A horse acylase mutant and its application

Abstract

The present invention belongs to the field of biotechnology and discloses a horse-derived acylase mutant and its application. The horse-derived acylase mutant of the present invention can efficiently and stably perform acylation reactions to produce lauroylglycine. Therefore, the horse-derived acylase mutant provided by the present invention can be used as an amino acid surfactant and has wide application potential in various fields, particularly in personal care products and the food industry.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a horse acylase mutant and application thereof. Background Art

[0002] Amino acid surfactants are highly surface-active amphiphilic molecules formed by the condensation of an amino acid as an acyl acceptor and one or more non-polar molecules, such as long-chain fatty acids, as acyl donors. Due to their low toxicity, high biodegradability, high ecocompatibility, and antibacterial properties, amino acid surfactants are widely used in the food, pharmaceutical, and cosmetics industries.

[0003] Sodium lauroyl glycine (2-(dodecanoylamino)aceticacid), with the chemical formula C14H27NO3, is an amino acid surfactant. It can be obtained from lauroyl glycine through a simple saponification reaction. The structural formula of lauroyl glycine is:

[0004]

[0005] Traditional methods for synthesizing lauroylglycine primarily rely on chemical acylation, where lauric acid and glycine are acylated in the presence of an acidic or alkaline catalyst. While this method is capable of producing lauroylglycine, it suffers from several significant drawbacks, including the requirement for high temperature and pressure, harsh reaction conditions, low product purity, and complex and costly subsequent processing. Furthermore, the chemical catalysis process often requires the use of large amounts of organic solvents and strong acid-base catalysts, which can easily lead to environmental pollution and safety hazards, making it incompatible with the requirements of modern green chemistry.

[0006] Currently, the most common method for producing amino acid surfactants is through the Schotten-Baumann condensation reaction. It is a process in which fatty acid chlorides and amino acids react in an alkaline aqueous solution or other organic reagents to obtain fatty acyl amino acid salts, which are then separated by neutralization with an inorganic acid to obtain crude fatty acyl amino acids, which are then neutralized with alkali to form purer fatty acyl amino acid salts. Since the process involves the use of phosgene to convert free fatty acids into the corresponding acyl chlorides, although this method produces a high yield of amides, it requires the use of stoichiometric amounts of harmful activation reagents and produces a large amount of waste, resulting in low atom economy. In addition, residual toxic irritants in the reaction, such as fatty acid chlorides and by-products, can cause quality and safety issues for end users of personal care and pharmaceuticals, and therefore require further purification steps to completely eliminate them.

[0007] Nowadays, more and more industries are trying to develop sustainable technologies to reduce the generation of organic waste, reduce environmental pollution problems and reduce post-processing costs. One of the most effective ways to achieve these goals is to use biologically mediated reactions, generally known as bioconversion.

[0008] Enzymatic synthesis is the most efficient and fastest method of biotransformation, that is, the amidation reaction of fatty acids and their derivatives with amino acids is catalyzed by biological enzymes. Compared with traditional chemical synthesis, enzymatic synthesis is green and safe.

[0009] There are currently several biological pathways for the synthesis of lauroylglycine, one of which is lipase synthesis. The mechanism is that glycine and C8-C18 free fatty acids are used as acyl donors for the synthesis of N-acylglycine, and then the ester-amide conversion with the amine donor is as follows:

[0010]

[0011] The Singaporean team of Professor Zhi Li (Glen Kai Bin Kua, Dr. Giang Kien Truc Nguyen, Professor Zhi Li, Enzyme Engineering for High-Yielding Amide Formation: Lipase-Catalyzed Synthesis of N-Acyl Glycines in Aqueous Media, Angewandte Chemie International Edition, 2023, Volume 62, Issue 14) has developed an enzymatic amidation method for the synthesis of N-acyl glycines by aminolysis of fatty acids with glycine. They modified an enzyme called proRML and, by reshaping its catalytic pocket, catalyzed the amidation of fatty acids with glycine, yielding N-lauroyl glycine with an 80% yield at 10 mM substrate concentration and a 45% yield at 100 mM substrate concentration.

[0012] Another synthetic pathway is the synthesis of lauroylglycine via the specific catalysis of aminoacylase I (ACY1).

[0013] ACY1 is an enzyme that catalyzes acylation reactions and has a wide range of applications in bioengineering and biotechnology. It specifically hydrolyzes N-acyl-L-amino acids to produce L-amino acids, while not reacting with their enantiomers, N-acyl-D-amino acids. Furthermore, it has a broad substrate range, catalyzing the hydrolysis of acetylated amino acids other than acetylproline and acetylaspartate. ACY1 is widely used in the industrial breakdown of amino acids.

[0014] ACY1 was discovered as early as 1881. It was initially shown that crude kidney homogenate catalyzed the hydrolysis of hippurate (N-benzoylglycine) into benzoate and glycine. It wasn't until 1922 that hippurase was shown to hydrolyze acyl derivatives of various amino acids, introducing the concept of aminoacylase.

[0015] ACY1 is present in many mammalian tissues, with the highest activity in the kidney. However, the physiological role and exact cellular localization of Acy1 remain a matter of debate. Margot er-Naumanna, Michael Mikoa, Lutz Konradb, Klaus-Heinrich The distribution of aminoacylase I among mammalian species and localization of the enzyme in porcine kidney, Biochimie 82 (2000) 129-137) found that, by comparing the activity of Acy1 in the kidney and liver homogenates of 11 mammalian species, the enzyme was most abundant in herbivores (such as sheep and cattle) and omnivores, while the activity in rodents and cats was very low, and no Acy1 activity was detected in the livers of five different breeds of dogs.

[0016] While research on acylases has primarily focused on common livestock sources such as pigs, cattle, sheep, and rabbits, there have been relatively few reports on equine acylase (Equus caballus aminoacylase-1). By searching for sequence-similar proteins based on the publicly available crystal diffraction structure of human acylase and verifying the predicted structure, the highly similar equine acylase has become a crucial entry point for acylase research. By combining computational design with wet lab experiments, the present invention screened and evolved a stable equine acylase with acylase activity.

[0017] The main challenges faced by the enzymatic pathway are that the known enzymes have low activity and poor stability when catalyzing acylation reactions, high enzyme costs, and low product yields. These are the main factors currently restricting the enzymatic synthesis of amino acid surfactants. Summary of the Invention

[0018] The present invention aims to develop a highly efficient equine acylase mutant for the catalytic synthesis of lauroylglycine. This enzyme efficiently acylates lauric acid with glycine under mild reaction conditions, exhibits high yield, high product purity, and is environmentally friendly. The lauroylglycine produced by this method has broad potential for application in various fields, particularly in personal care products and the food industry.

[0019] The first aspect of the present invention provides a horse-derived acylase mutant, the amino acid sequence of the wild-type acylase of which is shown in SEQ ID NO.1.

[0020] The acylase mutant contains one, two, three, four, five, six or more mutants selected from the following positions where the amino acid residues are mutated:

[0021] Q44V, Q44E, Q44K, Q44G, Q44Y, Q44L, Q44W, W65E, W65G, W65Y, K115R, K115M, K115L, K115E, K115F, G151A, G151S, L173R, L173M, L173S, L173T, T201N, T201L, T201P, T201K, T201S, T201E, T201W, G205S, G205L, G2 05A, G205Q, N252Q, N252S, G259A, G259L, G259E, E288M, E288P, E288F, E288Q, E288R, E288T, E288V, Q307 R, Q307G, Q307N, Q307E, Q307D, Q307P, W323Y, W323T, S328D, S328Q, S328I, S328V, S328L, S328M, S328A.

[0022] Preferably, the acylase mutant contains a mutant with amino acid residue mutations at the above two sites, more specifically containing the following mutations:

[0023] Q44W, W65E; Q44W, W65Y; Q44L, W65Y; Q44E, K115R; Q44E, K115M; Q44E, K115L; Q44E, K115E.

[0024] Preferably, the acylase mutant contains mutants with amino acid residue mutations at the above three sites, more specifically containing:

[0025] Q44W, W65Y, G151A; Q44W, W65Y, G151S; Q44W, W65Y, S328M; L173M, G205S, G259E; L173M, G205S, G259A.

[0026] Preferably, the acylase mutant contains mutants with amino acid residue mutations at the above four sites, more specifically containing:

[0027] Q44W, W65Y, G151A, L173M; Q44W, W65Y, G151A, Q307G; Q44W, K115E, S328M, W323Y; Q44W, K115E, S328A, N252Q.

[0028] Preferably, the acylase mutant contains mutants with amino acid residue mutations at the above five sites, more specifically containing:

[0029] Q44W, W65Y, G151A, L173M, G205S, G259E; Q44W, W65Y, G151A, L173M, G205S, G259A; Q44W, W65Y, G151A, L173M, G205S, G259L; Q44W, W65Y, G151A, L173M, G205S, E288R.

[0030] The acylase mutant containing the above mutation has an acylase with high catalytic activity and achieves a high synthesis conversion rate of lauroylglycine.

[0031] The second aspect of the present invention provides a nucleic acid encoding the above-mentioned acylase mutant.

[0032] The third aspect of the present invention provides a vector containing the nucleic acid encoding the acylase mutant, such as an expression vector, more specifically, the expression vector is a pET vector.

[0033] The fourth aspect of the present invention provides a genetically engineered bacterium containing the nucleic acid encoding the acylase mutant or the vector, such as bacteria or fungi, specifically, the starting bacterium is Escherichia coli, Bacillus subtilis or Pichia pastoris.

[0034] The fifth aspect of the present invention provides the use of the renal acylase mutant, its encoding nucleic acid, vector or genetically engineered bacteria in the biocatalytic synthesis of lauroylglycine.

[0035] The sixth aspect of the present invention provides a method for preparing lauroylglycine, which comprises using the aforementioned acylase mutant as a biocatalyst, lauric acid and amino acids as substrates to form a reaction system to generate lauroylglycine.

[0036] Specifically, the wet cell body, crude enzyme solution or pure enzyme obtained by fermentation culture of the acylase mutant or the genetically engineered bacteria is used as the biocatalyst. Preferably, the genetically engineered bacteria in the form of wet cell body is used as the whole cell catalyst.

[0037] More specifically, in the reaction system, the amount of the wet bacteria is 5-30 g / L, the concentration of lauric acid is 1.44-72 g / L, and the saturated glycine solution is 60%-100%;

[0038] The reaction conditions are as follows: the biocatalytic reaction is carried out at a temperature of 35-65° C., a pH of 5.5-8.5, and a stirring speed of 100-300 rpm.

[0039] The wet bacteria are obtained by fermenting and culturing the genetically engineered bacteria, centrifuging, discarding the supernatant, and collecting the precipitate.

[0040] More specifically, the wet cells are prepared as follows: recombinant Escherichia coli containing a gene encoding a mutant of horseradish acylase is inoculated into a LB liquid culture medium containing 50 μg / ml of kanamycin resistance, cultured at 37°C and 200 rpm for 12 hours, then inoculated into a fresh LB liquid culture medium containing 50 μg / ml of kanamycin resistance at a 1% (v / v) inoculum amount, cultured at 37°C and 200 rpm until the cell OD600 reaches 0.6-0.8, IPTG is added at a final concentration of 0.1 mM, and cultured at 20°C and 200 rpm for 15 hours; then the mixture is centrifuged at 4°C and 8000 rpm for 20 minutes, the supernatant is discarded, and the precipitate is collected to obtain the wet cells.

[0041] The present invention provides a series of horse-derived acylase mutants that can be used to improve the efficiency of industrial lauroylglycine production. The provided acylases have the following characteristics: High catalytic efficiency: Through enzyme design and screening of different mutation sites, acylases with high catalytic activity were obtained, achieving a high synthetic conversion rate of lauroylglycine. Environmental friendliness: The enzymatic reaction catalysis can replace traditional chemical production, conforming to the principles of green chemistry and reducing environmental pollution. Industrial application potential: Preliminary performance evaluation tests have demonstrated that the acylase can significantly reduce costs and improve product quality in actual production, making it suitable for large-scale production. DETAILED DESCRIPTION

[0042] The embodiments will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples.

[0043] It should be noted that the mutation site in the following examples refers to a position that differs from the wild-type acylase derived from horse having the amino acid sequence shown in SEQ ID NO: 1 in terms of the amino acid sequence.

[0044] Example 1. Obtaining wild-type acylase (1) from horse

[0045] The gene was synthesized based on the reported maleylase, the amino acid sequence of which is shown in SEQ ID NO: 1 (aminoacylase-1 isoform X1 [Equus caballus], XP_001492888.2), and the nucleic acid sequence of its cDNA coding region is shown in SEQ ID NO: 2 (ACY-1, XM_001492838.5).

[0046] After artificially synthesizing the wild-type acylase cDNA coding region, the gene was used as a template and primers sequence 3 and sequence 4 were used to amplify the fragment by PCR and introduce NdeⅠ and XhoⅠ endonuclease sites on both sides of the fragment. PCR reaction system: Takara Max DNA Polymerase 25 μL, ddH2O 20 μL, Sequence 3 primer 1.5 μL, Sequence 4 primer 1.5 μL, Sequence 2 template 2 μL. PCR reaction procedure: 98°C initial denaturation for 2 min, followed by 25 cycles of denaturation at 98°C for 10 s, annealing at 58°C for 15 s, and extension at 72°C for 10 s, followed by a final extension at 72°C for 5 min.

[0047] The PCR product was analyzed by 1% agarose gel electrophoresis, confirming the presence of an amplified product of approximately 1.2 kb.

[0048] Only a DNA fragment of about 1.2kb was cut out from the above agarose gel. The DNA in the gel strip was purified and recovered using the Tiangen ordinary agarose gel DNA recovery kit. The purified product was double-digested with restriction enzymes NdeⅠ and XhoⅠ. After 1% agarose gel electrophoresis confirmed that the band size was about 1.2kb, it was purified and recovered again to obtain the gene enzyme-digested fragment.

[0049] Similarly, the E. coli expression vector pET-28a(+) was cut using restriction enzymes NdeⅠ and XhoⅠ, and only a DNA fragment of about 5.3 kb was cut out from the agarose gel by agarose gel electrophoresis. The vector enzyme-digested fragment was obtained after purification and recovery.

[0050] The approximately 1.2 kb and approximately 5.3 kb fragments obtained above were ligated with T4 DNA ligase and then transformed into Escherichia coli BL21 (DE3) to obtain a transformant (1). Plasmids were prepared from the above-mentioned cells using the Tiangen Plasmid Miniprep Kit to obtain the recombinant plasmid pET-28a(+)-Acy. DNA sequencing confirmed that the nucleotide sequence of the coding region of the plasmid was consistent with Sequence 2. The transformant (1) produced acylase (1), which is a wild-type acylase derived from horse.

[0051] The recombinant E. coli BL21(DE3) / pAcy-pET-28a(+) obtained above was inoculated into LB liquid medium containing 50 μg / ml kanamycin and cultured at 37°C, 200 rpm for 12 hours. A 1% (v / v) inoculum was then inoculated into fresh LB liquid medium containing 50 μg / ml kanamycin and cultured at 37°C, 200 rpm until the OD600 of the cells reached 0.6-0.8. IPTG was then added to a final concentration of 0.1 mM and induced at 20°C, 200 rpm for 15 hours. The cells were centrifuged at 4°C, 8000 rpm for 20 minutes, the supernatant discarded, and the precipitate collected to obtain wet cells of the recombinant E. coli BL21(DE3) / pAcy-pET-28a(+) expressing the recombinant plasmid. These cells can be used directly as biocatalysts.

[0052] The wet cells obtained above were used to synthesize and hydrolyze lauroylglycine. The synthesis reaction system (10 mL) consisted of 360 mg of lauric acid, 10 ml of saturated glycine solution, and 0.2 g of wet cells. Reaction conditions included a temperature of 35-65°C and a pH of 5.5-8.5. Analytical conditions included a C18 column, 4.6 mm × 250 mm, 5 μm; detection wavelength was UV at 200 nm; mobile phase consisted of 85% acetonitrile and 15% 0.05% trifluoroacetic acid in water; column temperature was 30°C; flow rate was 1 mL / min; and detection time was 15 min.

[0053] Example 2: Obtaining acylase mutants (2) to (8) and determining their activity

[0054] Analyze sequence information, calculate PSSM matrix, observe amino acid conservation, and select less conserved sites for directed saturation mutagenesis.

[0055] After artificially synthesizing the recombinant plasmid pET-28a(+)-Acy, the plasmid was used as a template and the designed primer pair was used to amplify the fragment by PCR to introduce the mutation site required for the acylase mutant (2). PCR reaction system: Takara Max DNA Polymerase 25 μL, ddH2O 20 μL, Sequence 5 Primer F 1.5 μL, Primer R 1.5 μL, pET-28a(+)-Acy template 2 μL. PCR protocol: 98°C initial denaturation for 2 min, followed by 25 cycles of denaturation at 98°C for 10 s, annealing at 58°C for 15 s, and extension at 72°C for 40 s, followed by a final extension at 72°C for 5 min.

[0056] The PCR product was analyzed by 1% agarose gel electrophoresis, confirming the presence of an amplified product of approximately 6.5 kb.

[0057] Only a DNA fragment of about 6.5 kb was cut out from the above agarose gel, and the DNA in the gel strip was purified and recovered using the Tiangen ordinary agarose gel DNA recovery kit.

[0058] Escherichia coli BL21 (DE3) was transformed to obtain transformant (2). Transformant (2) produced a mutant (2) of the wild-type acylase derived from horse, and its culture and activity determination protocol were the same as in Example 1.

[0059] In order to obtain a transformant (3) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same operation as above was performed to obtain a plasmid to obtain a transformant (3). The transformant (3) produced an acylase (3) having the mutations described in the table.

[0060] To obtain a transformant (4) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same operations as above were performed to obtain a plasmid to obtain a transformant (4). The transformant (4) produced an acylase (4) having the mutations described in the table.

[0061] To obtain a transformant (5) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same operations as above were performed to obtain a plasmid to obtain a transformant (5). The transformant (5) produced an acylase (5) having the mutations described in the table.

[0062] To obtain a transformant (6) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same operations as above were performed to obtain a plasmid, thereby obtaining a transformant (6). The transformant (6) produced an acylase (6) having the mutations described in the table.

[0063] In order to obtain a transformant (7) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same operation as above was performed to obtain a plasmid to obtain a transformant (7). The transformant (7) produced an acylase (7) having the mutations described in the table.

[0064] To obtain a transformant (8) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same operations as above were performed to obtain a plasmid and a transformant (8). The transformant (8) produced an acylase (8) having the mutations described in the table.

[0065] The results of the activity assays of the acylase mutants (2) to (8) are as follows:

[0066]

[0067] For superimposed single-point mutations, a relative value of >1.5 for the synthetic conversion rate relative to the wild type is defined as a significant improvement, 1.2-1.5 as an improvement, and 1-1.2 as a slight improvement. As can be seen from the table above, mutations to amino acids at position 44 significantly improve the conversion rate, mutations to K, G, and L slightly improve, and mutations to E slightly improve activity, making this site a dominant site for improving conversion rate.

[0068] Example 3. Obtaining acylase mutants (9) to (11) and activity determination

[0069] In order to obtain a transformant (9) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid, thereby obtaining a transformant (9). The transformant (9) produced an acylase (9) having the mutations described in the table.

[0070] In order to obtain a transformant (10) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid and a transformant (10). The transformant (10) produced an acylase (10) having the mutations described in the table.

[0071] In order to obtain a transformant (11) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid and a transformant (11). The transformant (11) produced an acylase (11) having the mutations described in the table.

[0072] The results of the activity assays of the acylase mutants (9) to (11) are as follows:

[0073]

[0074] As can be seen from the above table, the conversion rate is significantly improved when the amino acid at position 65 is mutated to Y, and the activity is slightly improved when it is mutated to G. This point can be used as a dominant site for improving the conversion rate.

[0075] Example 4. Obtaining acylase mutants (12) to (16) and activity determination

[0076] In order to obtain a transformant (12) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid and a transformant (12) was obtained. The transformant (12) produced an acylase (12) having the mutations described in the table.

[0077] To obtain a transformant (13) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid and a transformant (13). The transformant (13) produced an acylase (13) having the mutations described in the table.

[0078] To obtain a transformant (14) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid and a transformant (14). The transformant (14) produced an acylase (14) having the mutations described in the table.

[0079] In order to obtain a transformant (15) expressing a mutant acylase having the amino acid residue substitutions shown in the table, a plasmid was obtained in the same manner as in Example 2, except that the primer of sequence number 18 was used instead of the primer of sequence number 5, thereby obtaining a transformant (15). The transformant (15) produced an acylase (15) having the mutations described in the table.

[0080] To obtain a transformant (16) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid and a transformant (16). The transformant (16) produced the acylase (16) having the mutations described in the table.

[0081] The results of the activity assays of the acylase mutants (12) to (16) are as follows:

[0082]

[0083] As can be seen from the above table, the conversion rate is significantly improved when the amino acid at position 115 is mutated into E, there is some improvement when it is mutated into F, M, and L, and the activity is slightly improved when it is mutated into R. This point can be used as a dominant site for improving the conversion rate.

[0084] Example 5. Obtaining acylase mutants (17) to (18) and activity determination

[0085] To obtain a transformant (17) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid and a transformant (17). The transformant (17) produced the acylase (17) having the mutations described in the table.

[0086] To obtain a transformant (18) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid and a transformant (18). The transformant (18) produced an acylase (18) having the mutations described in the table.

[0087] The results of the activity assays of the acylase mutants (17) to (18) are as follows:

[0088]

[0089] From the above table, it can be found that the conversion rate is improved when the amino acid at position 151 is mutated to S or A, and this point can be used as an advantageous site for improving the conversion rate.

[0090] Example 6. Obtaining acylase mutants (19) to (22) and activity determination

[0091] To obtain a transformant (19) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid and a transformant (19). The transformant (19) produced an acylase (19) having the mutations described in the table.

[0092] To obtain a transformant (20) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid and a transformant (20). The transformant (20) produced an acylase (20) having the mutations described in the table.

[0093] To obtain a transformant (21) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid and a transformant (21) was obtained. The transformant (21) produced the acylase (21) having the mutations described in the table.

[0094] To obtain a transformant (22) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid and a transformant (22) was obtained. The transformant (22) produced the acylase (22) having the mutations described in the table.

[0095] The results of the activity assays of the acylase mutants (19) to (22) are as follows:

[0096]

[0097] As can be seen from the above table, the conversion rate is significantly improved when the amino acid at position 173 is mutated into M or S, there is a slight improvement when it is mutated into T, and there is a slight improvement when it is mutated into R. This point can be used as a dominant site for improving the conversion rate.

[0098] Example 7. Obtaining acylase mutants (23) to (29) and activity determination

[0099] To obtain a transformant (23) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid and a transformant (23). The transformant (23) produced an acylase (23) having the mutations described in the table.

[0100] To obtain a transformant (24) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid and a transformant (24). The transformant (24) produced an acylase (24) having the mutations described in the table.

[0101] To obtain a transformant (25) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid and a transformant (25). The transformant (25) produced an acylase (25) having the mutations described in the table.

[0102] To obtain a transformant (26) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid and a transformant (26) was obtained. The transformant (26) produced the acylase (26) having the mutations described in the table.

[0103] To obtain a transformant (27) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid and a transformant (27). The transformant (27) produced the acylase (27) having the mutations described in the table.

[0104] To obtain a transformant (28) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid and a transformant (28). The transformant (28) produced the acylase (28) having the mutations described in the table.

[0105] To obtain a transformant (29) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid and a transformant (29). The transformant (29) produced an acylase (29) having the mutations described in the table.

[0106] The results of the activity assays of the acylase mutants (23) to (29) are as follows:

[0107]

[0108] As can be seen from the above table, the conversion rate is significantly improved when the amino acid at position 201 is mutated into S, E, K, L, or P, and the activity is improved when it is mutated into N or W. This point can be used as a dominant site for improving the conversion rate.

[0109] Example 8. Obtaining acylase mutants (30) to (29) and activity determination

[0110] To obtain a transformant (30) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid and a transformant (30) was obtained. The transformant (30) produced the acylase (30) having the mutations described in the table.

[0111] To obtain a transformant (31) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid and a transformant (31) was obtained. The transformant (31) produced the acylase (31) having the mutations described in the table.

[0112] To obtain a transformant (32) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid and a transformant (32) was obtained. The transformant (32) produced the acylase (32) having the mutations described in the table.

[0113] In order to obtain a transformant (33) expressing a mutant acylase having the amino acid residue substitutions shown in the table, a plasmid was obtained in the same manner as in Example 2, except that the primer of SEQ ID NO: 36 was used instead of the primer of SEQ ID NO: 5, thereby obtaining a transformant (33). The transformant (33) produced an acylase (33) having the mutations described in the table.

[0114] The results of the activity assays of the acylase mutants (30) to (33) are as follows:

[0115]

[0116]

[0117] As can be seen from the above table, the conversion rate is significantly improved when the amino acid at position 205 is mutated to Q, and the conversion rate is also improved when it is mutated to S, L, or A. This point can be used as a dominant site for improving the conversion rate.

[0118] Example 9: Obtaining acylase mutants (34) to (35) and activity determination

[0119] To obtain a transformant (34) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid and a transformant (34) was obtained. The transformant (34) produced the acylase (34) having the mutations described in the table.

[0120] To obtain a transformant (35) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid and a transformant (35) was obtained. The transformant (35) produced the acylase (35) having the mutations described in the table.

[0121] The results of the activity assays of the acylase mutants (34) to (35) are as follows:

[0122]

[0123] As can be seen from the above table, the conversion rate is significantly improved when the amino acid at position 252 is mutated to Q or S, and this point can be used as an advantageous site for improving the conversion rate.

[0124] Example 10: Obtaining acylase mutants (36) to (38) and determining their activity

[0125] To obtain a transformant (36) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid and a transformant (36) was obtained. The transformant (36) produced the acylase (36) having the mutations described in the table.

[0126] To obtain a transformant (37) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid and a transformant (37). The transformant (37) produced the acylase (37) having the mutations described in the table.

[0127] To obtain a transformant (38) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid and a transformant (38). The transformant (38) produced the acylase (38) having the mutations described in the table.

[0128] The results of the activity assays of the acylase mutants (36) to (38) are as follows:

[0129]

[0130] As shown in the table above, the conversion rate is significantly improved when the amino acid at position 259 is mutated to E, slightly improved when it is mutated to L, and slightly improved when it is mutated to A. This point can be used as a dominant site for improving conversion rate.

[0131] Example 11: Obtaining acylase mutants (39) to (45) and determining their activity

[0132] To obtain a transformant (39) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid and a transformant (39) was obtained. The transformant (39) produced an acylase (39) having the mutations described in the table.

[0133] To obtain a transformant (40) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid and a transformant (40). The transformant (40) produced an acylase (40) having the mutations described in the table.

[0134] To obtain a transformant (41) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid and a transformant (41) was obtained. The transformant (41) produced the acylase (41) having the mutations described in the table.

[0135] To obtain a transformant (42) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid and a transformant (42) was obtained. The transformant (42) produced the acylase (42) having the mutations described in the table.

[0136] To obtain a transformant (43) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid and a transformant (43) was obtained. The transformant (43) produced the acylase (43) having the mutations described in the table.

[0137] In order to obtain a transformant (44) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid and a transformant (44) was obtained. The transformant (44) produced an acylase (44) having the mutations described in the table.

[0138] To obtain a transformant (45) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid and a transformant (45) was obtained. The transformant (45) produced the acylase (45) having the mutations described in the table.

[0139] The results of the activity assays of the acylase mutants (39) to (45) are as follows:

[0140]

[0141] As can be seen from the above table, the conversion rate is significantly improved when the amino acid at position 288 is mutated into R, M, or V, while it is improved when it is mutated into F, Q, or T, and the activity is slightly improved when it is mutated into P. This point can be used as a dominant site for improving the conversion rate.

[0142] Example 12: Obtaining acylase mutants (46) to (51) and determining their activity

[0143] To obtain a transformant (46) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid and a transformant (46) was obtained. The transformant (46) produced the acylase (46) having the mutations described in the table.

[0144] To obtain a transformant (47) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid and a transformant (47) was obtained. The transformant (47) produced the acylase (47) having the mutations described in the table.

[0145] To obtain a transformant (48) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid and a transformant (48). The transformant (48) produced the acylase (48) having the mutations described in the table.

[0146] To obtain a transformant (49) expressing a mutant acylase having the amino acid residue substitutions shown in the table, a plasmid was obtained in the same manner as in Example 2, except that the primer of SEQ ID NO: 52 was used instead of the primer of SEQ ID NO: 5, thereby obtaining a transformant (49). The transformant (49) produced an acylase (49) having the mutations described in the table.

[0147] To obtain a transformant (50) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid and a transformant (50) was obtained. The transformant (50) produced the acylase (50) having the mutations described in the table.

[0148] To obtain a transformant (51) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid and a transformant (51) was obtained. The transformant (51) produced the acylase (51) having the mutations described in the table.

[0149] The results of the activity assays of the acylase mutants (46) to (51) are as follows:

[0150]

[0151] From the above table, it can be found that the conversion rate is significantly improved when the amino acid at position 307 is mutated into G or N, and is also improved when it is mutated into R, E, D, or P. This point can be used as a dominant site for improving the conversion rate.

[0152] Example 13: Obtaining acylase mutants (46) to (51) and determining their activity

[0153] To obtain a transformant (52) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid and a transformant (52) was obtained. The transformant (52) produced the acylase (52) having the mutations described in the table.

[0154] To obtain a transformant (53) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid and a transformant (53) was obtained. The transformant (53) produced the acylase (53) having the mutations described in the table.

[0155] The results of the activity assays of the acylase mutants (52) to (53) are as follows:

[0156]

[0157] As can be seen from the above table, the conversion rate is significantly improved when the amino acid at position 323 is mutated to Y or T, and this point can be used as an advantageous site for improving the conversion rate.

[0158] Example 14: Obtaining acylase mutants (55) to (60) and determining their activity

[0159] To obtain a transformant (54) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid and a transformant (54) was obtained. The transformant (54) produced the acylase (54) having the mutations described in the table.

[0160] To obtain a transformant (55) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid and a transformant (55) was obtained. The transformant (55) produced the acylase (55) having the mutations described in the table.

[0161] To obtain a transformant (56) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid and a transformant (56) was obtained. The transformant (56) produced the acylase (56) having the mutations described in the table.

[0162] To obtain a transformant (57) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid and a transformant (57) was obtained. The transformant (57) produced the acylase (57) having the mutations described in the table.

[0163] To obtain a transformant (58) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid and a transformant (58) was obtained. The transformant (58) produced the acylase (58) having the mutations described in the table.

[0164] To obtain a transformant (59) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid and a transformant (59) was obtained. The transformant (59) produced the acylase (59) having the mutations described in the table.

[0165] To obtain a transformant (60) expressing a mutant acylase having the amino acid residue substitutions shown in the table, corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid and a transformant (60) was obtained. The transformant (60) produced the acylase (60) having the mutations described in the table.

[0166] The results of the activity assays of the acylase mutants (55) to (60) are as follows:

[0167]

[0168] As can be seen from the above table, the conversion rate is significantly improved when the amino acid at position 328 is mutated into M, V, I, or L, the conversion rate is improved when it is mutated into Q, and the activity is slightly improved when it is mutated into D or A. This point can be used as a dominant site for improving the conversion rate.

[0169] Example 15: Obtaining acylase mutants (61) to (62) and determining their activity

[0170] To obtain a transformant (61) expressing a mutant acylase having the amino acid residue substitutions shown in the table, the vector obtained by plasmid extraction from transformant (8) was used to replace the original cloning plasmid, and the corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain transformant (61). Transformant (61) produced acylase (61) having the mutations described in the table.

[0171] [Example 62] Obtaining acylase mutant (62)

[0172] To obtain a transformant (62) expressing a mutant acylase having the amino acid residue substitutions shown in the table, the vector obtained by plasmid extraction from transformant (8) was used to replace the original cloning plasmid, and the corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid to obtain transformant (62). Transformant (62) produced acylase (62) having the mutations described in the table.

[0173] The results of the activity assays of the acylase mutants (61) to (62) are as follows:

[0174]

[0175] For stacked multiple mutations, a relative conversion rate of >1.3 relative to the parent transformant is defined as a significant improvement, 1.1-1.3 as an improvement, and 1-1.1 as a slight improvement. As shown in the table above, mutations at positions 44 and 65 to W and Y, respectively, significantly improve conversion rates. This combination of two mutations can improve conversion rates compared to a single mutation.

[0176] Example 16: Obtaining the acylase mutant (63) and determining its activity

[0177] To obtain a transformant (63) expressing a mutant acylase having the amino acid residue substitutions shown in the table, the same procedures as in Example 2 were followed, except that the vector obtained by plasmid extraction from transformant (7) was used to replace the original cloning plasmid and the primer of sequence number 10 was used instead of the primer of sequence number 5. This plasmid was obtained to obtain transformant (63). Transformant (63) produced acylase (63) having the mutations described in the table.

[0178] The results of the activity assay of the acylase mutant (63) are as follows:

[0179]

[0180] As can be seen from the table above, the conversion rate is slightly improved when amino acids at positions 44 and 65 are mutated to L and Y, respectively. This indicates that the combination of overlapping mutations is not limited to a single mutation form and that this two-point combination can improve the conversion rate compared to a single mutation.

[0181] Example 17: Obtaining acylase mutants (46) to (51) and determining their activity

[0182] To obtain a transformant (64) expressing a mutant acylase having the amino acid residue substitutions shown in the table, the vector obtained by plasmid extraction from transformant (3) was used to replace the original cloning plasmid, and the corresponding primers were designed. The same procedures as in Example 2 were followed to obtain a plasmid, resulting in transformant (64). Transformant (64) produced acylase (64) having the mutations described in the table.

[0183] To obtain a transformant (65) expressing a mutant acylase having the amino acid residue substitutions shown in the table, the vector obtained by plasmid extraction from transformant (3) was used to replace the original cloning plasmid, and the corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid to obtain transformant (65). Transformant (65) produced acylase (65) having the mutations described in the table.

[0184] To obtain a transformant (66) expressing a mutant acylase having the amino acid residue substitutions shown in the table, the vector obtained by plasmid extraction from transformant (3) was used to replace the original cloning plasmid, and the corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain transformant (66). Transformant (66) produced acylase (66) having the mutations described in the table.

[0185] To obtain a transformant (67) expressing a mutant acylase having the amino acid residue substitutions shown in the table, the vector obtained by plasmid extraction from transformant (3) was used to replace the original cloning plasmid, and the corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain transformant (67). Transformant (67) produced acylase (67) having the mutations described in the table.

[0186] The results of the activity assays of the acylase mutants (64) to (67) are as follows:

[0187]

[0188] As can be seen from the table above, the conversion rate is significantly improved when amino acids at positions 44 and 115 are mutated to E, E, or E, M, respectively. This indicates that the double-site combination is not limited to a specific site and can improve the conversion rate compared to single-site mutations.

[0189] Example 18: Obtaining acylase mutants (68) to (70) and determining their activity

[0190] From mutants 61 to 67, it can be seen that the combined mutation of two sites is helpful to improve the conversion rate, so more site mutations are superimposed.

[0191] To obtain a transformant (68) expressing a mutant acylase having the amino acid residue substitutions shown in the table, the vector obtained by plasmid extraction from transformant (62) was used to replace the original cloning plasmid, and the corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid to obtain transformant (68). Transformant (68) produced acylase (68) having the mutations described in the table.

[0192] To obtain a transformant (69) expressing a mutant acylase having the amino acid residue substitutions shown in the table, the vector obtained by plasmid extraction from transformant (62) was used to replace the original cloning plasmid, and the corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid to obtain transformant (69). Transformant (69) produced acylase (69) having the mutations described in the table.

[0193] To obtain a transformant (70) expressing a mutant acylase having the amino acid residue substitutions shown in the table, the vector obtained by plasmid extraction from transformant (62) was used to replace the original cloning plasmid, and the corresponding primers were designed and the same procedures as in Example 2 were followed to obtain a plasmid to obtain transformant (70). Transformant (70) produced acylase (70) having the mutations described in the table.

[0194] The results of the activity assays of the acylase mutants (68) to (70) are shown in the following table:

[0195]

[0196] As can be seen from the above table, the conversion rate is significantly improved when the amino acids at positions 44, 65, and 151 are mutated to W, Y, S or W, Y, A, respectively. This three-point combination can improve the conversion rate compared to the two-point mutation.

[0197] Example 19: Obtaining acylase mutants (71) to (72) and determining their activity

[0198] To obtain a transformant (72) expressing a mutant acylase having the amino acid residue substitutions shown in the table, the vector obtained by plasmid extraction from transformant (20) was used to replace the original cloning plasmid. Appropriate primers were designed and the corresponding fragments were amplified. Following the same amplification and recovery conditions as in Example 1, the fragments were ligated using the OK Clon DNA Seamless Cloning Kit. The plasmids were obtained in the same manner as in Example 1 to obtain transformant (71). Transformant (71) produced the acylase (71) having the mutations described in the table.

[0199] To obtain a transformant (72) expressing a mutant acylase having the amino acid residue substitutions shown in the table, the vector obtained by plasmid extraction from transformant (20) was used to replace the original cloning plasmid. Appropriate primers were designed and the corresponding fragments were amplified. The same procedures as above were used to obtain a plasmid to obtain transformant (72). Transformant (72) produced the acylase (72) having the mutations described in the table.

[0200] The results of the activity assays of the acylase mutants (71) to (72) are shown in the following table:

[0201]

[0202] As can be seen from the above table, the conversion rate is significantly improved when the amino acids at positions 173, 205, and 259 are mutated into M, S, E or M, S, A, respectively. This three-point combination can improve the conversion rate compared to single-point mutations.

[0203] Example 20: Obtaining acylase mutants (73) to (76) and determining their activity

[0204] It can be seen from mutants 68 to 72 that the combined mutation of three sites is helpful to improve the conversion rate, so more site mutations are superimposed.

[0205] To obtain a transformant (73) expressing a mutant acylase having the amino acid residue substitutions shown in the table, the vector obtained by plasmid extraction from transformant (67) was used to replace the original cloning plasmid. Appropriate primers were designed and the corresponding fragments were amplified. The plasmid was obtained using the same procedures as in Example 19 to obtain transformant (73). Transformant (73) produced acylase (73) having the mutations described in the table.

[0206] To obtain a transformant (74) expressing a mutant acylase having the amino acid residue substitutions shown in the table, the vector obtained by plasmid extraction from transformant (67) was used to replace the original cloning plasmid. Appropriate primers were designed and the corresponding fragments were amplified. The plasmid was obtained using the same procedures as in Example 19 to obtain transformant (74). Transformant (74) produced the acylase (74) having the mutations described in the table.

[0207] To obtain a transformant (75) expressing a mutant acylase having the amino acid residue substitutions shown in the table, the vector obtained by plasmid extraction from transformant (67) was used to replace the original cloning plasmid. Appropriate primers were designed and the corresponding fragments were amplified. The plasmid was obtained using the same procedures as in Example 19 to obtain transformant (75). Transformant (75) produced the acylase (75) having the mutations described in the table.

[0208] To obtain a transformant (76) expressing a mutant acylase having the amino acid residue substitutions shown in the table, the vector obtained by plasmid extraction from transformant (67) was used to replace the original cloning plasmid. Appropriate primers were designed and the corresponding fragments were amplified. The plasmid was obtained using the same procedures as in Example 19 to obtain transformant (76). Transformant (76) produced acylase (76) having the mutations described in the table.

[0209] The results of the activity assays of the acylase mutants (73) to (76) are shown in the following table:

[0210]

[0211] It can be found from the above table that the conversion rates are significantly improved when amino acids at positions 44, 115, 328, and 252 are mutated to W, E, A, and Q, or amino acids at positions 44, 65, 151, and 173 are mutated to W, Y, A, and M, or amino acids at positions 44, 65, 151, and 307 are mutated to W, Y, A, and G, or amino acids at positions 44, 115, 328, and 323 are mutated to W, E, M, and Y. This four-point combination can improve the conversion rate compared to two-point mutations.

[0212] Example 21. Obtaining acylase mutants (77) to (80) and determining their activity

[0213] It can be seen from acylase mutants 73 to 76 that the combined mutation of four sites is helpful to improve the conversion rate, so more site mutations are superimposed.

[0214] To obtain a transformant (77) expressing a mutant acylase having the amino acid residue substitutions shown in the table, the vector obtained by plasmid extraction from transformant (73) was used to replace the original cloning plasmid. Appropriate primers were designed and the corresponding fragments were amplified. The plasmid was obtained using the same procedures as in Example 19 to obtain transformant (77). Transformant (77) produced acylase (77) having the mutations described in the table.

[0215] To obtain a transformant (78) expressing a mutant acylase having the amino acid residue substitutions shown in the table, the vector obtained by plasmid extraction from transformant (73) was used to replace the original cloning plasmid. Appropriate primers were designed and the corresponding fragments were amplified. The plasmid was obtained using the same procedures as in Example 19 to obtain transformant (78). Transformant (78) produced the acylase (78) having the mutations described in the table.

[0216] To obtain a transformant (79) expressing a mutant acylase having the amino acid residue substitutions shown in the table, the vector obtained by plasmid extraction from transformant (73) was used to replace the original cloning plasmid. Appropriate primers were designed and the corresponding fragments were amplified. The same procedures as in Example 19 were used to obtain a plasmid to obtain transformant (79). Transformant (79) produced the acylase (79) having the mutations described in the table.

[0217] To obtain a transformant (80) expressing a mutant acylase having the amino acid residue substitutions shown in the table, the vector obtained by plasmid extraction from transformant (73) was used to replace the original cloning plasmid. Appropriate primers were designed and the corresponding fragments were amplified. The same procedures as in Example 19 were used to obtain a plasmid to obtain transformant (80). Transformant (80) produced acylase (80) having the mutations described in the table.

[0218] The results of the activity assays of the acylase mutants (77) to (80) are shown in the following table:

[0219]

[0220] It can be found from the above table that the conversion rate is significantly improved when amino acids at positions 44, 65, 151, 173, 205, and 259 are mutated to W, Y, A, M, S, E or W, Y, A, M, S, A or W, Y, A, M, S, L, or when amino acids 22, 65, 151, 173, 205, and 288 are mutated to W, Y, A, M, S, R. This six-point combination can improve the conversion rate compared to four-point mutations.

Claims

1. A horse acylase mutant, characterized in that: It is a mutant having the following amino acid residue mutations based on the wild-type acylase shown in the amino acid sequence of SEQ ID NO.1: Q44V;Q44Y;Q44W; Q44W and W65Y; Q44E and K115M; Q44E and K115E; Q44W, W65Y and G151A; Q44W, W65Y and G151S; Q44W, W65Y and S328M; Q44W, W65Y, G151A and L173M; Q44W, W65Y, G151A and Q307G; Q44W, K115E, S328M and W323Y; Q44W, K115E, S328A and N252Q; Q44W, W65Y, G151A, L173M, G205S and G259E; Q44W, W65Y, G151A, L173M, G205S and G259A; Q44W, W65Y, G151A, L173M, G205S and G259L; Or Q44W, W65Y, G151A, L173M, G205S and E288R.

2. A nucleic acid encoding the acylase mutant according to claim 1.

3. An expression vector containing the encoding nucleic acid according to claim 2.

4. The expression vector according to claim 3, wherein The starting vector is pET vector.

5. The genetically engineered bacteria of the encoding nucleic acid according to claim 2, or the expression vector according to claim 3 or 4, wherein the genetically engineered bacteria is a bacterium or a fungus.

6. The genetically engineered bacterium according to claim 5, wherein The starting bacteria are Escherichia coli, Bacillus subtilis or Pichia pastoris.

7. Use of the acylase mutant according to claim 1, its encoding nucleic acid, or an expression vector or genetically engineered bacteria containing the encoding nucleic acid in biocatalytic synthesis of lauroylglycine.

8. A method for preparing lauroylglycine, characterized in that: The method comprises using the acylase mutant as claimed in claim 1 as a biocatalyst, and using lauric acid and glycine as substrates to form a reaction system to generate lauroylglycine.

9. The preparation method according to claim 8, wherein The wet bacteria, crude enzyme solution or pure enzyme obtained by fermentation culture of the acylase mutant and the genetically engineered bacteria is used as a biocatalyst.

10. The preparation method according to claim 9, characterized in that The genetically engineered bacteria are used as whole-cell catalysts in the form of wet bacteria.

11. The preparation method according to claim 10, characterized in that In the reaction system, the amount of wet cells is 5-30 g / L, the concentration of lauric acid is 1.44-72 g / L, and the saturated glycine solution is 60%-100%; The reaction conditions are as follows: the biocatalytic reaction is carried out at a temperature of 35-65° C., a pH of 5.5-8.5, and a stirring speed of 100-300 rpm; The wet bacteria are obtained by fermenting and culturing the genetically engineered bacteria, centrifuging, discarding the supernatant, and collecting the precipitate.

12. The preparation method according to claim 11, wherein The wet cells were prepared as follows: recombinant Escherichia coli containing a gene encoding a mutant of horseradish acylase was inoculated into a LB liquid culture medium containing 50 μg / ml of kanamycin resistance, cultured at 37°C and 200 rpm for 12 hours, then inoculated into a fresh LB liquid culture medium containing 50 μg / ml of kanamycin resistance at a 1% (v / v) inoculum, cultured at 37°C and 200 rpm until the cell OD600 reached 0.6-0.8, and then IPTG was added to a final concentration of 0.1 mM, induced at 20°C and 200 rpm for 15 hours; then the mixture was centrifuged at 4°C and 8000 rpm for 20 minutes, the supernatant was discarded, and the precipitate was collected to obtain the wet cells.

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