GB2572398A

GB2572398A - Enzyme-polymer conjugate

Info

Publication number: GB2572398A
Application number: GB1805086.4A
Authority: GB
Inventors: Klumperman Lubertus; Cloete William; Swart Pieter; Hayward Stefan
Original assignee: Stellenbosch University
Current assignee: Stellenbosch University
Priority date: 2018-03-28
Filing date: 2018-03-28
Publication date: 2019-10-02
Also published as: GB201805086D0; WO2019186452A1

Abstract

An enzyme—polymer conjugate which has at least one hydrolase enzyme immobilised thereon is provided. The conjugate has the structure of Formula I: wherein R1 is a hydrolase enzyme and R2 is OH or O-, or R1 is OH or O- and R2 is a hydrolase enzyme; i is an integer from 1 to 19; and n is an integer from 5 to 1000. The SMA polymer may be suitable for use in a biotechnological process or for protecting a surface from biofouling. Preferably the hydrolase enzyme is chosen from amylase, protease and galactosidase and the enzyme forms a covalent bond between a lysine group of the enzyme and a maleic anhydride group of the polymer. In an embodiment, some subunits are conjugated to amylase and others to protease. Preferably the conjugate is in the form of nanofibres, coatings, nanoparticles or microparticles.

Description

This invention relates to an enzyme-polymer conjugate including a polymeric substrate having one or more hydrolase enzymes immobilised thereon. The enzyme-polymer conjugate may be for use in a biotechnological process or for protecting a surface from biofouling.

BACKGROUND TO THE INVENTION

Enzymes play a key role in catalysing biological reactions. As a result, they are often active under relatively mild conditions and their catalysis is highly specific. Due to these characteristics, enzymes are increasingly being used in modern biotechnological settings ranging from the food to petrochemical industries.

Progress has also been made in the field of immobilised enzymes. Enzymes may be immobilised by adsorption or covalent attachment to polymers and other high-surface-area materials that find application in fine-chemical synthesis, fabrication of biosensors, food processing, protein digestion and bioremediation.

Immobilised enzymes have an advantage over free enzymes in solution in that they can be used repeatedly and easily removed from the reaction mixture after completion. However, in certain cases the immobilisation substrate, also referred to as an immobilisation support, may negatively influence the catalytic activity of the immobilised enzymes. Enzyme immobilisation on mesoporous ceramics, for instance, can lead to a loss of catalytic activity because the enzyme is contained within the support. This containment prevents diffusion of the enzyme substrate (the chemical entity that reacts with the enzyme) and release of the product to and from the enzyme active site, respectively.

Furthermore, differences in structure, active site and enzyme substrate mean that not all enzymes are capable of being immobilised on a support with retention of activity. The process of immobilisation can alter the structure of the enzyme and its active site which can lead to a decrease in activity.

Oxidases are one class of enzymes that has been reported to be capable of being immobilised on a support with retention of some activity. In one example, horseradish peroxidase (HRP) and glucose oxidase (GOX) were immobilised on an electrospun nanofibre comprising a copolymer of alternating styrene and maleic anhydride residues and were successfully used to carry out a glucose-peroxide cascade reaction. However, the immobilisation process had a marked negative effect on the activity of the HRP, which was reduced by around 80% to that of the free enzyme.

There is therefore scope for an immobilised enzyme system capable of use in a biotechnological process which exhibits favourable catalytic activity.

The preceding discussion of the background to the invention is intended only to facilitate an understanding of the present invention. It should be appreciated that the discussion is not an acknowledgment or admission that any of the material referred to was part of the common general knowledge in the art as at the priority date of the application.

SUMMARY OF THE INVENTION

In accordance with a first aspect of the invention, there is provided an enzyme-polymer conjugate having the structure of Formula I

wherein Ri is a hydrolase enzyme and R2 is OH or O; or R1 is OH or O' and R2 is a hydrolase enzyme;

i is an integer from 1 to 19; and n is an integer from 5 to 1000.

The hydrolase enzyme may be selected from amylase, protease and galactosidase. In at least some of subunits n, the hydrolase enzyme may be an amylase, and in other subunits n, the hydrolase enzyme may be a protease. Where the hydrolase enzyme is a combination of amylase and protease enzymes, the conjugate may have an enhanced amylase activity relative to a conjugate in which only amylase enzyme is immobilised.

The amylase may be a-amylase, β-amylase ory-amylase.

The amylase may have an amino acid sequence with at least 80% sequence identity to SEQ ID NO. 1 or at least 80% sequence identity to SEQ ID NO. 2 and the protease may have an amino acid sequence with at least 80% sequence identity to SEQ ID NO. 3.

The hydrolase enzyme may be immobilised on the polymer by a covalent bond between a lysine group of the enzyme and a maleic anhydride (MA) group of the polymer.

The conjugate may be in the form of a nanofibre, a textile, a coating formulation, a nanoparticle, a microparticle, a solid bead or an extruded sheet, and the nanofibre may form part of an electrospun nanofibrous mat.

The conjugate may be for use in converting a hydrolysable substrate into a product. The hydrolysable substrate may be starch or a component thereof, a polypeptide or a galactoside. Where the substrate is starch or a component thereof, the product may be selected from maltotriose, maltose and glucose.

The conjugate may be for use in protecting a surface from biofouling.

In accordance with a second aspect of the invention, there is provided a method of converting a hydrolysable substrate into a product, the method comprising contacting the substrate with the conjugate defined above.

The hydrolysable substrate may be starch ora component thereof, a polypeptide ora galactoside. Where the substrate is starch or a component thereof the product may be selected from maltotriose, maltose and glucose.

In accordance with a third aspect of the invention, there is provided a method of protecting a surface from biofouling, the method comprising coating the surface with the conjugate defined above.

In accordance with a fourth aspect of the invention, there is provided a coating comprising the conjugate defined above.

The coating may be for use in converting a hydrolysable substrate into a product. The hydrolysable substrate may be starch or a component thereof, a polypeptide or a galactoside. Where the substrate is starch or a component thereof the product may be selected from maltotriose, maltose and glucose.

The coating may be for use in protecting a surface from biofouling.

In accordance with a fifth aspect of the invention, there is provided a method of enhancing the amylase activity of an enzyme-polymer conjugate including a polymeric support to which amylase enzyme is immobilised, the method comprising co-immobilising protease enzyme on the support.

BRIEF DESCRIPTION OF THE FIGURES

In the Figures:

Figure 1 is a schematic representation showing the immobilisation of protease or aamylase enzymes on poly(styrene-maleic anhydride) (SMA) nanofibres; and

Figure 2 is a graph showing activity progress curves obtained for β-galactosidase immobilised on a SMA nanofibrous mat individually, in combination with aamylase, and in combination with α-amylase and protease. Activity was spectrophotometrically determined as a function of time using orthonitrophenyl-p-galactopyranoside (ONPG) as substrate.

DETAILED DESCRIPTION OF THE INVENTION

Throughout the specification and claims unless the content requires otherwise the word “comprise” or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

“Biofouling” refers to the accumulation of microorganisms and/or biological material on a surface. The microorganisms can produce a biofilm comprising extracellular polymeric substances such as DNA, proteins and polysaccharides, which may be pathogenic to humans or animals.

“Hydrolase” means an enzyme that catalyses the hydrolysis of a chemical bond, such as an ester, sugar (glycosyl), ether, peptide, or acid anhydride.

“Identity” as used herein means the identity between two amino acid sequences compared to each other within a corresponding sequence region having approximately the same amount of amino acids. For example, the identity of a full-length sequence of two amino acid sequences may be determined. Furthermore, a shorter amino acid sequence can be compared with a longer sequence and the identity between the two sequences will relate to the identity between the short sequence and a section of the longer sequence of approximately the same number of amino acids. In this way the identity of a truncated fragment of a polypeptide can be compared to a full polypeptide over the length of the truncated fragment. The amino acid sequences to be compared may differ in several positions which do not alter the biological function or structure of the polypeptides. Such “variants” may include amino acid substitutions, deletions, combinations or insertions in one or more positions in the amino acid sequences, but they still function in a substantially similar manner to the reference polypeptide (SEQ ID NO. 1,2, 3 or 4).

The invention provides an enzyme-polymer conjugate having the structure of Formula I:

wherein Ri is a hydrolase enzyme and R₂ is OH or 0; or Ri is OH or O' and R₂ is a hydrolase enzyme; i is an integer from 1 to 19; and n is an integer from 5 to 1000.

The polymer is a styrene maleic anhydride (SMA) polymer in which the molar ratio of styrene monomers to maleic anhydride (MA) monomers can be from about 1:1 (that is about 50 mol% each) to about 19:1 (that is about 95 mol% styrene to 5 mol% MA). Each MA monomer is flanked by styrene monomers and the number of styrene monomers between consecutive MA monomers can vary.

The SMA polymer is a statistical polymer with a weight average molar mass of from about 1000 - 600,000 g.mol’¹, equivalent to approximately 10 - 6000 monomeric units of styrene and MA combined. The integer n in Formula I has the usual meaning of indicating the repetition of monomer residues defined within the square brackets to the indicated total number of 5 to 1000. In some embodiments, n can be 10 to 1000, 20 to 1000 or even 50 to 1000.

In Formula I, i can be an integer selected from 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19.

The hydrolase enzyme can be suitable for degrading starch or a component thereof, polypeptides, galactosides, or other hydrolysable substrates, and can be selected from one or more of an amylase, such as a-amylase, β-amylase or γ-amylase, a protease, such as a serine endopeptidase (e.g. Esperase™) and a galactosidase. In certain embodiments, a combination of two 5 or more of these enzymes can be immobilised on the polymer. In some of these embodiments, the two or more enzymes can be a combination of amylase and protease enzymes, where in at least some of subunits n, the hydrolase enzyme is an amylase, and in other subunits n, the hydrolase enzyme is a protease.

Where the hydrolase enzyme includes an amylase, the amylase can have an amino acid sequence with at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO. 1, as shown in Table 1, or at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO. 2, as shown in Table 2. The amylase can be capable of cleaving either or both of alpha-1,4glycosidic bonds and alpha-1,6-glycosidic bonds and can include a glucoamylase and a pullalase, such as the commercially available Dextrozyme ® E enzyme combination.

Table 1: Amino acid sequence of genetically modified Aspergillus niger glucoamylase (SEQ ID NO. 1)

MSFRSLLALSGLVCTGLANVISKRATWDSWLSNEATVARTAILNNIGADGAWVSGADSGIVVASPSTDN PDYFYTWTRDSGLVLKTLVDLFRNGDTSLLSTIENYISAQAIVQGISNPSGDLSSGAGLGEPKFNVDET AYTGSWGRPQRDGPALRATAMIGFGQWLLDNGYTSTATDIVWPLVRNDLSYVAQYWNQTGYDLWEVNGS SFFTIAVQHRALVEGSAFATAVGSSCSWCDSQAPEILCYLQSFWTGSFILANFDSSRSAKDANTLLLGS IHTFDPEAACDDSTFQPCSPRALANHKEVVDSFRSIYTLNDGLSDSEAVAVGRYPEDTYYNGNPWFLCT LAAAEQLYDALYQWDKQGSLEVTDVSLDFFKALYSDATGTYSSSSSTYSSIVDAVKTFADGFVSIVETH AASNGSMSEQYDKSDGEQLSARDLTWSYAALLTANNRRNVVPSASWGETSASSVPGTCAATSAIGTYSS VTVTSWPSIVATGGTTTTATPTGSGSVTSTSKTTATASKTSTSTSSTSCTTPTAVAVTFDLTATTTYGE NIYLVGSISQLGDWETSDGIALSADKYTSSDPLWYVTVTLPAGESFEYKFIRIESDDSVEWESDPNREY TVPQACGTSTATVTDTWR

Table 2: Amino acid sequence of genetically modified Bacillus cereus pullulanase (SEQ ID NO. 2)

MVQITKRLINKTVLLLTLIVMLSSVFSFQNVKAVSNSKTTEVIIHYKEQSGNTKDWNLWIWGENSSGKS

YEFTGEDEFGKYAKINIDGDYNRLGFIIRTNEWEKDGGDRWIENIKDGRAEVWILSGDEKVYNSKPSSD

LSIQKATIDSFHEITVTTNVPFHIKEKKIEMEGIKIKNITPYDINSGDITNKVKIITEQKIDLKQTYKV

KIENLADTHTEIGKVIRTEEFDKLFYYGGNDLGNIYTPQHTKFRVWAPTASEAKLVTYKKWNDKIGTEI

NMQQGEKGTWKAELKGNQKGLYYTYKVKIGDKWTEAVDPYVRAASVNGDKGAVVDLEETNPKRWNTNKK

PKLKNPEDAIIYELHVRDLSIQPESGIKQKGKYLGVTEKGTKGPEGVKTGLDHMKDLGVTHVQLLPIFD

YASVNEEKVNEPQYNWGYDPKNFNVPEGSYSTNPYEPTVRITELKQMIQTLHDNNLRVVMDVVYNHMYN

AVESNFHKLVPGYYYRYNEDGTFANGTGVGNDTASERKMMRKFMIDSVTYWAKEYNLDGFRFDLMGIHD

YETVNEIRKAVNQIDPSIILHGEGWNLNTPLAAELKANQKNAEKMKGIAHFNDNIRDGLKGSVFEEKEN

GFVNGKENMEDRIKKGITAGIDYDRNTSTYQDPEQVLTYVEAHDNHTLWDKLELTNPGDSEEARKQMHK

LSSSILLTSQGIPFLHAGQEFMRTKYGDHNSYKSPDSINQMDWLRRAAFNNEVDYMKGLIELRKKYPAF RMTSAEQIKTHVSFIDAPKNTVAYTIEGNKNEYFTVAHNANKEAGEITLPSKGPWKVLVDGKQAGSKPL YVVH DN KIKVPAL SSLVLKTEKPIK

Where the hydrolase enzyme includes a protease, the protease can have an amino acid sequence with at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO. 3, as shown in Table 3. In some embodiments, the protease can be a serine protease, such as the 5 commercially available enzyme, Esperase®.

Table 3: Amino acid sequence of genetically modified Bacillus pumilus subtilisin-like serine protease (SEQ ID NO. 3)

MKKKNVMTSVLLAVPLLFSAGFGGSMANAETVSKSASEKSYIVGFKASATTNSSKKQAVTQNGGKLEKQ YRLINAAQVKMSEQAAKKLEHDPSIAYVEEDHKAEAYAQTVPYGIPQIKAPAVHAQGYKGANVKVAVLD TGIHAAHPDLNVAGGASFVPSEPNATQDFQSHGTHVAGTIAALDNTIGVLGVAPSASLYAVKVLDRNGD GQYSWIISGIEWAVANNMDVINMSLGGPNGSTALKNAVDTANNRGVVVVAAAGNSGSTGSTSTVGYPAK YDSTIAVANVNSSNVRNSSSSAGPELDVSAPGTSILSTVPSSGYTSYTGTSMASPHVAGAAALILSKNP NLSNSQVRQRLENTAT PLGNS FYYGKGLINAQAASN

Where the hydrolase enzyme includes a galactosidase, the galactosidase can have an amino acid sequence with at least 80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO. 4, as shown in Table 4.

Table 4: Amino acid sequence of Aspergillus oryzae galactosidase (SEQ ID NO. 4)

MKLLSVAAVALLAAQAAGASIKHRLNGFTILEHPDPAKRDLLQDIVTWDDKSLFINGERIMLFSGEVHP

FRLPVPSLWLDIFHKIRALGFNCVSFYIDWALLEGKPGDYRAEGIFALEPFFDAAKEAGIYLIARPGSY

INAEVSGGGFPGWLQRVNGTLRSSDEPFLKATDNYIANAAAAVAKAQITNGGPVILYQPENEYSGGCCG VKYPDADYMQYVMDQARKADIVVPFISNDASPSGHNAPGSGTSAVDIYGHDSYPLGFDCANPSVWPEGK LPDNFRTLHLEQSPSTPYSLLEFQAGAFDPWGGPGFEKCYALVNHEFSRVFYRNDLSFGVSTFNLYMTF GGTNWGNLGHPGGYTSYDYGSPITETRNVTREKYSDIKLLANFVKASPSYLTATPRNLTTGVYTDTSDL AVTPLIGDSPGSFFVVRHTDYSSQESTSYKLKLPTSAGNLTIPQLEGTLSLNGRDSKIHVVDYNVSGTN IIYSTAEVFTWKKFDGNKVLVLYGGPKEHHELAIASKSNVTIIEGSDSGIVSTRKGSSVIIGWDVSSTR RIVQVGDLRVFLLDRNSAYNYWVPELPTEGTSPGFSTSKTTASSIIVKAGYLLRGAHLDGADLHLTADF NATTPIEVIGAPTGAKNLFVNGEKASHTVDKNGIWSSEVKYAAPEIKLPGLKDLDWKYLDTLPEIKSSY DDSAWVSADLPKTKNTHRPLDTPTSLYSSDYGFHTGYLIYRGHFVANGKESEFFIRTQGGSAFGSSVWL NETYLGSWTGADYAMDGNSTYKLSQLESGKNYVITVVIDNLGLDENWTVGEETMKNPRGILSYKLSGQD ASAITWKLTGNLGGEDYQDKVRGPLNEGGLYAERQGFHQPQPPSESWESGSPLEGLSKPGIGFYTAQFD

LDLPKGWDVPLYFNFGNNTQAARAQLYVNGYQYGKFTGNVGPQTSFPVPEGILNYRGTNYVALSLWALE

SDGAKLGSFELSYTTPVLTGYGNVESPEQPKYEQRKGA

The galactosidase can be encoded by a nucleic acid having a nucleotide sequence with at least

80%, 85%, 90%, 95% or 100% sequence identity to SEQ ID NO. 5, as shown in Table 5.

Table 5: Nucleic acid sequence encoding Aspergillus oryzae galactosidase (SEQ ID NO. 5)

ATGAAGCTCCTCTCTGTTGCTGCTGTTGCCTTGCTGGCGGCACAGGCAGCGGGTGCTTCCATCAAGCAC

GTCTCAATGGCTTCACGATCCTGGAACATCCGGATCCGGCGAAAAGAGACTTGCTGCAAGACATTGTTC

ATGGGATGACAAATCTCTGTTCATCAATGGAGAGAGGATTATGTTATTCAGCGGAGAAGTGCATCCTTC

AGATTGCCAGTACCTTCGCTTTGGCTTGATATCTTCCACAAGATCAGAGCTCTTGGTTTCAACTGTGTT

CTTTCTATATTGATTGGGCTCTTCTGGAGGGAAAGCCTGGCGACTACAGAGCAGAAGGCATCTTTGCTT

GGAACCCTTCTTTGATGCAGCCAAGGAAGCAGGCATTTATCTGATCGCCCGCCCCGGTTCGTACATCAT

GCCGAGGTCTCAGGCGGTGGCTTCCCTGGATGGTTGCAGAGGGTCAATGGCACTCTTCGCTCGTCTGAG

AGCCATTCCTTAAAGCTACTGATAACTATATCGCCAATGCCGCTGCTGCCGTGGCGAAGGCTCAAATCC

GAATGGAGGGCCAGTAATTCTCTACCAGCCCGAAAACGAATACAGCGGTGGCTGCTGCGGTGTCAAATC

CCCGATGCAGACTACATGCAGTATGTTATGGATCAGGCCCGGAAGGCTGACATTGTTGTACCTTTCATA

GCAACGATGCCTCACCTTCTGGGCACAATGCTCCTGGAAGTGGAACGAGCGCTGTTGATATTTATGGTA

CGATAGCTATCCCCTCGGCTTTGATTGCGCAAACCCATCCGTATGGCCCGAGGGTAAACTGCCCGACAC

TTCCGCACGCTCCATCTTGAGCAGAGCCCATCAACTCCGTATTCACTTCTTGAGTTCCAAGCGGGTGCT

TCGACCCATGGGGTGGACCCGGCTTTGAAAAATGCTATGCCCTCGTTAACCACGAATTCTCGAGAGTTT

CTATAGGAACGACTTGAGTTTCGGAGTTTCTACCTTTAACTTATACATGACTTTCGGCGGAACAAACTG

GGTAACCTCGGACATCCCGGTGGATATACATCCTACGACTACGGATCGCCTATAACTGAAACGCGAAAG

TTACGCGGGAGAAGTACAGCGACATAAAGCTCCTTGCCAACTTTGTCAAAGCATCGCCATCCTATCTCC

CGCTACTCCCAGAAACCTGACTACTGGTGTTTACACAGACACATCTGACCTGGCTGTCACCCCGTTAAT

GGTGATAGTCCAGGCTCATTCTTCGTGGTCAGACATACGGACTATTCCAGCCAAGAGTCAACCTCGTAA

AACTTAAGCTTCCTACCAGTGCTGGTAACCTGACTATTCCCCAGCTGGAGGGCACTCTAAGTCTCAACG

ACGTGACTCAAAAATTCATGTTGTTGATTATAATGTGTCTGGAACGAACATTATCTATTCGACAGCTGA

GTCTTCACCTGGAAGAAGTTTGACGGTAACAAGGTCCTGGTGTTATACGGCGGACCGAAGGAACACCAG

AATTGGCCATTGCCTCCAAGTCAAATGTGACCATCATCGAAGGTTCGGACTCTGGAATTGTCTCAACGG

GAAGGGCAGCTCTGTTATCATTGGCTGGGATGTCTCTTCTACTCGTCGCATCGTTCAAGTCGGTGACTG

AGAGTGTTCCTGCTTGATAGGAACTCTGCTTACAACTACTGGGTCCCCGAACTCCCCACAGAAGGTACT

CTCCCGGGTTCAGCACTTCGAAGACGACCGCCTCCTCCATTATTGTGAAGGCTGGCTACCTCCTCCGAG

CGCTCACCTTGATGGTGCTGATCTTCATCTTACTGCTGATTTCAATGCCACCACCCCGATTGAAGTGAC

GGTGCTCCAACAGGCGCTAAGAATCTGTTCGTGAATGGTGAAAAGGCTAGCCACACAGTCGACAAGAAG

GCATCTGGAGCAGTGAGGTCAAGTACGCGGCTCCAGAGATCAAGCTCCCCGGTTTGAAGGATTTGGACG

GAAGTATCTGGACACGCTTCCCGAAATTAAGTCTTCCTATGATGACTCGGCCTGGGTTTCGGCAGACCT

CCAAAGACAAAGAACACTCACCGTCCTCTTGACACACCAACATCGCTATACTCCTCTGACTATGGCTTC

ACACTGGCTACCTGATCTACAGGGGTCACTTCGTTGCCAACGGCAAGGAAAGCGAATTTTTTATTCGCC

ACAAGGCGGTAGCGCATTCGGAAGTTCCGTATGGCTGAACGAGACGTATCTGGGCTCTTGGACTGGTGC

GATTATGCGATGGACGGTAACTCTACCTACAAGCTATCTCAGCTGGAGTCGGGCAAGAATTACGTCATA

CTGTGGTTATTGATAACCTGGGTCTCGACGAGAATTGGACGGTCGGCGAGGAAACCATGAAGAATCCTG

TGGTATTCTTAGCTACAAGCTGAGCGGACAAGACGCCAGCGCAATCACCTGGAAGCTCACTGGTAACCC

GGAGGAGAAGACTACCAGGATAAGGTTAGAGGACCTCTCAACGAAGGTGGACTGTACGCAGAGCGCCAG

GCTTCCATCAGCCTCAGCCTCCAAGCGAATCCTGGGAGTCGGGCAGTCCCCTTGAAGGCCTGTCGAAGC

GGGTATCGGATTCTACACTGCCCAGTTCGACCTTGACCTCCCGAAGGGCTGGGATGTGCCGCTGTACTC

AACTTTGGCAACAACACCCAGGCGGCTCGGGCCCAGCTCTACGTCAACGGTTACCAGTATGGCAAGTTA

CTGGAAACGTTGGGCCACAGACCAGCTTCCCTGTTCCCGAAGGTATCCTGAACTACCGCGGAACCAACA

TGTGGCACTGAGTCTTTGGGCATTGGAGTCGGACGGTGCTAAGCTGGGTAGCTTCGAACTGTCCTACAC

ACCCCAGTGCTGACCGGATACGGGAATGTTGAGTCACCTGAGCAGCCCAAGTATGAGCAGCGGAAGGGG

CATACTAA

The enzyme can be immobilised on the SMA polymer by contacting the polymer with a solution of the enzyme for a predetermined time period, removing the polymer from the solution after elapse of the time period, and washing the polymer to remove non-immobilised enzyme. Where the enzyme has proteolytic activity (for example, where the enzyme is a protease), to prevent degradation of immobilised enzyme the proteolytic activity may be reversibly inhibited with phenylmethylsulfonyl fluoride (PMSF). The PMSF can be added to the enzyme solution. The proteolytic activity can be restored by washing the inhibited immobilised enzyme with a solution of phosphate buffered saline (PBS) containing a surfactant, such as Tween 20.

As illustrated schematically in Figure 1, the hydrolase enzyme can be conjugated to the SMA by a covalent bond between a lysine group of the enzyme and a maleic anhydride (MA) group of the SMA polymer. The ε-amine of the lysine group reacts with the anhydride to form an amide, which tethers or immobilises the hydrolase enzyme to the polymer backbone, and a pendant carboxylic acid group. The amine can react with either of the anhydride carbonyl groups and a mixture of structures can therefore be produced in which the amide is formed at either of the two carbonyls with the acid at the other.

The conjugate can be produced as a nanofibre, a yarn, a textile, a coating formulation, a nanoparticle, a microparticle, a solid bead or an extruded sheet. In some embodiments, the conjugate is produced as an electrospun nanofibre which may form part of a woven or non-woven textile, such as a mat.

The conjugate can be used in a manufacturing process for converting hydrolysable substrates, such as starch or a component thereof, polypeptides or galactosides, into products. The 9 component of starch can be partially hydrolysed starch, amylose or amylopectin. In some embodiments, the starch or component thereof is converted into maltotriose, maltose or glucose. Typically, a solution containing the hydrolysable substrate is contacted with the conjugate for a predetermined period of time to allow the immobilised enzyme to convert the substrate into the product. The conjugate can be stirred or agitated in the substrate solution to increase mixing and contact between the substrate and immobilised enzyme. Once the conversion is complete or has advanced to a sufficient extent, the polymer is separated from the solution and the product isolated. The polymer is preferably in the form of an electrospun nanofibre mat to provide a large surface area on which the catalytic reaction can take place and to facilitate ease of removal of the mat from the solution. Alternatively, the polymer can be coated onto a component of machinery used in the manufacturing process. In these embodiments, the polymer can be provided in the form of a resin, a slurry, a solution, a suspension or similar liquid which allows the polymer to be coated onto a surface of the component.

The conjugate is suitable for carrying out cascade reactions in which two (or more) enzymemediated reactions are performed in series such that the product of the first reaction forms the starting material for the second. Cascade reactions are usually carried out in separate bioreactors, sometimes requiring the product of the first reaction to be purified before being introduced into the second reaction. However, by using the conjugate of the present disclosure in which both enzymes involved in the cascade reaction are immobilised onto the same SMA polymer, the reaction can be carried out in a single bioreactor. This has the benefit of potentially reducing the number of processing steps required.

The conjugate can also be used for protecting a surface from biofouling. The surface can be coated or integrally formed with the conjugate so that it has antibiofouling properties. Surfaces requiring protection from biofouling which can be protected by the conjugate include surfaces of medical devices and membranes, surfaces used in paper manufacturing, surfaces used in the food and beverage industry for manufacturing, processing, transporting or storing food and beverages, underwater construction surfaces, and surfaces of desalination plants. Biofilm formation can be inhibited by activity of the immobilised enzyme on carbohydrate or peptide components thereof. In these embodiments, the conjugate can be provided in the form of a resin, a slurry, a solution, a suspension or similar liquid which allows the conjugate to be coated onto the surface to be protected.

In some embodiments where a combination of amylase and protease enzymes are immobilised on the SMA polymer, the amylase activity of the combination is greater than the amylase activity of a conjugate having only amylase enzyme immobilised thereon. The combination can have a 2 to 3 fold (200 % to 300 %) higher activity than the activity of the individually immobilised amylase. Co-immobilising amylase and protease enzymes on the SMA polymer can provide a useful means of reducing catalytic activity loss often associated with immobilising these enzymes individually on solid supports.

Enzyme immobilisation allows for greater control of the enzymatic reaction than regular batch solution processes since the contact time can be optimized to suit specific reaction demands. Furthermore, immobilised enzymes can be reused multiple times. This provides advantages over traditional enzymatic reactions involving single-use enzyme solutions in which the enzymes cannot be recovered or recycled. For instance, a major drawback of converting starch into maltose using α-amylase enzymes is that the conversion is performed in a batch reaction, limiting the possibility to recover the enzyme after use. In an industrial setting, this can result in increased production costs. Furthermore, as the enzyme ofthe conjugate can be easily separated from the reaction product, additional processing steps can be circumvented and costs of production further reduced.

The use of the conjugate in the form of a nonwoven nanofibrous mat which has a large surface area and high porosity permits mass transfer ofthe reaction substrates and products through the polymer support. This provides an advantage over enclosed polymer supports.

The invention will now be described in further detail with reference to the following non-limiting examples.

Examples

Poly(styrene-alt-maleic anhydride) was synthesised and electrospun into nanofibres according to procedures published by the inventors (Polym. Chem. 2011, 2 (7), 1479). Protein immobilisation was achieved by incubating a 4 cm² fibre mat of the nanofibrous SMA polymer in 5 mL protein solution (ca. 43 mg/mL for protease and ca. 296 mg/mL for α-amylase, used as supplied without dilution) for 1 hour at room temperature with gentle agitation. As a control, bovine serum albumin (10 mg/mL) was also concurrently immobilised on a separate mat individually and in combination with protease and α-amylase. When co-immobilisation with the protease was performed, the protease activity was reversibly inhibited using phenylmethylsulfonyl fluoride (PMSF) to prevent loss of activity via protein hydrolysis. Samples of each protein solution used (1 mL) were collected prior to incubation and the protein concentration was subsequently determined. After incubation, each mat was extensively washed with phosphate buffered saline (PBS, pH 7.0) containing 0.1% Tween 20 (4 x 5 min) to remove non-covalently bound protein. Four 1 mL aliquots of the PBS11

Tween wash solutions were collected and the protein content determined, together with the original protein solution collected prior to incubation with the membrane, using the Pierce BCA protein assay kit with bovine serum albumin as standard. The amount of immobilised protein was calculated as the difference in protein content prior to and following immobilisation. The same immobilisation procedure described above was used for all enzyme and control solutions.

During protease immobilisation studies, the protease was evaluated for retention of enzymatic activity using an assay adapted from Sheng-Feng Li et al. Azocasein substrate solution (2.5 mL of 2.5% stock in 50 mM borax buffer, pH 9.5) was added to the electrospun fibre mat containing 0.6 mg immobilised protein. The reaction was quenched with 2.5 mL of 10% trichloroacetic acid (TCA) in deionized water, after 5 min incubation at 30 °C. The solutions were held at a constant pH of 9.5 in order to simulate the conditions under which Esperase is used in laundry detergents. After centrifugation of each reaction mixture, the UV absorbance of the supernatant was read at 340 nm on a Cary 60 UV-Vis spectrophotometer. The rate at which the immobilised protein hydrolysed the azocasein substrate was calculated using Equation 1, where (ΔΑ) represents the change in absorbance at 340 nm, (V) the reaction volume (in mL), (ε) is the extinction coefficient of the product of azocasein hydrolysis at 340 nm and has a value of 38 and t is the reaction time of 5 min.

A Ceralpha method was used to quantify α-amylase activity of the immobilised and free enzyme using the Megazyme α-amylase kit (Megazyme International, Ireland) as per manufacturer instructions. Briefly, 10 mL of substrate solution, ONPG was equilibrated to 40 °C separate from the fibre mat samples. After 5 min, 800 pL of the substrate was added to each fibre mat and incubated for exactly 10 min at 40 °C. After incubation, enzyme activity was quenched by addition of 8 mL 1% Tris-base. A sample blank was prepared by incubating 200 pL liquid enzyme solution with 8 mL 1% Tris-base prior to addition of the substrate. Incubation was performed as with the fibre mat samples. The samples were subsequently thoroughly mixed and the absorbance of the supernatant was determined at 400 nm with a Cary 60 UV-Vis spectrophotometer (Agilent Technologies) against the sample blank. Total enzyme activity was calculated using Equation 1 where ΔΑ represents the change in absorbance at 400 nm following incubation (final absorbance - blank absorbance), V the total reaction volume in mL, ε is the millimolar extinction coefficient of para-nitrophenol (18.1 mM’Lcm’¹, as per kit instructions), t the total incubation time of 10 min for a specific area of the fibre mat.

U/mg protein/cm² = ---^(ΔΧ2_Είη] ........................Equation (1) ^{x ε x} I cm2 ))

Dextrozyme and Esperase, inhibited with PMSF (Thermo Scientific), were mixed in a buffered solution and added to a 4 cm² nanofibrous mat to immobilise the enzymes. PMSF reversibly inhibits the catalytic activity of the protease, preventing the degradation of the α-amylase during immobilisation. Subsequent to immobilisation, the PMFS was removed along with any noncovalently bound enzyme during the wash steps with PBS Tween-20 to restore protease activity. The enzymatic activity of Dextrozyme was assayed and calculated using Equation 1.

Co-immobilisation of Dextrozyme, Esperase and commercial β-galactosidase (β-gal) was also performed using nanofibrous mats obtained through a high-throughput industrial electrospinning process using commercially available poly(styrene-co-MA (SMA ) [XIRAN®, Polyscope], SMA was electrospun into nanofibrous mats. Protease and α-amylase activities were determined as described above. The activity of immobilised β-gal was determined using OPNG as substrate. The assay was performed discontinuously alongside a fibre mat containing immobilised BSA as the assay blank. The substrate solution was prepared by making up a solution of 100 mM sodium phosphate buffer (pH 7.0), 0.1 mM MgCI₂, 50 mM β-mercaptoethanol and 1.33 mg/mL ONPG. The final solution was equilibrated at 37 °C. 5 mL of the equilibrated substrate solution was then added to 4 cm² mats with immobilised β-gal. 100 pl_ aliquots were collected at 10 second intervals for the β-gal and amylase^-gal co-immobilised samples and at 1 minute intervals for the aamylase^-gal-protease co-immobilised samples. Each aliquot was added to a 100 μΙ_ 2% Tris base in the wells of a 96 well plate. The UV absorbance of the solution in each well was subsequently determined at 420 nm with a BioTek PowerWave HT plate reader.

The results are provided in Table 5 and show that both the protease and α-amylase enzymes were successfully immobilised on the electrospun nanofibrous mat, both individually and in combination. Both enzymes retained their catalytic activity, however, immobilisation did result in an overall decrease in enzyme activity. Interestingly, the activity of the α-amylase increased three fold (300%) compared to individually immobilised α-amylase when immobilised in combination with the protease. The activity of the protease decreased by ca. 98% when immobilised with aamylase.

Table 5: Enzymatic activity and protein loading of enzymes immobilised on poly(styrene-alt-maleic anhydride) nanofibrous mats

Enzyme	Enzyme loading³	Activity⁶	Free Enzyme Activity⁶	% Retention⁰
protease	7.54	7.78x1 O'⁴	8.87 x IO ⁴	87.00%
a-amylase	39.7	0.808x10°	8.81 x10°	9.00%
protease + a-amylase	ND	1.54x10-⁵	8.87 x IO ⁴	1.73 % (protease)
	ND	2.37x10°	8.81 x10°	27.0 % (a-amylase)

^a in mg/cm² (not determined for the combination) ^b in pmol.min.mg.cnr² ^c is the activity retained after immobilisation vs. the activity of the free enzyme

The experiments were extended to include the immobilisation of an additional enzyme, βgalactosidase (β-gal). The results are presented in Table 6 and Figure 2. Figure 2 shows that βgal activity was retained when immobilised on its own and in combination with protease and aamylase. However, β-gal activity decreased when co-immobilised with α-amylase. A further reduction in β-gal activity occurred when co-immobilised with a combination of α-amylase and protease. Determination of protease and α-amylase activities co-immobilised with β-gal indicated that enzyme activity was retained regardless of the combination of immobilised enzymes. In these experiments, α-amylase activity increased two fold (by 225 %) when immobilised in combination with protease relative to individually immobilised a-amylase.

Table 6: Enzymatic activity of α-amylase immobilised on its own and in combination with β-galactosidase and protease on poly(styrene-alt-maleic anhydride) nanofibrous mats

Enzyme	Total Enzyme loading	Activity (pmol.min.mg.cm¹)
a-amylase	35.2 mg	16x IO³
α-amylase + protease	10.5 mg	36x10³
α-amylase + protease + β-galactosidase	18.3 mg	5x10^-3
BSA	1.35 mg	N/A

The results demonstrate that co-immobilisation of protease and α-amylase on a nanofibrous SMA polymer mat leads to a marked increase in α-amylase activity albeit with a concurrent decrease in protease activity. Furthermore, α-amylase retains catalytic activity when co-immobilised with protease and β-gal on the same nanofibrous mat.

Claims

CLAIMS:

1. An enzyme-polymer conjugate having the structure of Formula I

wherein Ri is a hydrolase enzyme and R₂ is OH or O; or Ri is OH or O' and R₂ is a hydrolase enzyme;

i is an integer from 1 to 19; and n is an integer from 5 to 1000.
2. A conjugate as claimed in claim 1, wherein the hydrolase enzyme is selected from amylase, protease and galactosidase.
3. A conjugate as claimed in claim 1 or claim 2, wherein in at least some of subunits n, the hydrolase enzyme is an amylase, and in other subunits n, the hydrolase enzyme is a protease.
4. A conjugate as claimed in claim 3 having enhanced amylase activity relative to a conjugate in which only amylase enzyme is immobilised.
5. A conjugate as claimed in any one of claims 2 to 4, wherein the amylase is α-amylase, βamylase or y-amylase.
6. A conjugate as claimed in any one of claims 2 to 5, wherein the amylase has an amino acid sequence with at least 80% sequence identity to SEQ ID NO. 1 or SEQ ID NO. 2 and the protease has an amino acid sequence with at least 80% sequence identity to SEQ ID NO. 3.
7. A conjugate as claimed in any one of claims 1 to 6, wherein the enzyme is immobilised on the polymer by a covalent bond between a lysine group of the enzyme and a maleic anhydride (MA) group of the polymer.
8. A conjugate as claimed in any one of claims 1 to 7, which is in the form of a nanofibre, a textile, a coating formulation, a nanoparticle, a microparticle, a solid bead or an extruded sheet.
9. A conjugate as claimed in claim 8, wherein the nanofibre forms part of an electrospun nanofibrous mat.
10. A conjugate as claimed in any one of claims 1 to 9, for use in converting a hydrolysable substrate into a product.
11. A conjugate as claimed in claim 10, wherein the hydrolysable substrate is starch or a component thereof, a polypeptide or a galactoside.
12. A conjugate as claimed in claim 11, wherein when the substrate is starch or a component thereof, the product is selected from maltotriose, maltose and glucose.
13. A conjugate as claimed in any one of claims 1 to 8, for use in protecting a surface from biofouling.
14. A method of converting a hydrolysable substrate into a product, the method comprising contacting the substrate with the conjugate of any one of claims 1 to 12.
15. A method as claimed in claim 14, wherein the hydrolysable substrate is starch or a component thereof, a polypeptide or a galactoside.
16. A method as claimed in claim 15, wherein when the substrate is starch or a component thereof the product is selected from maltotriose, maltose and glucose.
17. A method of protecting a surface from biofouling, the method comprising coating the surface with the conjugate of any one of claims 1 to 8 or 13.
18. A coating comprising the conjugate of any one of claims 1 to 12.
19. A coating as claimed in claim 18, for use in converting a hydrolysable substrate into a product.
20. A coating as claimed in claim 19, wherein the hydrolysable substrate is starch or a component thereof, a polypeptide or a galactoside.
21. A coating as claimed in claim 20, wherein when the substrate is starch or a component thereof the product is selected from maltotriose, maltose and glucose.

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22. A coating comprising the conjugate or any one of claims 1 to 8 or 13, for use in protecting a surface from biofouling.
23. A method of enhancing the amylase activity of an enzyme-polymer conjugate including a polymeric support to which amylase enzyme is immobilised, the method comprising co10 immobilising protease enzyme on the support.