhypGal and MGalE4E6 write down during first 1000 seconds at gel formation respectively.Again, on the G residue, introduce the gel formation that side chain is unfavorable for LhypGal.On the contrary, employing enzyme method and the selectivity that is implemented on the non-gel formation M residue replace, and promptly for sample MGalE4E6, the gel formation character of polymkeric substance is unaffected.

Then, utmost point North Sea band, LhypGal and MGalE4E6 are carried out inner gelling gained gel be cured respectively, continue about 7 * 10 ⁴Second, under all three kinds of situations, obtain stable gel, shown in Figure 10 c.After being completed into gel, measure the mechanical collection of illustrative plates (Figure 11) of utmost point North Sea band, LhypGal and MGalE4E6 sample respectively.Under all three kinds of situations, in entire area ω, energy storage capacity (G ') always is higher than out-of-phase modulus (G "), satisfies primary demand so that this material becomes gel.Merit attention, in sample MGalE4E6, G ' and frequency-independent are in conjunction with G ' and G " between about 100 times difference, show that this system is strong gel.

In order further to estimate differences of physical properties from the hydrogel of three kinds of alginate sample, measure respectively from MGalE4E6, LhypGal and unmodified utmost point North Sea band alginate sample obtain gel-(Figure 12 a) for cylindrical Young's modulus.For three kinds of samples of quantitative comparison, making calcium ion and the constant mol ratio that the G residue of calcium chelating can take place is 0.59.Therefore, every kind of polymkeric substance is adopted different lime carbonate concentration respectively, promptly 13.3,16 and 22mM, preparation is from the gel right cylinder of MGalE4E6, LhypGal and utmost point North Sea band alginate.

It should be noted that value from unmodified alginate sample (～11kPa), but on the G residue, introduce galactosyl amine moiety remarkably influenced gel-strength, the Young's modulus of LhypGal is reduced to about 4.2kPa.Yet, on polymannuronic acid, introduce the side chain semi-lactosi, carry out twice epimerization reaction then, aspect gel-strength, produce result preferably.In fact, sample MGalE4E6 is recorded Young's modulus 8.7kPa, emphasized the importance of the polymer-modified chain of selectivity.

Sample MGalE4E6 also shows the calcium (CaCO of adding ₃) the remarkable dehydrating condensation of amount institute's inductive, shown in Figure 12 b.The gel dehydrating condensation is a kind of macroscopic appearance, it is characterized in that slowly, changes in time, shrinks, causes partially liq to ooze out.Having proposed dehydrating condensation is because the horizontal integration of gel formation post polymerization thing chain produces, relevant with the amount of the alternate sequence that exists in the alginate sample.In Figure 12 b, respectively to sample MGalE4E6, LhypGal and utmost point North Sea band, with the ratio mapping of dehydrating condensation (%) with calcium/polymer repeat unit.It should be noted that and compare from the unmodified sample of utmost point North Sea band, the epimerization material is that MGalE4E6 shows that dehydrating condensation is to being dispersed in the CaCO in the solution ₃Measurer has higher dependency.Consider the alternately MGM sequence that has more amount in the MGalE4E6 polymkeric substance, the soluble behavior.On the contrary, alginate sample (being LhypGal) from the G-modification of utmost point North Sea band does not show that dehydrating condensation has any dependency to calcium concn: under latter event, the existence of large volume galactose moiety has spatially hindered the horizontal integration of polymer chain in the gel on the G residue, thereby has hindered the dissipate-swelling effect.

Capsule forms and stability

Especially pay close attention to sample MGalE4E6 and form capsular ability.It should be noted that the 2%MGalE4E6 aqueous solution is added drop-wise in the 50mM calcium chloride solution, obtain stable capsule.Use static pearl producer control capsule diameter (referring to the materials and methods part), find that capsule diameter is 404 ± 19 μ m (n=20).

By measuring the variation of handling back size (diameter) with salt brine solution (NaCl 0.9%), the capsular stability that test is obtained by sample MGalE4E6.In order to compare, also considered the capsular stability that obtains by unmodified utmost point North Sea band and sample LhypGal.

Capsule is an ionic gel, and its volume is mainly controlled by infiltration malleation (swelling), and the negative pressure that infiltration malleation and network resilience cause contends with, and negative pressure is relevant with the crosslinked quantity in the gel.

Use excessive Na ⁺Counter ion, promptly salt brine solution is handled capsule, competes between unit price and divalent cation, finally causes the displacement of calcium ion in the capsule.Total effect of this processing is that the quantity and the length of the G connection that causes the capsule diameter increase are reduced.Therefore, the variation that given salt solution is replaced quantity is big more, and capsular stability is low more.

Figure 13 has reported that salt brine solution repeats to replace respectively by utmost point North Sea band alginate, LhypGal and the capsular influence of MGalE4E6 gained.By introducing the sample (being LhypGal) of 14% semi-lactosi on more unmodified utmost point North Sea band and the G residue, emphasize under latter event, the clean reduction of stability takes place, as mentioned above.In fact, after 2 salt brine solutions are changed, show that from the capsule of sample LhypGal diameter increases 2-doubly, and only increase by 1.1 times from the capsule of the unmodified alginate of utmost point North Sea band.This effect can be traced back to the existence of pendant moiety on the uronic acid residue of alginate Lip river in the middle ancient times, causes significantly damaging its calcium in conjunction with character.

On the contrary, the capsule from MGalE4E6 shows remarkable stability, 1.3 times of 2 salt solution replacing back diameter increases.Consider that in the MGalE4E6 polymkeric substance, the introducing of side-chain radical only influences the M residue, this has explained that MGalE4E6 compares advantages of higher stability with the material LhypGal of G-modification.Selectivity modification on this residue does not relate to gel formation, can not damage the calcium combination of alginate sample, causes more stable capsule.In addition, the effect of alternately long sequence in capsule stability also proposed, as mentioned above.

Conclusion

Obtain new departure that polymannuronic acid pure on the structure and different C-5 epimerization enzymes make it possible to design a kind of preparation alginate sample molecule of selectivity modification on the M residue.Preparing this enzyme method of test in the new biologic activity biomaterial that only on the mannuronic acid part, has galactose residue.By ¹H-NMR analyzes the effect of epimerization enzyme to the material of semi-lactosi modification, records limiting viscosity, SEC-MALLS and the circular dichroism spectrogram of resulting polymers.

Rheology measurement to the material of modification and epimerization is pointed out, especially compares with the alginate sample of carrying out similar modification on the G residue, and selectivity is introduced the benefit of side-chain radical to mechanical properties on the M residue.

By galactose moiety is provided, the material of modification and epimerization becomes and is used to seal hepatocellular new biological activity biomaterial, and wherein, with respect to the modification alginate from utmost point North Sea carry sample, the machinery and the swelling property of alginate jelly improve.Yet, it should be noted that this enzyme method provides applicability widely, be the especially attractive and open novel method of preparation novel biomaterial.As a result, to polymannuronic acid carry out modification then epimerization become reliable novel method, with the material of the selectivity modification that obtains to have customization structure and physical properties.

Table 2: polymkeric substance MGal, MGalE4 and MGalE4E6 composition, limiting viscosity and the molecular weight aspect monad and dyad content

1. sample	F G	F M	F GG	F GM/ MG	F MM	[η] (dL/g) a	k’	k”	Mw (g/mol) b	D (Mw/M N) c
											MGal MGalE4 MGalE4E6	0 0.33 0.45	1 0.67 0.55	0 0 0.16	0 0.33 0.29	1 0.34 0.26	11.98 9.34 8.85	0.424 0.393 0.372	0.12 0.130 0.141	448000 236200 183200	1.54 1.68 1.73

F _GThe ratio of the alginate that expression is made of guluronic acid.F _GGThe ratio of the alginate that expression is made of the guluronic acid of dimer block form, and F _MMThe ratio of the alginate that expression is made of the mannuronic acid dyad.F _GM/ _MGThe ratio of the alginate that expression is made of blended guluronic acid and mannuronic acid sequence. ^aSolvent: NaCl 0.1M, T=20 ℃, k ' and k " represent Huggins and Kraemer constant respectively. ^bWeight-average molecular weight, ^cThe heterogeneity index that HPSEC-RI-MALLS measures.

Claims (20)

1. method for preparing the alginate polymer of modification said method comprising the steps of:

A) by connector or not by connector with the covalently bound one or more unmodified monomer subunits of modification part to alginate polymer; With

B) epimerization reaction by enzyme makes one or more unmodified mannuronic acid (M) monomer subunits of alginate polymer be converted into one or more unmodified guluronic acids (G) monomer subunits.

2. the method for claim 1 is characterized in that, described modification partly is selected from: monose, oligosaccharides, mononucleotide, oligonucleotide, amino acid, peptide and protein.

3. the method for claim 1 is characterized in that, described modification partly is selected from: semi-lactosi and oligomer thereof, seminose and oligomer thereof, sLe ^x(NeuAc α 2-3Gal β 1-[4Fuc α 1-3] GlcNAc), GlcNAc, HA-oligomer (transparent attachment proteins; Hyaluronic acid binding protein), RDG peptide, YIGSR peptide, REDV peptide, IKVAV peptide, KHIFSDDSSE peptide and KRSR peptide.

4. the method for claim 1 is characterized in that, the epimerization reaction of described enzyme uses the epimerization enzyme that derives from azotobacter vinelandii, pseudomonas syringae or palmate sea-tangle.

5. the method for claim 1, it is characterized in that the epimerization reaction of described enzyme uses the epimerization enzyme that is selected from down group: azotobacter vinelandii AlgE1, azotobacter vinelandii AlgE2, azotobacter vinelandii AlgE3, azotobacter vinelandii AlgE4, azotobacter vinelandii AlgE5, azotobacter vinelandii AlgE6 and azotobacter vinelandii AlgE7.

6. the method for claim 1 is characterized in that, step a) was carried out before step b).

7. the method for claim 1 is characterized in that, the unmodified monomer subunits of described alginate polymer all is unmodified M monomer subunits before step a).

8. the method for claim 1 is characterized in that, step a) and b) afterwards, the alginate polymer of modification comprises the unmodified G monomer subunits of 40-50%.

9. method for preparing alginate jelly or fiber, described method comprises: mix multiple alginate polymer as claimed in claim 1 and divalence gelling ion in solvent.

10. method as claimed in claim 9 is characterized in that, described divalence gelling ion is calcium, strontium or barium.

11. the method for preparing alginate jelly as claimed in claim 9 is characterized in that described alginate jelly also comprises one or more viable cell.

12. method as claimed in claim 11, it is characterized in that described alginate jelly also comprises one or more viable cell that is selected from down group: islet cells, liver cell, neurocyte, renal cortical cell, vascular endothelial cell, Tiroidina and parathyroid gland cell, adrenal cells, thymocyte, gonad cell, chondrocyte, myocyte, heart cell, stem cell, inoblast, keratinocyte or derive from established cell line as 293, the cell of MDCK and C2C12 clone.

13. alginate polymer that comprises the modification of unmodified mannuronic acid (M) monomer subunits and unmodified guluronic acid (G) monomer subunits; Wherein, only the M monomer subunits is modified, and modification comprises that the modification part that makes except that ethanoyl is by connector or be not attached to one or more mannuronic acids (M) monomer subunits of alginate polymer by connector.

14. the alginate polymer of modification as claimed in claim 1 is characterized in that, described modification partly is selected from: monose, oligosaccharides, mononucleotide, oligonucleotide, amino acid, peptide and protein.

15. the alginate polymer of modification as claimed in claim 1 is characterized in that, described modification partly is selected from: semi-lactosi and oligomer thereof, seminose and oligomer thereof, sLe ^x(NeuAc α 2-3Gal β 1-[4Fuc α 1-3] GlcNAc), GlcNAc, HA-oligomer (transparent attachment proteins; Hyaluronic acid binding protein), RDG peptide, YIGSR peptide, REDV peptide, IKVAV peptide, KHIFSDDSSE peptide and KRSR peptide.

16. the alginate polymer of modification as claimed in claim 1 is characterized in that, the alginate polymer of described modification comprises the unmodified G monomer subunits of 40-50%.

17. alginate jelly or fiber, it comprises multiple alginate polymer as claimed in claim 1 by divalence gelling ionomer.

18. alginate jelly as claimed in claim 6 or fiber is characterized in that, described divalence gelling ion is calcium, strontium or barium.

19. alginate jelly as claimed in claim 6 is characterized in that, described alginate jelly also comprises one or more viable cell.

20. alginate jelly as claimed in claim 8, it is characterized in that described alginate jelly also comprises one or more viable cell that is selected from down group: islet cells, liver cell, neurocyte, renal cortical cell, vascular endothelial cell, Tiroidina and parathyroid gland cell, adrenal cells, thymocyte, gonad cell, chondrocyte, myocyte, heart cell, stem cell, inoblast, keratinocyte or derive from established cell line as 293, the cell of MDCK and C2C12 clone.

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