Novel colanic acid hydrolase variant and use thereof

Abstract

Provided is a colanic acid hydrolase variant, wherein the colanic acid hydrolase variant comprises an amino acid sequence in which one to several amino acid residues are deleted, substituted, and / or inserted in the amino acid sequence of SEQ ID NO: 1. The amino acid sequence of the colanic acid hydrolase variant has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. Moreover, compared with the colanic acid hydrolase having the amino acid sequence of SEQ ID NO: 1, the colanic acid hydrolase variant has colanic acid hydrolysis efficiency and / or hydrolysis activity that is not significantly reduced, the same, or improved. Compared with the wild type, the activity of the colanic acid hydrolase variant for catalyzing the hydrolysis of macromolecular CA and the activity for catalyzing the hydrolysis of small molecular CA are both significantly improved.

Description

A novel colanic acid hydrolase variant and application thereof

[0001] Related Applications

[0002] The present disclosure claims priority to Chinese application CN202410837085.2, filed on June 25, 2024, the entire contents of which are expressly incorporated herein by reference in their entirety. TECHNICAL FIELD

[0003] The present application belongs to the field of genetic engineering and enzyme engineering, and specifically relates to a novel colanic acid hydrolase variant and application thereof. BACKGROUND

[0004] Colanic acid (CA) is an exopolysaccharide in certain bacteria, especially in enteric 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 bacterial body in a mucous state, which helps to protect the bacteria from dry, low pressure and low pH environments, and enhances the survival ability of the strain. Studies have shown that feeding purified CA or CA-secreting E. coli can prolong the lifespan of Caenorhabditis elegans. As an active biopolymer, CA has a porous cellulose structure and abundant hydrophilic groups, and can be used as a natural hydrogel, which has a wide application prospect, including in the fields of cosmetics and healthcare.

[0005] Colanic acid (CA) is composed of D-glucose, L-fucose, D-galactose, D-glucuronic acid, and non-stoichiometric modifications of O-acetyl and pyruvic acid on the side chain, with a high content of L-fucose of about 30%. Studies have shown that fucose-containing polysaccharides or oligosaccharides have anti-inflammatory function in zebrafish models. In addition, fucose or fucose-containing polysaccharides or oligosaccharides are more easily penetrated into the dermis, increasing skin thickness and promoting the fine and delicate structure of collagen, thereby slowing down skin aging. This highlights the great application potential of CA in the fields of anti-inflammatory, immune enhancement and anti-aging.

[0006] The molecular weight of colanic acid synthesized by E. coli usually exceeds 5 million Daltons, but high molecular weight colanic acid has some problems, such as high viscosity and difficulty in flowing. In skincare products, high molecular weight colanic acid has water retention function, but its large molecular weight may hinder its penetration into the dermis, affecting its function. Currently, there is no research report on the specific effects of colanic acid with different molecular weights. In contrast, the research on hyaluronic acid (HA) is more extensive. High molecular weight HA is used for moisturizing and lubrication, and low molecular weight HA penetrates deep into the skin, promotes cell metabolism, antioxidant, and increases collagen synthesis, etc. It is speculated that colanic acid with different molecular weights may have similar effects, but further research is needed to study its mechanism of action and application in different fields.

[0007] Preparation of polysaccharides with different molecular weight sizes involves various methods, including enzymatic hydrolysis, acid-base hydrolysis, chemical modification, ultrafiltration separation, biological fermentation and fractional separation method, etc. Among them, the enzymatic hydrolysis method is relatively superior in green environmental protection, but the high efficiency and specificity of the enzyme have always been a challenge. CN116334039A patent provides a kind of pull acid hydrolytic enzyme, which makes it possible to prepare small molecules of pull acid by enzymatic hydrolysis. However, the soluble expression amount, hydrolysis efficiency, etc. of the enzyme still need to be improved, which limits its application in scale-up production. The present application provides a brand new pull acid hydrolytic enzyme, which has improved soluble expression amount and hydrolysis efficiency, and has been successfully applied to the efficient preparation of pull acid with different molecular weights.

[0008] The present application aims to develop a new type of pull acid hydrolytic enzyme to efficiently prepare pull acid with different molecular weights and to stabilize and control the industrial production. SUMMARY

[0009] The present disclosure provides a new type of pull acid hydrolytic enzyme, which has high expression and high enzyme catalytic activity. After modification of the enzyme, compared with the wild type, the activity of catalyzing the hydrolysis of large molecule CA is increased by more than 2 times, and the activity of catalyzing the hydrolysis of small molecule CA is significantly improved (about 1.8 times).

[0010] The present disclosure provides a method for specifically hydrolyzing pull acid, comprising contacting a protein comprising SEQ ID NO: 1 or as shown in SEQ ID NO: 1 or a variant thereof with pull acid, preferably obtaining 6 sugar units and / or 12 sugar units products, wherein more preferably, the site of the protein comprising SEQ ID NO: 1 or a variant thereof hydrolyzing pull acid is located between (1→3,4)-Fuc and (1→3)-Glc.

[0011] In another aspect, the present disclosure provides the use of a protein comprising SEQ ID NO: 1 or a protein as shown in SEQ ID NO: 1 or a variant thereof in the preparation of a kit for hydrolyzing pull acid.

[0012] In certain embodiments, the variant of SEQ ID NO: 1 comprises an amino acid sequence that has one to several amino acid residues deleted, substituted, and / or inserted in the amino acid sequence of SEQ ID NO: 1; for example, a truncation of SEQ ID NO: 1 that has one or more amino acids deleted from its N-terminus or C-terminus, but still retains its activity. For another example, a tag sequence is added to the N-terminus or C-terminus of SEQ ID NO: 1 for protein purification or identification. The variant that has one or more amino acids deleted or the variant that has a tag sequence added has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 1; and the koralic acid hydrolase variant has not significantly decreased, the same or increased koralic acid hydrolysis efficiency and / or hydrolytic activity compared to the koralic acid hydrolase of the amino acid sequence of SEQ ID NO: 1.

[0013] In some embodiments, the variant of SEQ ID NO: 1 comprises an amino acid sequence that has one to ten amino acid residues deleted, substituted, and / or inserted in the amino acid sequence of SEQ ID NO: 1, for example, one to eight amino acid residues, one to seven amino acid residues, one to six amino acid residues, one to five amino acid residues, one to four amino acid residues, one to three amino acid residues, one to two amino acid residues.

[0014] In another aspect, the present disclosure provides a koralic acid hydrolase variant, wherein the koralic acid hydrolase variant comprises an amino acid sequence that has one to several amino acid residues deleted, substituted, and / or inserted in the amino acid sequence of SEQ ID NO: 1; wherein the koralic acid hydrolase variant has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 1; and the koralic acid hydrolase variant has not significantly decreased, the same or increased koralic acid hydrolysis efficiency and / or hydrolytic activity compared to the koralic acid hydrolase of the amino acid sequence of SEQ ID NO: 1.

[0015] In some embodiments, the variant of SEQ ID NO: 1 comprises an amino acid sequence that has one to ten amino acid residues deleted, substituted, and / or inserted in the amino acid sequence of SEQ ID NO: 1, for example, one to eight amino acid residues, one to seven amino acid residues, one to six amino acid residues, one to five amino acid residues, one to four amino acid residues, one to three amino acid residues, one to two amino acid residues.

[0016] In some embodiments, the deletion and / or substitution corresponds 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.

[0017] In some specific embodiments, the substitution is selected from one or more of S276A, S276K, M296A, M296K, S298R, C302A, C312A, Y313R, K315A, C321A, S333K, Q340A, Q340K, C358A, N420A, Y433F, C456A, C459A, C490A, Y495F, C497A, R518K, D523A, W524A, N527A, N528A, Y531A, S549R, E551A, E551L, C571A, V573K, V573R, K575A, K575L, R577A, C590A, D594A, C623A, C643A, and C694A.

[0018] In some embodiments, the substitutions are selected from one of 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, S298R+S333K, 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, E551L+K575L+V573K+S549R, S298R+S276K+M296K+E551L+K575L+V573K+S549R, C490A+E551L+K575L+V573K, C490A+E551L+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.

[0019] 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.

[0020] In some embodiments, the 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.

[0021] In some embodiments, the substitutions are selected from one of 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.

[0022] In another aspect, the disclosure provides a polynucleotide encoding the colanic acid hydrolyase variant as described above.

[0023] In another aspect, the disclosure provides a vector comprising the polynucleotide as described above.

[0024] In another aspect, the disclosure provides a cell comprising the vector as described above.

[0025] In another aspect, the disclosure provides a method of hydrolyzing colanic acid, comprising contacting the colanic acid hydrolyase variant as described above or the colanic acid hydrolyase variant encoded by the polynucleotide as described above with colanic acid.

[0026] In another aspect, the disclosure provides a composition comprising the colanic acid hydrolyase variant as described above, the polynucleotide as described above, the vector as described above, the cell as described above.

[0027] In yet another aspect, the present disclosure provides use of a colanic acid hydrolase variant as described herein, a polynucleotide as described herein, a vector as described herein, a cell as described herein, a composition as described herein in the manufacture of a kit for hydrolyzing colanic acid.

[0028] In yet another aspect, the present disclosure provides use of a colanic acid hydrolase comprising SEQ ID NO: 1 in the manufacture of a kit for hydrolyzing colanic acid.

[0029] In yet another aspect, the present disclosure provides use of a colanic acid hydrolase as set forth in SEQ ID NO: 1 in the manufacture of a kit for hydrolyzing colanic acid. BRIEF DESCRIPTION OF DRAWINGS

[0030] The present application can be more completely understood in consideration of the following description.

[0031] FIG. 1 shows the HPLC detection results of the products after wild-type CAE2 completely enzymatically hydrolyzed CA.

[0032] FIG. 2 shows the NMR identification of the enzymatic hydrolysis products 1 (a) and 2 (b), which are 6-sugars (1CA) and 12-sugars (2CA), respectively.

[0033] FIG. 3 shows the molecular formula of the enzymatic hydrolysis products 1 (a) and 2 (b) inferred from the results of NMR.

[0034] FIG. 4 shows the crystal structure of CAE2 (PDB ID: 6e0w) and the molecular docking results with the substrate 1CA.

[0035] FIG. 5 shows the sequence alignment of CAE2 and CAE1.

[0036] FIG. 6 shows the structure alignment of CAE2 (PDB ID: 6e0w) and CAE1 (from alphafold prediction), RMSD = 1.674.

[0037] FIG. 7 shows the SDS-PAGE results of different mutants after purification, with a purity greater than or equal to 95%.

[0038] M: marker; 2: CAE2; 4: C456A; 5: C694A; 6: C643A; 11: C459A; 12: C321A; 13: C571A; 18: C497A; 20: Y495F; 26: Y433F; 27: C302A; 29: N420A; 30: C312A; 34: W524A.

[0039] FIG. 8 shows the SDS-PAGE results of different mutants after purification, with a purity greater than or equal to 95%.

[0040] 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.

[0041] Figure 9 shows SDS-PAGE results of different mutants after purification, with purity greater than or equal to 95%.

[0042] 2: CAE2; 11: C459A; 13: C571A; 14: C623A; 15: C590A; 26: Y433F; 19: N527A; 34: W524A; 29: N420A; 42: K315A; 46: N528A; M: marker.

[0043] Figure 10 shows SDS-PAGE results of different mutants after purification, with purity greater than or equal to 95%. 8: E390S; 17: E390A; 30: C312A; 40: E551A; 16: H461R; 41: R518A; 49: D523A; M: marker.

[0044] Figure 11 shows SDS-PAGE results of different mutants after purification, with purity greater than or equal to 95%.

[0045] M: marker; 1: CAE2; 2: E551A+S549R; 3: E551+AS549R+V573R.

[0046] Figure 12 shows SDS-PAGE results of different mutants after purification, with purity greater than or equal to 95%.

[0047] M: marker; 1: CAE2; 2: Q458A; 3: Y360A; 4: Y531A; 5: E551A; 6: Q340A+K315A; 7: Q340A+K315A+S276A; 8: Q340A+K315A+S276A+M296A.

[0048] Figure 13 shows SDS-PAGE results of different mutants after purification, with purity greater than or equal to 95%.

[0049] M: marker; 1: CAE2; 2: S276K; 3: M296K; 4: S276K+M296K; 5: Q340K.

[0050] Figure 14 shows SDS-PAGE results of different mutants after purification, with purity greater than or equal to 95%.

[0051] M: marker; 1 : S298R; 2: Y313R; 3: S333K; 4: S298R+333K; 5: Y313R+S333K; 6: S298R+Y313R; 7: S298R+Y313R+S333K; 8: V573K; 9: S549R; 10: V573K+S549R; 11 : S298R+Y313R+S333K+V573K+S549R; 12: CAE2.

[0052] Figure 15 shows SDS-PAGE results of different mutants after purification, with purity greater than or equal to 95%.

[0053] 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.

[0054] Figure 16 shows different molecular weight CA prepared. DETAILED DESCRIPTION

[0055] Various features and aspects of the present application are discussed in more detail below. It should be appreciated that the particular embodiments described herein are merely exemplary of the present application and are not intended to limit the present application.

[0056] DEFINITIONS

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application.

[0058] All numerical designations, e.g., pH, temperature, time, concentration, amount, and molecular weight, include ranges depending on the context in which the numerical designation is used, and are approximations that are variable depending, as would be understood by those skilled in the art, on a great variety of factors, including the definition of the term being used and / or the description of the embodiment being discussed, in some embodiments, the numerical values are in increments of 0.1 or 1.0, unless otherwise specified. It is to be understood that the numerical designations are preceded by the term "about" unless otherwise specified.

[0059] The terms "about" and "approximately" include amounts within ±10% of the stated value.

[0060] As will be appreciated by those of skill in the art, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof, for any and all purposes, particularly in providing written description art. Any listed range can be easily recognized as sufficiently describing and enabling the same range, and can be divided into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be easily divided in thirds, into a first third, a middle third, and a last third, etc. of the same size. As another non-limiting example, each range discussed herein can be easily divided in half, into a first half, a middle half, and a last half, etc. of the same size.

[0061] The term "amino acid" includes a compound having a -COOH group and a -NH2 group. In the present disclosure, the amino acid is a natural amino acid or a non-natural amino acid, including but not limited to glycine (Gly, G), alanine (Ala, A), valine (Val, V), leucine (Leu, L), isoleucine (lie, 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 (Gin, Q), threonine (Thr, T), aspartic acid (Asp, D), glutamic acid (Glu, E), lysine (Lys, K), arginine (Arg, R), and histidine (His, H).

[0062] The meaning of the term "protein" is well known in the art and is used accordingly in the context of the present application.

[0063] The term "hydrolase" refers to a polypeptide that catalyzes hydrolysis. Specifically, the hydrolases of the present disclosure are used interchangeably with "hydrolytic enzymes" or "degrading enzymes."

[0064] The term "corresponding to" refers to an amino acid residue at a listed position in a polypeptide or amino acid residue that is analogous, identical, or homologous to a listed amino acid residue in a polypeptide. Identifying an amino acid at a corresponding position can be determining a particular amino acid in a sequence designated as a particular sequence.

[0065] "Mutant" as used in the present disclosure is equivalent to the term "variant" and can have a single point mutation or a combination of point mutations.

[0066] The term "identity" as used herein is equivalent to the term "homology" and means sequence similarity to the amino acid sequence of the mutant uronic acid hydrolase SEQ ID NO: 1.

[0067] The term "hydrolase activity" means the amount of product generated per unit time by a particular enzyme.

[0068] The term "hydrolase efficiency" means the ratio of product to substrate generated per unit time by a particular enzyme.

[0069] The technical solution is to construct the gene (GeneBank Accession Number: YP_009012482.1) of caerulic acid hydrolase (CAE2) and its mutant through codon optimization into an expression vector (construction of an engineering strain) by genetic engineering technology. Then the expression vector carrying the CAE2 gene is transformed into a host cell, and the commonly used host cells include but are not limited to Escherichia coli, yeast, Bacillus subtilis, mold, mammalian cells, etc. (expression of the engineering strain). Through the sequence design of CAE2, the application in different scenarios is successfully realized, including but not limited to the efficient preparation of macromolecular or small molecular caerulic acid. Finally, the optimized CAE2 successfully obtains high-quality and batch-stable products through process flow optimization, production equipment design and optimization, and production process monitoring, etc. The industrial production of caerulic acid with different molecular weights is realized.

[0070] The wild-type CAE2 can specifically hydrolyze caerulic acid, and this ability exists in its natural state. The specificity of its hydrolysis site is crucial for the activity and selectivity of the enzyme, reflecting the specific interaction mode between CAE2 and caerulic acid. By cutting the caerulic acid molecule at a specific position, CAE2 can effectively decompose it into smaller fragments to realize the hydrolysis function.

[0071] According to certain embodiments of the present disclosure, various engineered CAE2s with at least one amino acid substitution are designed relative to the wild-type CAE2. The example sequences of wild-type and engineered CAE2s are shown in Table 1. The wild-type CAE2 is modified by different methods to improve or reduce the hydrolysis efficiency and thermal stability of CAE2, especially in the preparation of caerulic acid with different molecular weights.

[0072] In one aspect of the present disclosure, a caerulic acid hydrolase variant is provided, wherein the caerulic acid hydrolase variant comprises an amino acid sequence in which one to several amino acid residues are deleted, substituted, and / or inserted in the amino acid sequence of SEQ ID NO: 1;

[0073] wherein the amino acid sequence of the caerulic acid hydrolase variant has at least 90% identity to the amino acid sequence of SEQ ID NO: 1; and

[0074] Compared with the caerulic acid hydrolase of the amino acid sequence of SEQ ID NO: 1, the caerulic acid hydrolase variant has no significantly reduced, the same or improved caerulic acid hydrolysis efficiency and / or hydrolysis activity.

[0075] According to some embodiments of the disclosure, the foregoing coralic acid hydrolyase variant comprises an amino acid sequence that is 1 to 10 amino acid residues, e.g., 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, 1 to 2 amino acid residues, deleted, substituted, and / or inserted in the amino acid sequence of SEQ ID NO: 1. Specifically, the foregoing coralic acid hydrolyase variant can be an amino acid sequence that is 1 amino acid residue, 2 amino acid residues, 3 amino acid residues, 4 amino acid residues, 5 amino acid residues, 6 amino acid residues, 7 amino acid residues, 8 amino acid residues, deleted, substituted, and / or inserted in the amino acid sequence of SEQ ID NO: 1.

[0076] According to some embodiments of the disclosure, the foregoing deletion and / or substitution corresponds 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.

[0077] According to some embodiments of the disclosure, the foregoing substitution is selected from one or more of S276A, S276K, M296A, M296K, S298R, C302A, C312A, Y313R, K315A, C321A, S333K, Q340A, Q340K, C358A, N420A, Y433F, C456A, C459A, C490A, Y495F, C497A, R518K, D523A, W524A, N527A, N528A, Y531A, S549R, E551A, E551L, C571A, V573K, V573R, K575A, K575L, R577A, C590A, D594A, C623A, C643A, and C694A.

[0078] According to some embodiments of the disclosure, the foregoing substitution is selected from one of 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, S298R+S333K, 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, E551L+K575L+V573K+S549R, S298R+S276K+M296K+E551L+K575L+V573K+S549R, C490A+E551L+K575L+V573K, C490A+E551L+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.

[0079] According to some embodiments of the disclosure, the foregoing deletion and / or substitution corresponds to one or more of S276, M296, S298, C302, C312, Y313, C321, S333, C456, C459, C490, C497, D523, S549, E551, C571, V573, K575, C590, D594, C623, C643, C694 of SEQ ID NO: 1.

[0080] According to some embodiments of the disclosure, the foregoing 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.

[0081] According to some embodiments of the disclosure, the foregoing 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.

[0082] Another aspect of the disclosure provides a polynucleotide encoding the foregoing colanic acid hydrolyase variant.

[0083] Another aspect of the disclosure also provides a vector comprising the foregoing polynucleotide.

[0084] Another aspect of the disclosure also provides a cell comprising the foregoing vector.

[0085] Another aspect of the disclosure also provides a method of hydrolyzing colanic acid, comprising contacting the foregoing colanic acid hydrolyase variant or the colanic acid hydrolyase variant encoded by the foregoing polynucleotide with colanic acid.

[0086] Another aspect of the disclosure also provides a composition comprising the foregoing colanic acid hydrolyase variant, the foregoing polynucleotide, the foregoing vector, or the foregoing cell.

[0087] Another aspect of the present disclosure also provides use of the aforementioned colanic acid hydrolase variant, the aforementioned polynucleotide, the aforementioned vector, the aforementioned cell, or the aforementioned composition in the preparation of a kit for hydrolyzing colanic acid.

[0088] Another aspect of the present disclosure also provides use of the colanic acid hydrolase comprising SEQ ID NO: 1 in the preparation of a kit for hydrolyzing colanic acid.

[0089] The enzyme catalytic pocket is an important biomolecular structure, in which key amino acids play a crucial role in hydrolyzing colanic acid. During the process of hydrolyzing colanic acid, specific amino acids in the enzyme catalytic pocket interact with the colanic acid molecule, thereby affecting its hydrolysis efficiency. The modification of the enzyme catalytic pocket is a key bioengineering technology aimed at adjusting the catalytic performance of the enzyme to meet specific application needs. The present disclosure utilizes computational chemistry combined with knowledge and technology in multiple fields such as structural biology and bioinformatics to modify the enzyme catalytic pocket, in order to achieve precise control and optimization of the enzyme catalytic pocket, thereby improving the catalytic efficiency, selectivity and stability of the enzyme, and expanding its application prospects in the fields of biological industry, medicine and environmental protection, etc.

[0090] The percentage of catalytic activity exhibited by the present disclosure is the ratio of the activity detected by the mutant to the activity detected by the wild type under the same conditions. Greater than 100% indicates that the activity of the mutant is better than that of the wild type, and less than 100% indicates that the activity of the mutant is less than that of the wild type. The method used to detect enzyme activity in the present disclosure is the DNS reducing sugar detection method (substrate is macromolecular CA, MW: 5000 kDa), except as otherwise specified. This indicates that the amino acids in the key catalytic region of CAE2 in the catalytic pocket must have high specificity and accuracy, and any slight change can affect its catalytic activity.

[0091] The interaction between colanic acid and the enzyme is complex, involving multiple molecular level effects. As a polysaccharide substance, colanic acid has multiple functional groups such as hydroxyl and carboxyl, enabling it to interact with proteins. The interaction between the enzyme and colanic acid can be achieved through physical adsorption, hydrogen bonding, hydrophobic interaction, ionic interaction, etc. In this process, the active site of the enzyme plays a key role. The molecular structure of colanic acid binds to the active site of the enzyme, affecting the conformation and catalytic activity of the enzyme. In addition, the polysaccharide structure of colanic acid may affect the interaction between the enzyme and the substrate through spatial arrangement and charge distribution, etc., leading to changes in the conformation of the enzyme and affecting its catalytic efficiency.

[0092] Examples

[0093] The following examples are merely illustrative and are not intended to limit the scope or content of the present disclosure in any way.

[0094] Experimental Methods

[0095] 1. Commonly used expression vectors include, but are not limited to, pET-24a, pLMAR, pALTER-Exl, 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-1 λT, 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, pProEX HT, 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, and the like.

[0096] 2. Expression of engineered strains: Expression vectors carrying enzyme genes are transformed into host cells, commonly used host cells include but are not limited to E. coli, yeast, Bacillus subtilis, mold, mammalian cells, etc. Commonly used E. coli cells include but are not limited to: E. coli BL21(DE3), E. coli BL21, and E. coli ArhaB (B0002). And sub-series: 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. coli AD494, 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 ArcticExpress, E. coli ArcticExpress(DE3). Mold cells: Streptomyces lividans. Lactococcus lactis cells: Lactoccocus 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, Baby hamster kidney (BHK) cells, CAD, DUKX-X11, HeLa, Hep G2, HT1080, J558L, L929, MCF-7, N2a, NIH 3T3, P19, SO-Rb50, U2OS, Y79, etc.

[0097] Example 1 Hydrolysis of wild-type CAE2 on colanic acid

[0098] After CAE2 was allowed to react with the substrate colanic acid, the resulting hydrolysis products were detected by high-performance liquid chromatography (HPLC), as shown in Figure 1, and two distinct peaks were found. Subsequently, through a series of purification and isolation operations, two samples with relatively high purity were obtained (Figure 1): enzyme product 1 (a) and enzyme product 2 (b). Further nuclear magnetic resonance (NMR) detection of these two samples found that one sample contained 6 sugar units, while the other sample contained 12 sugar units, as shown in Figure 2.

[0099] These findings reveal the position specificity of CAE2 hydrolyzing colanic acid. According to the results, it is inferred that the hydrolysis site is located between (1→3,4)-Fuc and (1→3)-Glc, as shown in Figure 3. This conclusion is based on the analysis of the number of sugar units in different samples and is obtained through quantitative and structural identification by nuclear magnetic resonance technology. These results are of great significance for determining the hydrolysis mechanism of CAE2 and its interaction mode with the colanic acid substrate. It is speculated that the key amino acid sequence near the enzyme catalytic pocket of CAE2 has the same hydrolysis site as other potential colanic acid hydrolytic enzymes similar to CAE2.

[0100] Example 2 Design of engineered colanic acid hydrolytic enzyme (CAE2)

[0101] (1) Related amino acids constituting the enzyme catalytic pocket

[0102] Through analysis of the crystal structure of the colanic acid-CAE2 complex (PDB ID: 6e0w), it is speculated that the enzyme catalytic pocket is located as shown in Figure 4, and the related amino acids constituting the catalytic pocket are: Y360, D387, A388, E390, N392, V393, N420, I423, A428, Y433, Q458, H461, E463, L487, T488, C490, Y495, D523, etc.

[0103] Through experiments, it was found that during the hydrolysis of CAE2 on colanic acid, amino acids such as H461, E463, D387, and E390 are crucial for enzyme activity. These amino acids were respectively mutated into amino acids with the same charge or no charge (such as H461A (0%), H461K (0%), H461R (2%), E463A (3%), E463D (0%), E463S (0%), D387A (0%), D383S (4%), D387E (3%), E390A (11%), E390D (3%), E390S (12%)), and the results showed that these mutations almost completely lost the activity of CAE2 (see Table 3).

[0104] Table 1. Sequence information

[0105] Table 2. Amino acid mutants related to protein aggregation

[0106] * Negative values represent background interference, which can be understood as 0, no hydrolysis.

[0107] Table 3. Key amino acid mutants of enzyme catalytic pocket

[0108] * Negative values represent background interference, which can be understood as 0, no hydrolysis.

[0109] Table 4. Amino acid mutants related to substrate binding

[0110] * Negative values represent background interference, which can be understood as 0, no hydrolysis.

[0111] Table 5. Efficiency of mutant hydrolysis of 2CA to generate 1CA

[0112] After sequence alignment analysis with the previously reported CAE1 hydrolase with hydrolysis of colanic acid, it was found that the amino acid sequence similarity between CAE2 and CAE1 was 30.04% (Figure 5), and the structural similarity RMSD was 1.27 (Figure 6). Moreover, the key amino acid types in the catalytic pocket area (H461, E463, D387 and E390) were the same (Figure 5). Based on this finding, it can be speculated that other enzymes with the same or similar amino acid sequences at the key amino acid positions (H461, E463, D387 and E390) in the catalytic pocket of CAE2, with an overall amino acid sequence similarity greater than 30% and a structural similarity RMSD less than 5, will also specifically hydrolyze colanic acid. These similar enzymes may be ubiquitous in bacteria or other microorganisms, and have similar biological functions for hydrolyzing similar substrates. This speculation provides an important clue for further exploring the functions and catalytic mechanisms of other enzymes, and helps to better understand the functions and regulation of bacterial metabolic pathways and microbial ecosystems.

[0113] (2) Interaction of colanic acid with the enzyme

[0114] Through analysis of the crystal structure of the colanic acid-CAE2 complex (PDB ID: 6e0w), it was found that the left side of the CAE2 structure that interacts with colanic acid has T274SSI277, G294TMIS298, Y313VKML317, S333TAAFLMQA341, Y360AILQQGT367, D387AIELNV393, Q417GANWGIGIGVAGSGPY433, R457QCLHVEM464, T479, G484TGLTTCGVALYG496, etc. Through analysis of the docking results of CAE2 with a unit of colanic acid molecules, it was found that the right side of the CAE2 structure that interacts with colanic acid has V515STRMVFIDWGVNNGRYA532, E551, K575, S605, etc. (Table 1 and Table 2).

[0115] By designing the above amino acids interacting with the substrate, we observed that single-point mutations N392A (0%), Q365A (1.8%), W421A (8%), Q458A (1%), Y360A (1%), S333K (0.5%) almost completely impaired the activity of CAE2. These results indicate that these amino acids are essential for the interaction between CAE2 and colanic acid. It is worth noting that mutating these amino acids to other similar amino acids (such as Q, F, T, N, Y, W, S, etc.) or charged amino acids (such as D, E, R, K, H, etc.) may slightly affect the catalytic efficiency, but it is also possible that it will have no effect or improve the catalytic efficiency. In the experiment, we also observed that the combined mutations N420A + E390A (3%) and N420A + E390S (2%) almost completely impaired the activity of CAE2, which may be due to the effect of 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.

[0116] Single-point mutations N420A (34%), W524A (55%), R577A (76%), K315A (78%), N528A (81%), Q340A (85%), N527A (88%), Y433F (97%), Y531A (97%), Y495F (99%), E551A (102%), etc. also affect the catalytic efficiency of CAE2, indicating that these amino acids are related to the interaction of the substrate. However, the combined mutations Q340A + K315A (6%), Q340A + K315A + S276A (14%), and Q340A + K315A + S276A + M296A (9%) almost completely lost the activity of CAE2, which may be that the single-point mutations change the ability of the substrate to interact with the enzyme is weak, but the combined mutations can greatly enhance this change, thereby having a significant impact on the catalytic efficiency of the enzyme. It can be reasonably speculated that the combination of single-point mutations with less impact on the catalytic efficiency of the enzyme (> 50%) in the present application may have a significant impact on the activity of CAE2.

[0117] The single point mutants M296A (111%), S276A (111%), D594A (122%), D523A (135%) etc. have significant improvement in catalytic efficiency towards CAE2, indicating that these amino acids are related to the interaction with the substrate, more likely to be non-specific binding with the substrate, and then affect its catalytic efficiency; after mutation, the non-specific binding is weakened, thus improving the catalytic efficiency. Therefore, mutating the amino acids at these sites to other amino acids without side chains, short side chains or with opposite charges (G, P, S, T, I, V, L, D, E, R, K, H) may have similar or better effects.

[0118] By analyzing the results of the docking of CAE2 and colanic acid molecules, it is speculated that the interaction of R518 site with colanic acid may affect the catalytic activity of CAE2. Mutating the R518 site to different amino acids, it is found that it has a great influence on its catalytic activity, among which R518A (10%), R518D (3%), R518E (4%), R518H (5%) almost lost all activity, and R518K (101%) has no effect on the activity. It is shown that the charge type of the amino acid at this site is very important, when it is mutated to no charge or opposite charge, the enzyme activity almost loses all, but when it becomes the same charge, the enzyme activity almost does not change, thus it is speculated that the amino acids at this site and its vicinity are positively charged, which may enhance the interaction with colanic acid. Similarly, it can be speculated that at the similar position on the left side of the structure of CAE2, the charge type of the positively charged amino acid and the interaction with colanic acid will have a similar relationship.

[0119] Therefore, amino acids near these two regions were selected and mutated to positively charged or uncharged amino acids, respectively, to enhance the interaction between CAE2 and substrate coumaric 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+S549R (41%), S298R+Y313R+S333K+V573K+S549R (65%), S298R+S276K+M296K (38%), E551L+K575L+V573K (105%), E551L+K575L+S549R, E551L+K575L+V573K+S549R, S298R+S276K+M296K+E551L+K575L+V573K+S549R. Among them, the catalytic activities of E551A+S549R (122%), M296K (122%), S276K (120%) and S276K+M296K (117%) were improved to different degrees, which might be because these mutants enhanced the binding ability to the substrate while not affecting or weakly affecting the speed of substrate release, thereby improving the overall catalytic activity of CAE2. The catalytic activities of the remaining mutants remained unchanged or were weakened, which might be because these mutants enhanced the binding ability to the substrate while also weakening the speed of substrate release. When the speed of substrate release is greater than the speed of substrate binding, the overall catalytic activity of the mutant will show a weakening effect. It is speculated that mutating these mutants to uncharged, uncharged side chain, similar charge or opposite charge amino acids (G, P, S, T, I, V, L, D, E, R, K, H) might have similar or better effects.

[0120] To further verify the interaction of these mutants with the substrate, the present application sets the substrate as a small molecule 2CA (6 sugars of 2 units, 12 sugars) to carry out the hydrolysis reaction, and detects the proportion of one unit CA (6 sugars) generated, while comparing 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 single point or combined mutant mutants significantly improve the efficiency of CAE2 hydrolysis of 2CA to generate 1CA. It can be seen that changing the charge type 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 molecules CA.

[0121] (3) Weaken the random interaction between enzyme molecules

[0122] Through sequence analysis, it is found that CAE2 contains 13 cysteines (Cys), which are extremely likely to cause random aggregation between protein molecules, and then affect the solubility, catalytic activity and other properties of the protein molecules. After the cysteine is respectively mutated to Ala, the enzyme activity of CAE2 is improved to different degrees, and the highest can reach 124%, C302A (110%), C312A (120%), C321A (103%), C456A (112%), C459A (100%), C490A (123%), C497A (71%), C571A (110%), C590A (117%), C623A (124%), C643A (115%), C694A (117%), CAE2. It can be inferred that mutating the cysteines at these sites to other amino acids without side chains or short side chains (G, P, S, T, I, V, L) may have similar or better effects. At the same time, combining single point mutations to carry out combined mutations, two-by-two combined or more combined mutations, will also have similar or better effects.

[0123] (4) Superimpose (2) and (3)

[0124] The C490A (123%) in selection (3) was selected to be combined with the mutants in superposition (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 (66%), C490A+S298R+S276K+M296K+V573K+S549R, C490A+S298R+S276K+M296K+E551L+K575L+V573K+S549R, and it was found that the catalytic activity of the combined mutant enzymes was significantly improved, with the highest C490A+S298R+V573K combination mutant reaching 218%, followed by the C490A+E551L+K575L+V573K combination mutant reaching 178%. It is speculated that the combination of other mutants in (3), especially C623A (124%), C312A (120%), C590A (117%), C694A (117%), C643A (115%), C456A (112%), C571A (110%), C302A (110%), etc. single point or combination mutation with the mutants in (2) will also have similar or better effects.

[0125] Example 3: Expression and purification of enzymes

[0126] The CAE2 gene or its mutants were 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, 0.1 mM inducer IPTG was added, and the culture temperature was adjusted to 20°C, and the protein expression was induced overnight. Then, the induced cells were collected, resuspended with buffer A1 (PBS, pH 7.0), and then high-pressure broken. The unbroken cells and larger membrane fragments were removed by high-speed centrifugation at 20000 rpm, the supernatant was collected, and it was added to a pre-equilibrated nickel column. The column was washed with buffer A1, and then the target protein was eluted using buffer B1 containing 300 mM imidazole and 20 mM PB (pH 7.0). Finally, the eluted protein was dialyzed overnight at 4°C using a buffer of 20 mM PB (pH 7.0). After purification, the purity of the protein was greater than 95%, as shown in FIGS. 7-15.

[0127] Example 4: Enzyme activity test

[0128] The activity of CAE2 was quantified by determining the content of reducing sugar using 3,5-dinitrosalicylic acid (DNS) method with glucose as the standard substance. Briefly, 900 μL of CA (final concentration: 2 g / L) was mixed with CAE at a ratio of 9:1 by volume, and incubated at 55°C for 15 min. Then, 1 mL of DNS reagent was added, and the mixture was boiled for 5 min. After cooling, 3 mL of distilled water was added to the mixture, and the absorbance was detected at 540 nm. Finally, the content of reducing sugar in the sample was determined by comparing the absorbance value of the sample with the standard curve.

[0129] Small molecule enzymatic efficiency detection: The 2CA sample was completely dissolved in an appropriate amount of water to ensure its solubility was sufficient. After reaching a concentration of 2 g / L in the solution, the temperature was adjusted to 30°C. Then, CAE2 and its mutants were added to different solutions at a final concentration of 50 ug / ml, respectively, and the reaction was carried out for 24 hours. After the reaction was completed, the reaction solution was heated at 100°C for 10 min to terminate the reaction. Finally, the proportion of 1CA in the reaction solution was detected and analyzed by high performance liquid chromatography (HPLC) technology.

[0130] Example 5: Preparation of CA with different molecular weights

[0131] The macromolecular CA sample was completely dissolved in an appropriate amount of water to ensure its solubility was sufficient. After reaching a concentration of 10 g / L in the solution, the temperature was adjusted to 25°C. Then, CAE2 was added to different solutions at a final concentration of 5-100 ug / mL, 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 min to terminate the reaction. Subsequently, the reaction solution was diluted 5 times with water to make the final concentration of the sample reach 2 g / L. Finally, the weight average molecular weight of CA in the reaction solution was detected and analyzed by high performance liquid chromatography (HPLC) technology. The results showed that CA with different molecular weights was successfully prepared, realizing the industrial scale-up production, and the results are shown in Figure 16.

[0132] INCORPORATED BY REFERENCE

[0133] The entire contents of each patent and scientific document referred to herein is incorporated by reference herein for all purposes.

[0134] EQUIVALENCIES

[0135] The present application can be embodied in other specific ways without departing from the spirit or essential characteristics thereof. Therefore, the above embodiments are merely illustrative, not a limitation of the application described herein. The scope of the application is indicated by the appended claims rather than by the description above, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

1. A method for specifically hydrolyzing colacid, comprising contacting a protein comprising SEQ ID NO:1 or a variant thereof as shown in SEQ ID NO:1 with colacid, preferably obtaining a product of 6 glycounits and / or 12 glycounits, wherein more preferably, the site for hydrolyzing colacid in the protein comprising SEQ ID NO:1 or a variant thereof is located between (1→3,4)-Fuc and (1→3)-Glc.

2. Use of the protein comprising SEQ ID NO:1 or a protein as shown in SEQ ID NO:1 or a variant thereof in the preparation of a kit for hydrolyzing colacid.

3. A variant of colacid hydrolase, wherein, The colacid hydrolase variant comprises an amino acid sequence in which one or more amino acid residues are missing, substituted, and / or inserted in the amino acid sequence of SEQ ID NO:

1. The amino acid sequence of the colacid hydrolase variant is at least 90% identical to the amino acid sequence of SEQ ID NO:1; and Compared with the colacid hydrolase of the amino acid sequence of SEQ ID NO:1, the colacid hydrolase variant has the same or improved colacid hydrolysis efficiency and / or hydrolysis activity without significant reduction.

4. The colacid hydrolase variant according to claim 3, wherein the colacid hydrolase variant comprises an amino acid sequence in which 1 to 10 amino acid residues are deleted, substituted, and / or inserted, 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.

5. The colacid hydrolase variant according to claim 3 or 4, wherein the deletion and / or substitution corresponds to one or more of the following positions in 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.

6. The collamylate hydrolase variant according to claim 5, wherein the substitution is selected from S276A, S276K, M296A, M296K, S298R, C302A, C312A, Y313R, K315A, C321A, S333K, Q340A, Q340K, C358A, N420A, Y433F, C456A, C459A, C490 One or more of the following: A, Y495F, C497A, R518K, D523A, W524A, N527A, N528A, Y531A, S549R, E551A, E551L, C571A, V573K, V573R, K575A, K575L, R577A, C590A, D594A, C623A, C643A, and C694A.

7. The collamylate hydrolase variant according to claim 5, wherein 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, S276A, D594A, D523A, E551A, E551A+S549R, E551A+S549R+V573R, M296K, Q340K, S276K, S276K+M296K, S298R, Y313R , S298R+S333K, 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+S549 R、E551L+K575L+V573K+S549R、S298R+S276K+M296K+E551L+K575L+V573K+S549R、C490A+E551L+K575L+V573K、C490A+E551 One of L+K575L+S549R, C490A+E551L+K575L+V573K+S549R, C490A+S298R+V573K, C490A+S298R+S549R, C490A+S298R+V573K+S549R, C490A+S298R+S276K+M293K+S549R, C490A+S298R+S276K+M296K+E551L+K575L+V573K+S549R, Y531A, and K575A.

8. The colacid hydrolase variant according to any one of claims 3 to 5, wherein the deletion and / or substitution corresponds to one or more of the following positions in 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.

9. The colacid hydrolase variant according to claim 8, wherein 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.

10. The colacid hydrolase variant according to claim 9, wherein the substitution is 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+K575 One of L+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.

11. A polynucleotide encoding a variant of the colacid hydrolase according to any one of claims 3 to 10.

12. A vector comprising the polynucleotide according to claim 11.

13. A cell comprising the carrier according to claim 12.

14. A composition comprising a colacid hydrolase variant according to any one of claims 3 to 10, a polynucleotide according to claim 11, a carrier according to claim 12, or a cell according to claim 13.

15. Use of the colacid hydrolase variant according to any one of claims 3 to 10, the polynucleotide according to claim 11, the vector according to claim 12, the cell according to claim 13, or the composition according to claim 14 in the preparation of a kit for hydrolyzing colacid.

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