A colanate hydrolase variant and its application

Abstract

The present disclosure provides a colanate hydrolase variant, wherein the colanate hydrolase variant comprises an amino acid sequence in which one to several amino acid residues are deleted, substituted, and / or inserted into the amino acid sequence of SEQ ID NO: 1; wherein the amino acid sequence of the colanate hydrolase variant is at least 90% identical to the amino acid sequence of SEQ ID NO: 1; and compared to the colanate hydrolase having the amino acid sequence of SEQ ID NO: 1, the colanate hydrolase variant has the same or improved colanate hydrolysis efficiency and / or hydrolysis activity as that of the colanate hydrolase having the amino acid sequence of SEQ ID NO: 1. Compared to the wild-type colanate hydrolase, the colanate hydrolase variant disclosed herein has significantly improved activity in catalyzing the hydrolysis of both large-molecule CA and small-molecule CA.

Description

Technical Field

[0001] The present invention belongs to the fields of genetic engineering and enzyme engineering, and particularly relates to a colanic acid hydrolase variant and application thereof. Background Art

[0002] Colanic acid (CA) is an exopolysaccharide found in certain bacteria, especially intestinal bacteria such as Escherichia coli. It has a complex structure and plays an important role in bacterial biology. CA has a large molecular weight and covers the surface of the bacteria to form a slime state, which helps protect the bacteria from the effects of dryness, low pressure and low pH environments, and enhances the survival ability of the strain. Studies have shown that feeding purified CA or CA-secreting Escherichia coli can extend the lifespan of Caenorhabditis elegans. As an active biopolymer, CA has a porous cellulose structure and abundant hydrophilic groups. It can be used as a natural hydrogel and has broad application prospects, including in the fields of cosmetics and healthcare.

[0003] Coranoic acid (CA) is composed of D-glucose, L-fucose, D-galactose, D-glucuronic acid, and non-stoichiometric side chain modifications of O-acetyl and pyruvic acid. L-fucose accounts for approximately 30% of the total. Studies have shown that polysaccharides or oligosaccharides containing fucose exhibit anti-inflammatory properties in zebrafish models. Furthermore, fucose or fucose-containing polysaccharides or oligosaccharides more readily penetrate the dermis, increasing skin thickness and promoting collagen refinement, thereby slowing skin aging. This highlights the enormous potential of CA in anti-inflammatory, immune-enhancing, and anti-aging applications.

[0004] The molecular weight of colanic acid synthesized by Escherichia coli typically exceeds 5 million daltons, but high-molecular-weight colanic acid presents several challenges, such as high viscosity and difficulty flowing. In skincare products, while high-molecular-weight colanic acid retains moisture, its excessive molecular weight may hinder its penetration into the dermis, impacting its effectiveness. Currently, no studies have examined the specific effects of colanic acid of varying molecular weights. In contrast, hyaluronic acid (HA) has been more extensively studied. High-molecular-weight HA is used for moisturizing and lubrication, while low-molecular-weight HA penetrates deeper into the skin to promote cellular metabolism, anti-oxidation, and increase collagen synthesis. It is speculated that colanic acid of varying molecular weights may have similar effects, but further research is needed into their mechanisms of action and their applications in different fields.

[0005] The preparation of polysaccharides of varying molecular weights involves a variety of methods, including enzymatic hydrolysis, acid-base hydrolysis, chemical modification, ultrafiltration separation, biofermentation, and fractionation. Enzymatic hydrolysis is relatively superior in terms of environmental friendliness, but the efficiency and specificity of the enzyme remain challenges. Patent CN116334039A provides a colanic acid hydrolase, enabling the enzymatic preparation of small-molecule colanic acid. However, the enzyme's soluble expression level and hydrolysis efficiency still need to be improved, limiting its application in scale-up production. The present invention provides a novel colanic acid hydrolase with improvements in both soluble expression level and hydrolysis efficiency, and has been successfully applied to the efficient preparation of colanic acid of varying molecular weights.

[0006] The present invention aims to develop a colanic acid hydrolase to efficiently prepare colanic acid of different molecular weights, and to ensure stability and controllability in industrial production. Summary of the Invention

[0007] The present disclosure provides a colanic acid hydrolase with high expression level and high catalytic activity. After the enzyme is modified, compared with the wild type, its activity in catalyzing the hydrolysis of large-molecule CA is increased by more than 2 times, and its activity in catalyzing the hydrolysis of small-molecule CA is significantly increased (about 1.8 times).

[0008] The present disclosure provides a method for specifically hydrolyzing colanic acid, comprising contacting a protein comprising or as shown in SEQ ID NO: 1, or a variant thereof, with colanic acid, preferably obtaining a product having 6 sugar units and / or 12 sugar units, wherein more preferably, the site at which the protein comprising SEQ ID NO: 1 or its variant hydrolyzes colanic acid is located between (1→3,4)-Fuc and (1→3)-Glc.

[0009] In another aspect, the present disclosure provides use of a protein comprising SEQ ID NO: 1 or a protein as shown in SEQ ID NO: 1 or a variant thereof in preparing a kit for hydrolyzing colanic acid.

[0010] In certain embodiments, the variant of SEQ ID NO: 1 comprises an amino acid sequence in which one or more amino acid residues are deleted, substituted, and / or inserted within the amino acid sequence of SEQ ID NO: 1; for example, a truncation of SEQ ID NO: 1 having one or more amino acids deleted from its N- or C-terminus while still retaining its activity. Another example is a tag sequence added to the N- or C-terminus of SEQ ID NO: 1 for protein purification or labeling. Variants with one or more amino acid deletions or tag sequences have an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 1; and compared to the colanate hydrolase having the amino acid sequence of SEQ ID NO: 1, the colanate hydrolase variant has no significantly decreased, the same, or improved colanate hydrolysis efficiency and / or hydrolysis activity.

[0011] In some embodiments, the colanate hydrolase variant comprises an amino acid sequence in which 1 to 10 amino acid residues are deleted, substituted, and / or inserted into the amino acid sequence of SEQ ID NO: 1, for example, 1 to 8 amino acid residues, 1 to 7 amino acid residues, 1 to 6 amino acid residues, 1 to 5 amino acid residues, 1 to 4 amino acid residues, 1 to 3 amino acid residues, or 1 to 2 amino acid residues.

[0012] In another aspect, the present disclosure provides a colanate hydrolase variant, wherein the colanate hydrolase variant comprises an amino acid sequence in which one to several amino acid residues are deleted, substituted, and / or inserted into the amino acid sequence of SEQ ID NO: 1; wherein the amino acid sequence of the colanate hydrolase variant is at least 90% identical to the amino acid sequence of SEQ ID NO: 1; and compared to the colanate hydrolase having the amino acid sequence of SEQ ID NO: 1, the colanate hydrolase variant has the same or improved colanate hydrolysis efficiency and / or hydrolysis activity that is not significantly reduced.

[0013] In some embodiments, the colanate hydrolase variant comprises an amino acid sequence in which 1 to 10 amino acid residues are deleted, substituted, and / or inserted into the amino acid sequence of SEQ ID NO: 1, for example, 1 to 8 amino acid residues, 1 to 7 amino acid residues, 1 to 6 amino acid residues, 1 to 5 amino acid residues, 1 to 4 amino acid residues, 1 to 3 amino acid residues, or 1 to 2 amino acid residues.

[0014] In some embodiments, the deletions and / or substitutions correspond to one or more of the following positions of SEQ ID NO: 1: S276, M296, S298, C302, C312, Y313, K315, C321, S333, Q340, C358, N420, Y433, C456, C459, C490, Y495, C497, R518, D523, W524, N527, N528, Y531, S549, E551, C571, V573, K575, R577, C590, D594, C623, C643, and C694.

[0015] In some embodiments, the substitution is selected from S276A, S276K, M296A, M296K, S298R, C302A, C312A, Y313R, K315A, C321A, S333K, Q340A, Q340K, C358A, N420A, Y433F, C456A, C459A, C490A, Y495 F, one or more of C497A, R518K, D523A, W524A, N527A, N528A, Y531A, S549R, E551A, E551L, C571A, V573K, V573R, K575A, K575L, R577A, C590A, D594A, C623A, C643A and C694A.

[0016] In some embodiments, the substitution is selected from C302A, C312A, C321A, C358A, C456A, C459A, C490A, C497A, C571A, C590A, C623A, C643A, C694A, D383S, D387A, D387E, E463A, E463D, S333K, R518K, N420A, W524A, R577A, K315A, N528A, Q340A, N527A, Y433F, Y495F, M296A, S27 6A, D594A, D523A, E551A, E551A+S549R, E551A+S549R+V573R, M296K, Q340K, S276K, S276K+M296K, S298R, Y313R, S298R+S 333K, Y313R+S333K, S298R+Y313R, S298R+Y313R+S333K, V573K, S549R, V573K+S549R, S298R+Y313R+S333K+V573K+S549R , S298R+S276K+M296K, C490A+V573K, C490A+S549R, C490A+V573K+S549R, E551L+K575L+V573K, E551L+K575L+S549R, E55 1L+K575L+V573K+S549R, S298R+S276K+M296K+E551L+K575L+V573K+S549R, C490A+E551L+K575L+V573K, C490A+E551L+K One of 575L+S549R, C490A+E551L+K575L+V573K+S549R, C490A+S298R+V573K, C490A+S298R+S549R, C490A+S298R+V573K+S549R, C490A+S298R+S276K+M296K+V573K+S549R, C490A+S298R+S276K+M296K+E551L+K575L+V573K+S549R, Y531A and K575A.

[0017] In some embodiments, the deletions and / or substitutions correspond to one or more of the following positions of SEQ ID NO: 1: S276, M296, S298, C302, C312, Y313, C321, S333, C456, C459, C490, C497, D523, S549, E551, C571, V573, K575, C590, D594, C623, C643, C694.

[0018] In some embodiments, the substitution is selected from one or more of S276A, S276K, M296A, M296K, S298R, C302A, C312A, Y313R, C321A, S333K, C456A, C459A, C490A, C497A, D523A, S549R, E551A, E551L, C571A, V573K, K575L, C590A, D594A, C623A, C643A, and C694A.

[0019] In some embodiments, the substitutions are selected from M296A, S276A, D594A, D523A, E551A+S549R, M296K, S276K, S276K+M296K, S298R, Y313R, S298R+S333K, Y313R+S333K, V573K, S549R, V573K+S549R, S298R+S276K+M296K, E551L+K575L+V573K , C302A, C312A, C321A, C456A, C459A, C490A, C497A, C571A, C590A, C623A, C643A, C694A, CAE2, C490A+S298R+V573K, C490A+E551L+K575L+V573K, C623A, C312A, C590A, C694A, C643A, C456A, C571A and C302A.

[0020] In yet another aspect, the present disclosure provides a polynucleotide encoding the colanate hydrolase variant as described above.

[0021] In another aspect, the present disclosure provides a vector comprising the polynucleotide as described above.

[0022] In yet another aspect, the present disclosure provides a cell comprising the vector as described above.

[0023] In yet another aspect, the present disclosure provides a method for hydrolyzing colanic acid, comprising contacting the colanic acid hydrolase variant as described above or the colanic acid hydrolase variant encoded by the polynucleotide as described above with colanic acid.

[0024] In yet another aspect, the present disclosure provides a composition comprising the aforementioned colanate hydrolase variant, the aforementioned polynucleotide, the aforementioned vector, and the aforementioned cell.

[0025] In yet another aspect, the present disclosure provides use of the aforementioned colanate hydrolase variant, the aforementioned polynucleotide, the aforementioned vector, the aforementioned cell, or the aforementioned composition in preparing a kit for hydrolyzing colanic acid.

[0026] In yet another aspect, the present disclosure provides use of a colanate hydrolase comprising SEQ ID NO: 1 in preparing a kit for hydrolyzing colanic acid.

[0027] In yet another aspect, the present disclosure provides use of the colanate hydrolase shown in SEQ ID NO: 1 in preparing a kit for hydrolyzing colanic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The present invention can be more fully understood with reference to the following drawings.

[0029] Figure 1 The HPLC detection results of the products after complete enzymatic digestion of CA by wild-type CAE2 are shown.

[0030] Figure 2 The NMR identifications of enzymatic hydrolysis products 1 (a) and 2 (b) are shown, which are 6 sugars (1CA) and 12 sugars (2CA), respectively.

[0031] Figure 3 The molecular formulas of the enzymatic hydrolysis product 1 (a) and the enzymatic hydrolysis product 2 (b) estimated from the NMR results are shown.

[0032] Figure 4 Shown are the crystal structure of CAE2 (PDB ID: 6e0w) and the molecular docking results with the substrate 1CA.

[0033] Figure 5 Shown is a sequence alignment of CAE2 and CAE1.

[0034] Figure 6 The structural alignment of CAE2 (PDB ID: 6e0w) and CAE1 (from alphafold prediction) is shown, with RMSD = 1.674.

[0035] Figure 7 The SDS-PAGE results of different mutants after purification are shown, and the purity is greater than or equal to 95%.

[0036] M: marker; 2: CAE2; 4: C456A; 5: C694A; 6: C643A; 11: C459A; 12: C321A; 13: C571 A; 18: C497A; 20: Y495F; 26: Y433F; 27: C302A; 29: N420A; 30: C312A; 34: W524A.

[0037] Figure 8 The SDS-PAGE results of different mutants after purification are shown, and the purity is greater than or equal to 95%.

[0038] M: marker; 36: M296A; 37: R577A; 38: D594A; 40: E551A; 41: R518A; 43: Q340A; 49: D523A; 50: S276A; 51: R518K; 52: R518D; 53: R518H; 54: R518E; 55: C490A.

[0039] Figure 9 The SDS-PAGE results of different mutants after purification are shown, and the purity is greater than or equal to 95%.

[0040] 2: CAE2; 11: C459A; 13: C571A; 14: C623A; 15: C590A; 26: Y433F; 19: N527A; 34: W524A; 29: N420A; 42: K315A; 46: N528A; M: marker.

[0041] Figure 10 The SDS-PAGE results after purification of different mutants are shown, with a purity greater than or equal to 95%. 8: E390S; 17: E390A; 30: C312A; 40: E551A; 16: H461R; 41: R518A; 49: D523A; M: marker.

[0042] Figure 11 The SDS-PAGE results of different mutants after purification are shown, and the purity is greater than or equal to 95%.

[0043] M: marker; 1: CAE2; 2: E551A+S549R; 3: E551+AS549R+V573R.

[0044] Figure 12 The SDS-PAGE results of different mutants after purification are shown, and the purity is greater than or equal to 95%.

[0045] M: marker; 1: CAE2; 2: Q458A; 3: Y360A; 4: Y531A; 5: E551A; 6: Q340A+K315A; 7: Q340A+K315A+S276A; 8: Q340A+K315A+S276A+M296A.

[0046] Figure 13 The SDS-PAGE results of different mutants after purification are shown, and the purity is greater than or equal to 95%.

[0047] M: marker; 1: CAE2; 2: S276K; 3: M296K; 4: S276K+M296K; 5: Q340K.

[0048] Figure 14 The SDS-PAGE results of different mutants after purification are shown, and the purity is greater than or equal to 95%.

[0049] M: marker; 1: S298R; 2: Y313R; 3: S333K; 4: S298R+333K; 5: Y313R+S333K; 6: S298R+Y313R; 7: S298R+Y 313R+S333K; 8: V573K; 9: S549R; 10: V573K+S549R; 11: S298R+Y313R+S333K+V573K+S549R; 12: CAE2.

[0050] Figure 15 The SDS-PAGE results of different mutants after purification are shown, and the purity is greater than or equal to 95%.

[0051] M: marker; 1: CAE2; 2: S298R+S276K+M296K; 3: C490A+V573K; 4: C490A+S549R; 5: C490A+V573K+S549R; 6: E551L+K575L+ V573K; 7: C490A+E551L+K575L+V573K; 8: C490A+S298R+V573K; 9: C490A+S298R+S549R; 10: C490A+S298R+V573K+S549R.

[0052] Figure 16 Different molecular weight CAs prepared are shown. DETAILED DESCRIPTION

[0053] The various features and aspects of the present invention are discussed in more detail below. It should be understood that the specific embodiments described herein are only used to illustrate the present invention and are not intended to limit the present invention.

[0054] definition

[0055] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0056] All numerical designations used herein, such as pH values, temperatures, times, concentrations, amounts, and molecular weights, including ranges, are approximate and, where appropriate, vary by increments of 0.1 or 1.0 (+) or (-). It will be understood that, although not always explicitly stated, all numerical designations may be preceded by the term "about."

[0057] The terms "about" and "approximately" include amounts within ±10% of the associated numerical value.

[0058] As will be understood by those skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be readily identified as fully described, and the same range can be broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, a middle third, an upper third, etc.

[0059] The term "amino acid" includes compounds having a -COOH group and an -NH2 group. In the present disclosure, amino acids are natural or non-natural amino acids, including but not limited to glycine (Gly, G), alanine (Ala, A), valine (Val, V), leucine (Leu, L), isoleucine (Ile, I), methionine (Met, M), proline (Pro, P), tryptophan (Trp, W), serine (Ser, S), tyrosine (Tyr, Y), cysteine (Cys, C), phenylalanine (Phe, F), asparagine (Asn, N), glutamine (Gln, Q), threonine (Thr, T), aspartic acid (Asp, D), glutamic acid (Glu, E), lysine (Lys, K), arginine (Arg, R) and histidine (His, H).

[0060] The meaning of the term "protein" is well known in the art and is used accordingly in the context of the present invention.

[0061] The term "hydrolase" refers to a polypeptide that catalyzes hydrolysis. Specifically, the hydrolase of the present disclosure can be used interchangeably with "hydrolyzing enzyme" or "degrading enzyme".

[0062] The term "corresponding to" refers to an amino acid residue at a listed position in a polypeptide or amino acid residue that is similar, identical or homologous to the listed amino acid residue in the polypeptide. Identifying an amino acid at a corresponding position can be determining a specific amino acid in a sequence called a specific sequence.

[0063] As used in this disclosure, "mutant" is equivalent to the term "variant" and may have a single point mutation or a combination of point mutations.

[0064] As used herein, the term "identity" is equivalent to the term "homology" and means sequence similarity to the amino acid sequence of the mutant colanate hydrolase SEQ ID NO: 1.

[0065] The term "hydrolytic activity" refers to the amount of product generated per unit time by a specific amount of enzyme.

[0066] The term "hydrolysis efficiency" refers to the ratio of product to substrate produced per unit time by a specific amount of enzyme.

[0067] This technical solution uses genetic engineering technology to construct the colanic acid hydrolase (CAE2) gene (GeneBank Accession Number: YP_009012482.1) and its mutants into an expression vector after codon optimization (construction of engineered strains). The expression vector carrying the CAE2 gene is then transformed into a host cell. Common host cells include but are not limited to Escherichia coli, yeast, Bacillus subtilis, mold, mammalian cells, etc. (expression of engineered strains). Through the sequence design of CAE2, applications in different scenarios have been successfully achieved, including but not limited to the efficient preparation of large or small molecules of colanic acid. Finally, the optimized CAE2 successfully obtains high-quality and batch-to-batch stable products through process optimization, production equipment design and optimization, and production process monitoring. Industrial production of colanic acid of different molecular weights is achieved.

[0068] Wild-type CAE2 is able to specifically hydrolyze colanic acid, a capability inherent in its natural state. This specificity in the hydrolysis site is crucial for the enzyme's activity and selectivity, reflecting the unique interaction between CAE2 and colanic acid. By cleaving the colanic acid molecule at a specific location, CAE2 effectively breaks it down into smaller fragments, achieving its hydrolysis function.

[0069] According to certain embodiments described herein, various engineered CAE2s were designed with at least one amino acid substitution relative to wild-type CAE2. Example sequences of wild-type and engineered CAE2s are shown in Table 1. Wild-type CAE2 was modified using various methods to increase or decrease its hydrolysis efficiency and thermal stability, particularly for the preparation of colanic acid of varying molecular weights.

[0070] In one aspect of the present disclosure, a colanate hydrolase variant is provided, wherein the colanate hydrolase variant comprises an amino acid sequence in which one to several amino acid residues are deleted, substituted, and / or inserted into the amino acid sequence of SEQ ID NO: 1;

[0071] wherein the amino acid sequence of the colanate hydrolase variant is at least 90% identical to the amino acid sequence of SEQ ID NO: 1; and

[0072] Compared to the colanate hydrolase having the amino acid sequence of SEQ ID NO: 1, the colanate hydrolase variant has a colanate hydrolysis efficiency and / or hydrolysis activity that is not significantly reduced, is the same as, or is improved.

[0073] According to some specific embodiments of the present disclosure, the aforementioned colanate hydrolase variant comprises an amino acid sequence in which 1 to 10 amino acid residues are deleted, substituted, and / or inserted into the amino acid sequence of SEQ ID NO: 1, for example, 1 to 8 amino acid residues, 1 to 7 amino acid residues, 1 to 6 amino acid residues, 1 to 5 amino acid residues, 1 to 4 amino acid residues, 1 to 3 amino acid residues, or 1 to 2 amino acid residues. Specifically, the aforementioned colanate hydrolase variant may comprise an amino acid sequence in which 1 amino acid residue is deleted, substituted, and / or inserted into the amino acid sequence of SEQ ID NO: 1, an amino acid sequence in which 2 amino acid residues are deleted, substituted, and / or inserted into the amino acid sequence of SEQ ID NO: 1, an amino acid sequence in which 3 amino acid residues are deleted, 4 amino acid residues are inserted, an amino acid sequence in which 5 amino acid residues are inserted, an amino acid sequence in which 6 amino acid residues are inserted, an amino acid sequence in which 7 amino acid residues are inserted, or an amino acid sequence in which 8 amino acid residues are inserted.

[0074] According to some specific embodiments of the present disclosure, the aforementioned deletions and / or substitutions correspond to one or more of the following positions of SEQ ID NO: 1: S276, M296, S298, C302, C312, Y313, K315, C321, S333, Q340, C358, N420, Y433, C456, C459, C490, Y495, C497, R518, D523, W524, N527, N528, Y531, S549, E551, C571, V573, K575, R577, C590, D594, C623, C643 and C694.

[0075] According to some specific embodiments of the present disclosure, the aforementioned substitution is selected from S276A, S276K, M296A, M296K, S298R, C302A, C312A, Y313R, K315A, C321A, S333K, Q340A, Q340K, C358A, N420A, Y433F, C456A, C459A, C490A, Y4 One or more of: 95F, C497A, R518K, D523A, W524A, N527A, N528A, Y531A, S549R, E551A, E551L, C571A, V573K, V573R, K575A, K575L, R577A, C590A, D594A, C623A, C643A and C694A.

[0076] According to some specific embodiments of the present disclosure, the aforementioned substitutions are selected from C302A, C312A, C321A, C358A, C456A, C459A, C490A, C497A, C571A, C590A, C623A, C643A, C694A, D383S, D387A, D387E, E463A, E463D, S333K, R518K, N420A, W524A, R577A, K315A, N528A, Q340A, N527A, Y433F, Y495F, M296A, S276A, D594A, D523A, E551A, E551A+S549R, E551A+S549R+V573R, M296K, Q340K, S276K, S276K+M296K, S298R, Y313R, S298 R+S333K, Y313R+S333K, S298R+Y313R, S298R+Y313R+S333K, V573K, S549R, V573K+S549R, S298R+Y313R+S333K+V573K+S54 9R, S298R+S276K+M296K, C490A+V573K, C490A+S549R, C490A+V573K+S549R, E551L+K575L+V573K, E551L+K575L+S549R, E 551L+K575L+V573K+S549R, S298R+S276K+M296K+E551L+K575L+V573K+S549R, C490A+E551L+K575L+V573K, C490A+E551L+ One of K575L+S549R, C490A+E551L+K575L+V573K+S549R, C490A+S298R+V573K, C490A+S298R+S549R, C490A+S298R+V573K+S549R, C490A+S298R+S276K+M296K+V573K+S549R, C490A+S298R+S276K+M296K+E551L+K575L+V573K+S549R, Y531A and K575A.

[0077] According to some specific embodiments of the present disclosure, the aforementioned deletions and / or substitutions correspond to one or more of the following positions of SEQ ID NO: 1: S276, M296, S298, C302, C312, Y313, C321, S333, C456, C459, C490, C497, D523, S549, E551, C571, V573, K575, C590, D594, C623, C643, and C694.

[0078] According to some specific embodiments of the present disclosure, the aforementioned substitutions are selected from one or more of S276A, S276K, M296A, M296K, S298R, C302A, C312A, Y313R, C321A, S333K, C456A, C459A, C490A, C497A, D523A, S549R, E551A, E551L, C571A, V573K, K575L, C590A, D594A, C623A, C643A and C694A.

[0079] According to some specific embodiments of the present disclosure, the aforementioned substitutions are selected from M296A, S276A, D594A, D523A, E551A+S549R, M296K, S276K, S276K+M296K, S298R, Y313R, S298R+S333K, Y313R+S333K, V573K, S549R, V573K+S549R, S298R+S276K+M296K, E551L+K575L+V576 One of 3K, C302A, C312A, C321A, C456A, C459A, C490A, C497A, C571A, C590A, C623A, C643A, C694A, CAE2, C490A+S298R+V573K, C490A+E551L+K575L+V573K, C623A, C312A, C590A, C694A, C643A, C456A, C571A and C302A.

[0080] Another aspect of the present disclosure provides a polynucleotide encoding the aforementioned colanate hydrolase variant.

[0081] Another aspect of the present disclosure provides a vector comprising the aforementioned polynucleotide.

[0082] Another aspect of the present disclosure provides a cell comprising the aforementioned vector.

[0083] Another aspect of the present disclosure provides a method for hydrolyzing colanic acid, comprising contacting the aforementioned colanic acid hydrolase variant or the colanic acid hydrolase variant encoded by the aforementioned polynucleotide with colanic acid.

[0084] Another aspect of the present disclosure further provides a composition comprising the aforementioned colanate hydrolase variant, the aforementioned polynucleotide, the aforementioned vector, or the aforementioned cell.

[0085] Another aspect of the present disclosure further provides use of the aforementioned colanate hydrolase variant, the aforementioned polynucleotide, the aforementioned vector, the aforementioned cell, or the aforementioned composition in preparing a kit for hydrolyzing colanic acid.

[0086] Another aspect of the present disclosure further provides use of a colanate hydrolase comprising SEQ ID NO: 1 in preparing a kit for hydrolyzing colanic acid.

[0087] The enzyme catalytic pocket is an important biomolecular structure, where key amino acids play a crucial role in the hydrolysis of colanic acid. During the hydrolysis of colanic acid, specific amino acids in the enzyme catalytic pocket interact with the colanic acid molecule, thereby affecting its hydrolysis efficiency. Modification of the enzyme catalytic pocket is a key bioengineering technology aimed at adjusting the catalytic performance of enzymes to meet specific application requirements. This disclosure utilizes computational chemistry combined with knowledge and techniques from multiple fields, such as structural biology and bioinformatics, to modify the enzyme catalytic pocket. This allows for precise control and optimization of the catalytic pocket, thereby improving the enzyme's catalytic efficiency, selectivity, and stability, and expanding its application prospects in fields such as bioindustry, medicine, and environmental protection.

[0088] The percentage of catalytic activity shown in this disclosure is the ratio of the activity detected by the mutant to the activity detected by the wild-type under the same conditions. A percentage greater than 100% indicates that the mutant has better activity than the wild-type, while a percentage less than 100% indicates that the mutant has less activity than the wild-type. The method used in this disclosure to detect enzyme activity is the DNS reducing sugar assay (substrate is the macromolecule CA, MW: 5000kDa), unless otherwise specified. This indicates that the amino acids in the key catalytic region of CAE2 in the catalytic pocket must be highly specific and accurate, and any slight change may affect its catalytic activity.

[0089] The interaction between colanic acid and enzymes is complex, involving multiple molecular-level influences. As a polysaccharide, colanic acid possesses multiple functional groups, such as hydroxyl and carboxyl groups, enabling it to interact with proteins. Enzyme-colanic acid interactions can occur through physical adsorption, hydrogen bonding, hydrophobic interactions, and ionic interactions. The enzyme's active site plays a key role in this process. The molecular structure of colanic acid binds to the enzyme's active site, influencing the enzyme's conformation and catalytic activity. Furthermore, the polysaccharide structure of colanic acid may influence the enzyme-substrate interaction through spatial arrangement and charge distribution, leading to conformational changes in the enzyme and affecting its catalytic efficiency.

[0090] Example

[0091] The following examples are illustrative only and are not intended to limit the scope or content of the invention in any way.

[0092] Experimental methods

[0093] 1. Commonly used expression vectors include, but are not limited to, pET-24a, pLMAR, pALTER-Ex1, pALTER-Ex2, pBAD / His, pBAD / Myc-His, pBAD / gIII, pBacPAK, pBAC, pBact-myc, pCAL-n, pCAL-n-EK, pCAL-c, pCAL-kc, pcDNA 2.1, pDUAL, pET-3a-c, pET-9a-d, pET-11a-d, pET-12a-c, pET-14b, pET-15b, pET-16b, pET-17b, pET-19b, pET-20b(+), pET-21a-d(+), pET-22b(+), pET-23a-d(+), pET-24b-d(+), pET-25b(+), pET-26b(+), pET-27b(+), pET-28a-c(+), pET-29a-c(+), pET-30a-c(+), pET-31b(+), pET-32a-c(+), pET-33b(+), pET-34b(+), pET-35b(+), pET-36b(+), pET-37b(+), pET-38b(+), pET-39b(+), pET-40b(+), pET-41a-c(+), pET-42a-c(+), pET-43a-c(+), pETBlue-1, pETBlue-2, pETBlue-3, pGEMEX-1, pGEMEX-2, pGEX-1λT, pGEX-2T, pGEX-2TK, pGEX-3X, pGEX-4T, pGEX-5X, pGEX-6P, pHAT 10 / 11 / 12, pHAT20, pHAT-GFPuv, pKK223-3, pLEX, pMAL-c2X, pMAL-c2E, pMAL-c2G, pMAL-p2X, pMAL-p2E, pMAL-p2G, pProEXHT, pPROLar.A, pPROTet.E, pQE-9, pQE-16, pQE-30 / 31 / 32, pQE-40, pQE-60, pQE-70, PQE-80 / 81 / 82L, pQE-100, pRSET, pSE280, pSE380, pSE420, pThioHis, pTrc99A, pTrcHis, pTrcHis2, pTriEx-1, pTriEx-2, pTrxFus, pBP26, pBP27, pBQ200, pGP380, pGP382, pGP3273, pGM1202, etc.

[0094] 2. Expression in engineered strains: The expression vector carrying the enzyme gene is transformed into a host cell. Common host cells include, but are not limited to, E. coli, yeast, Bacillus subtilis, mold, and mammalian cells. Commonly used E. coli cells include, but are not limited to, E. coli BL21 (DE3), E. coli BL21, and E. coli ArhaB (B0002). and subseries: E.coli BL21(DE3)-pLysS, E.coli BL21 Star-pLysS, E.coli BL21-SI, E.coli BL21-AI, E.coli Tuner, E.coli Tuner pLysS, E.coli Origami, E.coli Origami B, E.coli Origami B pLysS, E.coli Rosetta, E.coli Rosetta pLysS, E.coli Rosetta-gami-pLysS, E.coli Rosetta2, E.coli Rosetta2 pLysS, E.coli BL21 CodonPlus, E.coliAD494, E.coli BL21trxB, E.coli HMS174, E.coli NovaBlue(DE3), E.coli BLR, E. coli C41(DE3), E. coli C43(DE3), E. coli Lemo21(DE3), E. coli SHuffle T7, E. coli Arctic Express, E. coli Arctic Express(DE3). Fungal cells: Streptomyces lividans. Lactobacillus cells: Lactococcus lactis. Bacillus subtilis cells: Bacillus subtilis. Yeast cells: Saccharomyces cerevisiae. Insect cells: Spodoptera frugiperda (e.g., Sf9 or Sf21). Mammalian cells: Human Embryonic Kidney 293, Chinese hamster ovary cells, A549, Babyhamster kidney (BHK) cells, CAD, DUKX-X11, HeLa, Hep G2, HT1080, J558L, L929, MCF-7, N2a, NIH 3T3, P19, SO-Rb50, U2OS, Y79, etc.

[0095] Example 1: Hydrolysis of colanic acid by wild-type CAE2

[0096] After CAE2 and the substrate colanic acid were fully reacted, the resulting hydrolyzed product was detected by high performance liquid chromatography (HPLC). Figure 1 As shown in Figure 2, two obvious peaks were found. Subsequently, through a series of purification and separation operations, two samples with higher purity were obtained ( Figure 1 ): Enzyme hydrolysis product 1 (a) and enzyme hydrolysis product 2 (b). After further nuclear magnetic resonance (NMR) detection of these two samples, it was found that one sample contained 6 sugar units, while the other sample contained 12 sugar units, such as Figure 2 shown.

[0097] These findings reveal the positional specificity of CAE2 in hydrolyzing colanic acid. Based on these results, it is inferred that the hydrolysis site is located between (1→3,4)-Fuc and (1→3)-Glc, such as Figure 3 This conclusion was based on an analysis of the number of sugar units in different samples, as well as quantitative and structural characterization using nuclear magnetic resonance (NMR) techniques. These results are crucial for determining the hydrolysis mechanism of CAE2 and its interaction with the colanic acid substrate. It is speculated that the key amino acid sequence near the catalytic pocket of the enzyme shares the same hydrolysis site with other potential colanic acid hydrolases similar to CAE2.

[0098] Example 2 Design of Engineered Colanate Hydrolase (CAE2)

[0099] (1) Amino acids that make up the enzyme catalytic pocket

[0100] By analyzing the crystal structure of the complex of colanic acid and CAE2 (PDB ID: 6e0w), it is speculated that the catalytic pocket of the enzyme is located in the Figure 4 As shown in the positions, the relevant amino acids that make up the catalytic pocket are: Y360, D387, A388, E390, N392, V393, N420, I423, A428, Y433, Q458, H461, E463, L487, T488, C490, Y495, D523, etc.

[0101] Experimental verification revealed that amino acids H461, E463, D387, and E390 are crucial for the enzymatic activity of CAE2 during its hydrolysis of colanic acid. Mutations of these amino acids to either equally charged or uncharged amino acids (e.g., H461A (0%), H461K (0%), H461R (2%), E463A (3%), E463D (0%), E463S (0%), D387A (0%), D383S (4%), D387E (3%), E390A (11%), E390D (3%), and E390S (12%)) demonstrated that these mutations nearly completely abolished CAE2 activity (Table 3).

[0102] Table 1. Sequence information

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134] Table 2. Amino acid mutants associated with protein aggregation

[0135]

[0136] *Negative values represent background interference and can be interpreted as 0, indicating no hydrolysis.

[0137] Table 3. Mutants of key amino acids in the enzyme catalytic pocket

[0138]

[0139] *Negative values represent background interference and can be interpreted as 0, indicating no hydrolysis.

[0140] Table 4. Amino acid mutants related to substrate binding

[0141]

[0142]

[0143] *Negative values represent background interference and can be interpreted as 0, indicating no hydrolysis.

[0144] Table 5. Efficiency of mutants in hydrolyzing 2CA to 1CA

[0145]

[0146] After sequence alignment analysis with the previously reported CAE1 hydrolase that hydrolyzes colanic acid, it was found that the amino acid sequence similarity between CAE2 and CAE1 was 30.04% ( Figure 5 ), the structural similarity RMSD is 1.27 ( Figure 6 ). And the key amino acid species in the catalytic pocket region (H461, E463, D387 and E390) are the same ( Figure 5 Based on this finding, it can be speculated that other enzymes with identical or similar amino acid sequences to CAE2 at key amino acid positions in the catalytic pocket (H461, E463, D387, and E390), with overall amino acid sequence similarity greater than 30% and structural similarity less than 5 RMSD, will also specifically hydrolyze colanic acid. These similar enzymes may be ubiquitous in bacteria or other microorganisms and have similar biological functions, hydrolyzing similar substrates. This speculation provides important clues for further exploration of the functions and catalytic mechanisms of other enzymes and contributes to a deeper understanding of the function and regulation of bacterial metabolic pathways and microbial ecosystems.

[0147] (2) Interaction between colanic acid and enzymes

[0148] Analysis of the crystal structure of the colanic acid-CAE2 complex (PDB ID: 6e0w) revealed that the left-side amino acids of CAE2 that interact with colanic acid include T274SSI277, G294TMIS298, Y313VKML317, S333TAAFLMQA341, Y360AILQQGT367, D387AIELNV393, Q417GANWGIGIGVAGSGPY433, R457QCLHVEM464, T479, G484TGLTTCGVALYG496. Analysis of the docking results of CAE2 with a single colanic acid molecule revealed that the right-side amino acids of CAE2 that interact with colanic acid include V515STRMVFIDWGVNNGRYA532, E551, K575, and S605 (Tables 1 and 2).

[0149] By designing the amino acids interacting with the substrate, we observed that single point mutations N392A (0%), Q365A (1.8%), W421A (8%), Q458A (1%), Y360A (1%), and S333K (0.5%) almost completely abolished CAE2 activity. These results indicate that these amino acids are crucial for the interaction between CAE2 and colanic acid. Notably, mutating these amino acids individually to other similar amino acids (e.g., Q, F, T, N, Y, W, S, etc.) or charged amino acids (e.g., D, E, R, K, H, etc.) may slightly affect catalytic efficiency, but may also have no effect or even improve catalytic efficiency. We also observed that the combined mutations N420A+E390A (3%) and N420A+E390S (2%) almost completely abolished CAE2 activity, likely due to the effects of the single key amino acid mutations E390S and E390A on CAE2. These findings are of great significance for understanding the interaction mechanism between CAE2 and colanic acid and developing related applications.

[0150] Single point mutants such as N420A (34%), W524A (55%), R577A (76%), K315A (78%), N528A (81%), Q340A (85%), N527A (88%), Y433F (97%), Y531A (97%), Y495F (99%), and E551A (102%) also affect the catalytic efficiency of CAE2, indicating that these amino acids are related to the interaction with the substrate. However, the combined mutations Q340A+K315A (6%), Q340A+K315A+S276A (14%), and Q340A+K315A+S276A+M296A (9%) almost completely inactivated CAE2. This is likely because single-point mutations have a weak ability to alter substrate-enzyme interactions, but combined mutations can significantly enhance these alterations, thus significantly affecting the enzyme's catalytic efficiency. Furthermore, it is reasonable to speculate that combined mutations at sites where single-point mutations have a minimal effect on enzyme catalytic efficiency (>50%) may also significantly affect CAE2 activity.

[0151] Single-point mutants such as M296A (111%), S276A (111%), D594A (122%), and D523A (135%) significantly improved the catalytic efficiency of CAE2, suggesting that these amino acids interact with the substrate, likely through nonspecific binding, which in turn affects catalytic efficiency. Mutations reduce this nonspecific binding, thereby improving catalytic efficiency. Therefore, mutating the amino acids at these sites to other amino acids with no side chains, short side chains, or oppositely charged amino acids (G, P, S, T, I, V, L, D, E, R, K, H) may have similar or even better effects.

[0152] Analysis of docking results between CAE2 and colanic acid suggests that the interaction between R518 and colanic acid may influence the catalytic activity of CAE2. Mutating R518 to different amino acids significantly affected its catalytic activity. Mutations to R518A (10%), R518D (3%), R518E (4%), and R518H (5%) nearly completely abolished its activity, while R518K (101%) had no effect. This suggests that the charge of the amino acid at this site is crucial. Mutations to uncharged or oppositely charged residues almost completely abolished enzymatic activity, while mutations to the same charge maintained almost no activity. This suggests that positively charged amino acids at this site and nearby may enhance the interaction with colanic acid. Similarly, it is speculated that similar interactions between positively charged amino acids and colanic acid may be associated with similar interactions at similar positions on the left side of the CAE2 structure.

[0153] Therefore, amino acids near these two regions were selected and mutated individually or in combination to positively charged or uncharged amino acids in the hope of enhancing the interaction between CAE2 and the substrate colanic acid. The following mutants were designed: E551A+S549R (122%), E551A+S549R+V573R, M296K (122%), S276K (120%), S276K+M296K (117%), S298R (86%), Q340K (89%), Y313R (97%), S298R+S333K (97%), Y313R+S333K (84%), S298R+Y313R (0. 5%), S298R+Y313R+S333K (41%), V573K (90%), S549R (48%), V573K+S54 9R(41%), S298R+Y313R+S333K+V573K+S549R(65%), S298R+S276K+M29 6K(38%), E551L+K575L+V573K(105%), E551L+K575L+S549R, E551L+K5 75L+V573K+S549R, S298R+S276K+M296K+E551L+K575L+V573K+S549R. The catalytic activities of E551A+S549R (122%), M296K (122%), S276K (120%), and S276K+M296K (117%) all increased to varying degrees. This may be because these mutants enhance substrate binding while having little or no effect on substrate release, thereby improving the overall catalytic activity of CAE2. The catalytic activities of the remaining mutants remained unchanged or decreased. This may be because these mutants, while enhancing substrate binding, also decrease substrate release. When the substrate release rate exceeds the substrate binding rate, the overall catalytic activity of the mutant is weakened. It is speculated that single-point mutations or combinations of these mutants to uncharged amino acids, amino acids without side chains, or amino acids with similar or opposite charges (G, P, S, T, I, V, L, D, E, R, K, H) may have similar or even better effects.

[0154] To further verify the interaction between these mutants and substrates, the present invention set the substrate as a small molecule 2CA (2 units of 6 sugars, 12 sugars) for hydrolysis reaction, detected the proportion of one unit CA (6 sugars) generated, and compared it with the proportion of wild-type CAE2 under the same conditions. The results are shown in Table 5: S298R (161%), Y313R (181%), S298R+S333K (134%), Y313R+S333K (127%), V573K (111%), V573K+S549R (121%), S298R+S276K+M296K (123%), E551L+K575L+V573K (109%). These mutants, either single-point or combined, significantly improved the efficiency of CAE2 in hydrolyzing 2CA to 1CA. It can be seen that changing the charged species of amino acids in these two regions of CAE2 can significantly improve its catalytic activity, including but not limited to increasing the positively charged amino acids to improve the efficiency of hydrolyzing small molecule CA.

[0155] (3) Weakening the random interactions between enzyme molecules

[0156] Sequence analysis revealed that CAE2 contains 13 cysteines (Cys), which are highly likely to cause random aggregation between protein molecules, thereby affecting protein solubility, catalytic activity, and other properties. The present invention mutates these cysteines to Ala, and the enzymatic activity of CAE2 is improved to varying degrees, reaching as high as 124%. Therefore, it can be speculated that mutating the cysteine residues at these sites to other amino acids with no or short side chains (G, P, S, T, I, V, L) may have similar or even better effects. At the same time, combining single-point mutations, or combining them in pairs or more, may also produce similar or even better effects.

[0157] (4) Superposition of (2) and (3)

[0158] C490A (123%) in (3) was selected to superimpose the mutants in (2), C490A+E551L+K575L+V573K (178%), C490A+E551L+K575L+S549R, C490A+E551L+K575L+V573K+S549R, C490A+S298R+V573K (218%), C490A+S298R+S549R (70%), C490A+S298R+V573K+S549R 49R (66%), C490A+S298R+S276K+M296K+V573K+S549R, C490A+S298R+S276K+M296K+E551L+K575L+V573K+S549R, and found that the catalytic activity of the superimposed mutant enzymes was significantly improved, with the highest C490A+S298R+V573K combination mutant reaching 218%, followed by C490A+E551L+K575L+V573K combination mutant reaching 178%. It is speculated that superimposing and combining other mutants in (3), especially single-point or combined mutations such as C623A (124%), C312A (120%), C590A (117%), C694A (117%), C643A (115%), C456A (112%), C571A (110%), and C302A (110%), with the mutants in (2) will also have similar or better effects.

[0159] Example 3: Enzyme expression and purification

[0160] The CAE2 gene or its mutant was codon-optimized for E. coli and then cloned into the E. coli expression vector pet28a. Next, the recombinant vector was transformed into E. coli BL21, and the cell optical density (OD) was monitored during the culture process until it reached 0.6-1. After reaching the target optical density, 0.1mM inducer IPTG was added, and the culture temperature was adjusted to 20°C to induce protein expression overnight. Subsequently, the induced cells were collected, resuspended with buffer A1 (PBS, pH 7.0), and then crushed under high pressure. Unbroken cells and larger membrane fragments were removed by high-speed centrifugation at 20,000rpm, the supernatant was collected, and added to a pre-equilibrated nickel column. The column was washed with buffer A1, and the target protein was eluted with buffer B1 containing 300mM imidazole and 20mMPB (pH 7.0). Finally, the eluted protein was dialyzed with 20mM PB (pH 7.0) buffer at 4°C overnight. After purification, the purity of the protein was greater than 95%, as shown in Figure 2. Figure 7-Figure 15 shown.

[0161] Example 4: Enzyme activity test

[0162] CAE2 activity was quantified by measuring the reducing sugar content using the 3,5-dinitrosalicylic acid (DNS) method, using glucose as a standard. Briefly, 900 μL of CA (final concentration: 2 g / L) was mixed with CAE in a 9:1 volume ratio and incubated at 55°C for 15 minutes. Then, 1 mL of DNS reagent was added, and the mixture was boiled for 5 minutes. After cooling, 3 mL of distilled water was added to the mixture, and the absorbance was measured at 540 nm. Finally, the reducing sugar content in the sample was determined by comparing the absorbance value of the sample with the standard curve.

[0163] Small molecule enzymatic hydrolysis efficiency test: The 2CA sample was completely dissolved in an appropriate amount of water to ensure its sufficient solubility. After reaching a concentration of 2g / L in the solution, the temperature was adjusted to 30°C. Next, CAE2 and its mutants were added to different solutions at a final concentration of 50ug / ml, and the reaction was continued for 24 hours. After the reaction was completed, the reaction solution was heated at 100°C for 10 minutes to terminate the reaction. Finally, the proportion of 1CA in the reaction solution was detected and analyzed using high performance liquid chromatography (HPLC) technology.

[0164] Example 5: Preparation of colanic acid with different molecular weights

[0165] The macromolecular CA sample was completely dissolved in an appropriate amount of water to ensure its sufficient solubility. After the concentration reached 10g / L in the solution, the temperature was adjusted to 25°C. Then, CAE2 with a final concentration of 5-100ug / mL was added to different solutions respectively, and the reaction was carried out for 1 hour. After the reaction was completed, the reaction solution was heated at 100°C for 5 minutes to terminate the reaction. Subsequently, the reaction solution was diluted 5 times with water to make the final concentration of the sample reach 2g / L. Finally, the weight-average molecular weight of CA in the reaction solution was detected and analyzed using high-performance liquid chromatography (HPLC) technology. The results showed that CAs of different molecular weights were successfully prepared, and industrial scale-up production was achieved. The results are as follows Figure 16 .

[0166] Incorporated by Reference

[0167] Each patent and scientific document mentioned herein is incorporated by reference in its entirety for all purposes.

[0168] Equivalence

[0169] The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Therefore, the above embodiments should be considered in all cases as illustrative rather than limiting of the invention described herein. The scope of the present invention is therefore indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalence of the claims are intended to be embraced therein.

Claims

1. A method for specifically hydrolyzing colanic acid, comprising contacting a colanic acid hydrolase variant with colanic acid to obtain a product having 6 sugar units and / or 12 sugar units, wherein the site at which the colanic acid hydrolase variant hydrolyzes colanic acid is located between (1→3,4)-Fuc and (1→3)-Glc, wherein: The amino acid sequence of the colanate hydrolase variant is an amino acid sequence in which one to several amino acid residues are substituted in the amino acid sequence of SEQ ID NO: 1; and the substitution is selected from one of Y313R, C490A+S298R+V573K, and C490A+E551L+K575L+V573K.

2. A colanate hydrolase variant, wherein: The amino acid sequence of the colanate hydrolase variant is an amino acid sequence in which one to several amino acid residues are substituted in the amino acid sequence of SEQ ID NO: 1; wherein the substitution is selected from one of Y313R, C490A+S298R+V573K, and C490A+E551L+K575L+V573K; and Compared to the colanate hydrolase having the amino acid sequence of SEQ ID NO: 1, the colanate hydrolase variant has a colanate hydrolysis efficiency and / or hydrolysis activity that is not significantly reduced, is the same as, or is improved.

3. A polynucleotide encoding the colanate hydrolase variant according to claim 2. A vector comprising the polynucleotide according to claim 3 . A cell comprising the vector according to claim 4 .

6. A composition comprising the colanate hydrolase variant according to claim 2, the polynucleotide according to claim 3, the vector according to claim 4, or the cell according to claim 5.

7. Use of the colanate hydrolase variant according to claim 2, the polynucleotide according to claim 3, the vector according to claim 4, the cell according to claim 5, or the composition according to claim 6 in preparing a kit for hydrolyzing colanic acid.

Images