Horse-derived acylase mutant and application thereof

Abstract

The invention belongs to the technical field of biology, and discloses a horse-derived acylase mutant and application thereof. The horse-derived acylase mutant disclosed by the invention can efficiently and stably act on acylation reaction to generate lauroyl glycine. Therefore, the horse-derived acylase mutant provided by the invention can be used as an amino acid surfactant, can be widely applied in different fields, and particularly has huge application potential in personal care products and 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 amphiphilic molecules with high surface activity, which are formed by condensing an amino acid as an acyl acceptor and one or more non-polar molecules such as long-chain fatty acids as acyl donors. Amino acid surfactants have the characteristics of low toxicity, high biodegradability, high eco-compatibility and antibacterial properties, so they are widely used in the food, pharmaceutical and cosmetic 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] The traditional method for synthesizing lauroylglycine is mainly through chemical acylation reaction, that is, acylation of lauric acid and glycine under the action of acidic or alkaline catalysts. Although this method can achieve the preparation of lauroylglycine, it has some obvious defects, such as the need for high temperature and high pressure, harsh reaction conditions, low product purity, complex subsequent treatment and high cost. More importantly, a large amount of organic solvents and strong acid-base catalysts are often required in the chemical catalysis process, which is easy to cause environmental pollution and safety hazards, and does not meet 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 alkaline aqueous solution or other organic reagents to obtain fatty acyl amino acid salts, and then the crude fatty acyl amino acids are separated by neutralization with inorganic acids, and 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, with low atom economy. In addition, residual toxic irritants such as fatty acyl chlorides and by-products in the reaction can cause quality and safety issues for end users of personal care and pharmaceuticals, so further purification steps are required 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 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. It involves the amidation reaction of fatty acids and their derivatives with amino acids catalyzed by biological enzymes. Compared with traditional chemical synthesis, enzymatic synthesis is green and safe.

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

[0010]

[0011] The team of Prof. Zhi Li in Singapore (Glen Kai Bin Kua, Dr. Giang Kien Truc Nguyen, Prof. 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) synthesized N-acyl glycine by aminolysis of fatty acids and glycine. An enzyme called proRML was modified to catalyze the amidation of fatty acids and glycine by reshaping its catalytic pocket, and N-lauroyl glycine was obtained with a yield of 80% at a substrate concentration of 10mM, and a yield of 45% at a substrate concentration of 100mM.

[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 can specifically hydrolyze N-acyl-L-amino acids to generate L-amino acids, but does not act on its enantiomer N-acyl-D-amino acids. In addition, it has a wide range of substrates and can catalyze the hydrolysis of other acetylated amino acids except acetylproline and acetylaspartic acid. ACY1 is widely used in the industry to disassemble amino acids.

[0014] ACY1 was discovered as early as 1881. The initial discovery was that crude kidney homogenate could catalyze the hydrolysis of hippurate (N-benzoylglycine) into benzoate and glycine. It was not until 1922 that it was demonstrated that hippurase could hydrolyze the acyl derivatives of various amino acids, introducing the concept of aminoacylase for the first time.

[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 localizationof the enzyme in porcine kidney, Biochimie 82 (2000) 129-137) found that when comparing the activity of Acy1 in the kidney and liver homogenates of 11 mammals, 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] Since the research on acylase is mainly focused on common livestock sources such as pigs, cattle, sheep, rabbits, etc., there are few reports on horse acylase (Equus caballus aminoacylase-1). Through the public human acylase crystal diffraction structure, the sequence similarity protein is searched, and through the re-examination of the predicted structure, the horse acylase with high similarity has become an entry point for acylase research that cannot be ignored. By combining computer design methods with wet experiments, the present invention screened and evolved a horse acylase with acylase activity and stable expression.

[0017] The main challenges faced by the enzyme catalysis 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 horse-derived acylase mutant that efficiently catalyzes the synthesis of lauroylglycine. The enzyme can achieve efficient acylation of lauric acid and glycine, and has the advantages of mild reaction conditions, high yield, high product purity, and environmental friendliness. The lauroylglycine prepared by the method can be widely used in different fields, especially in personal care products and the food industry, and has great application potential.

[0019] The first aspect of the present invention provides a horse acylase mutant, wherein the amino acid sequence of the wild-type acylase is as 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:

[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:

[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:

[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 higher 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 vector is a pET vector.

[0033] The fourth aspect of the present invention provides a genetically engineered bacterium containing the above-mentioned nucleic acid encoding the acylase mutant or the above-mentioned vector, for example, a bacterium or a fungus, and 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 lauroyl glycine, which comprises using the above-mentioned acylase mutant as a biocatalyst, and using lauric acid and amino acids as substrates to form a reaction system to react and generate lauroyl glycine.

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

[0037] More specifically, in the reaction system, the amount of 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: 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 bacterial body is obtained by fermenting and culturing the genetically engineered bacteria, centrifuging, discarding the supernatant, and collecting the precipitate.

[0040] More specifically, the wet bacteria 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 kanamycin resistance, and cultured at 37°C and 200 rpm for 12 hours; then inoculated into a fresh LB liquid culture medium containing 50 μg / ml kanamycin resistance at a 1% (v / v) inoculation amount, and cultured at 37°C and 200 rpm until the OD600 of the bacteria reaches 0.6-0.8; IPTG with a final concentration of 0.1 mM is added, and the culture is induced at 20°C and 200 rpm for 15 hours; then the culture is centrifuged at 4°C and 8000 rpm for 20 minutes, the supernatant is discarded, and the precipitate is collected to obtain the wet bacteria.

[0041] The present invention provides a series of horse-derived acylase mutants that can be used to improve the efficiency of industrial production of lauroylglycine. The provided acylase has the following characteristics: High catalytic efficiency: By designing and screening enzymes at different mutation sites, an acylase with high catalytic activity is obtained, achieving a higher synthetic conversion rate of lauroylglycine. Environmental friendliness: The enzyme reaction catalysis can replace traditional chemical production, conform to the principles of green chemistry, and reduce environmental pollution. Industrial application potential: After preliminary performance evaluation tests, it is proved that the acylase can significantly reduce costs and improve product quality in actual production, and is suitable for large-scale production. DETAILED DESCRIPTION

[0042] The embodiments are described in more detail by 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 is different 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] Gene synthesis was performed based on the reported maleylase, whose amino acid sequence is shown in Sequence 1 (aminoacylase-1isoform X1 [Equus caballus], XP_001492888.2), and the nucleic acid sequence of its cDNA coding region is shown in Sequence 2 (ACY-1, XM_001492838.5).

[0046] After artificially synthesizing the wild-type acylase cDNA coding region, the gene was used as a template, using primer pairs sequence 3 and sequence 4 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 25μL, ddH2O 20μl, sequence 3 primer 1.5μl, sequence 4 primer 1.5μl, sequence 2 template 2μl. PCR reaction program: after 98℃ pre-denaturation for 2min, repeat 98℃ denaturation for 10s, 58℃ annealing for 15s, 72℃ extension for 10s for a total of 25 cycles, and finally 72℃ final extension for 5min.

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

[0048] Only a DNA fragment of about 1.2 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. The purified product was double-digested with restriction enzymes NdeⅠ and XhoⅠ. After 1% agarose gel electrophoresis confirmed that the band size was about 1.2 kb, 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-cut fragment was obtained after purification and recovery.

[0050] Under the action of T4 DNA ligase, the above-obtained enzyme fragments of about 1.2 kb and about 5.3 kb were connected and transformed into Escherichia coli BL21 (DE3) to obtain transformant (1). Plasmids were prepared from the above-mentioned bacteria using the Tiangen plasmid mini-extraction kit to obtain recombinant plasmid pET-28a (+) -Acy. DNA sequencing confirmed that the nucleotide sequence of the coding region of the plasmid was consistent with sequence 2. 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 a LB liquid medium containing 50 μg / ml kanamycin resistance, and cultured at 37 ° C, 200 rpm for 12 hours, and then inoculated into a fresh LB liquid medium containing 50 μg / ml kanamycin resistance at a 1% (v / v) inoculation amount, and cultured at 37 ° C, 200 rpm until the bacterial OD600 reached 0.6-0.8, and IPTG with a final concentration of 0.1mM was added, and induced culture was carried out at 20 ° C, 200 rpm for 15 hours. Centrifuged at 4 ° C, 8000 rpm for 20 minutes, the supernatant was discarded, and the precipitate was collected to obtain the wet bacterial cells of the recombinant E. coli BL21 (DE3) / pAcy-pET-28a (+) containing the expression recombinant plasmid. The bacterial cells can be directly used as biocatalysts.

[0052] The wet cells obtained above were used to synthesize lauroylglycine and determine its hydrolysis activity. Synthesis reaction system (10 mL): 360 mg of lauric acid, 10 ml of saturated glycine solution, 0.2 g of wet cells. Reaction conditions: temperature 35-65°C, pH 5.5-8.5. Analysis conditions: chromatographic column: C18, 4.6 mm × 250 mm, 5 μm; detection wavelength: ultraviolet (UV) = 200 nm; mobile phase: 85% acetonitrile and 15% 0.05% trifluoroacetic acid aqueous solution; column temperature: 30°C; flow rate: 1 mL / min; detection time: 15 min.

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

[0054] Analyze sequence information, calculate PSSM matrix, observe amino acid conservation, and select less conservative 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 25μL, ddH2O 20μl, sequence 5 primer F 1.5μl, primer R 1.5μl, pET-28a(+)-Acy template 2μl. PCR reaction program: after 98℃ pre-denaturation for 2min, repeat 98℃ denaturation for 10s, 58℃ annealing for 15s, 72℃ extension for 40s for a total of 25 cycles, and finally 72℃ final extension for 5min.

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

[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] E. 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 measurement protocol were the same as in Example 1.

[0059] In order to obtain a transformant (3) expressing a mutant acylase having an amino acid residue substitution as shown in the table, the 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 mutation described in the table.

[0060] In order to obtain a transformant (4) expressing a mutant acylase having an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as above was performed to obtain a plasmid to obtain a transformant (4). The transformant (4) produced an acylase (4) having the mutation described in the table.

[0061] In order to obtain a transformant (5) expressing a mutant acylase having an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as above was performed to obtain a plasmid to obtain a transformant (5). The transformant (5) produced an acylase (5) having the mutation described in the table.

[0062] In order to obtain a transformant (6) expressing a mutant acylase having an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as above was performed to obtain a plasmid to obtain a transformant (6). The transformant (6) produced an acylase (6) having the mutation described in the table.

[0063] In order to obtain a transformant (7) expressing a mutant acylase having an amino acid residue substitution as shown in the table, the 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 mutation described in the table.

[0064] In order to obtain a transformant (8) expressing a mutant acylase having an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as above was performed to obtain a plasmid to obtain a transformant (8). The transformant (8) produced an acylase (8) having the mutation 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, the relative value of the synthetic conversion rate relative to the wild type is defined as a significant improvement of >1.5, 1.2-1.5 is an improvement, and 1-1.2 is a slight improvement. From the above table, it can be found that the conversion rate of the 44th amino acid mutation to Y, W, V is significantly improved, the mutation to K, G, L has an improvement, and the mutation to E has a slight improvement in activity. This point can be used as a dominant site for improving the 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 an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (9). The transformant (9) produced an acylase (9) having the mutation described in the table.

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

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

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

[0073]

[0074] From the above table, it can be found that 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 an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (12). The transformant (12) produced an acylase (12) having the mutation described in the table.

[0077] In order to obtain a transformant (13) expressing a mutant acylase having an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (13). The transformant (13) produced an acylase (13) having the mutation described in the table.

[0078] In order to obtain a transformant (14) expressing a mutant acylase having an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (14). The transformant (14) produced an acylase (14) having the mutation described in the table.

[0079] In order to obtain a transformant (15) expressing a mutant acylase having an amino acid residue substitution as 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 having the mutation described in the table (15)

[0080] In order to obtain a transformant (16) expressing a mutant acylase having an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (16). The transformant (16) produced an 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] From the above table, it can be found that the conversion rate is significantly improved when the amino acid at position 115 is mutated into E, there is an improvement when it is mutated into F, M, and L, 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.

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

[0085] In order to obtain a transformant (17) expressing a mutant acylase having an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (17). The transformant (17) produced an acylase (17) having the mutations described in the table.

[0086] In order to obtain a transformant (18) expressing a mutant acylase having an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain 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] It can be found from the above table 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 a dominant site for improving the conversion rate.

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

[0091] In order to obtain a transformant (19) expressing a mutant acylase having an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (19). The transformant (19) produced an acylase (19) having the mutations described in the table.

[0092] In order to obtain a transformant (20) expressing a mutant acylase having an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (20). The transformant (20) produced an acylase (20) having the mutation described in the table.

[0093] In order to obtain a transformant (21) expressing a mutant acylase having an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (21). The transformant (21) produced an acylase (21) having the mutation described in the table.

[0094] In order to obtain a transformant (22) expressing a mutant acylase having an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (22). The transformant (22) produced an 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] From the above table, it can be found that 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] In order to obtain a transformant (23) expressing a mutant acylase having an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (23). The transformant (23) produced an acylase (23) having the mutation described in the table.

[0100] In order to obtain a transformant (24) expressing a mutant acylase having an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (24). The transformant (24) produced an acylase (24) having the mutation described in the table.

[0101] In order to obtain a transformant (25) expressing a mutant acylase having an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (25). The transformant (25) produced an acylase (25) having the mutations described in the table.

[0102] In order to obtain a transformant (26) expressing a mutant acylase having an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (26). The transformant (26) produced an acylase (26) having the mutations described in the table.

[0103] In order to obtain a transformant (27) expressing a mutant acylase having an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (27). The transformant (27) produced an acylase (27) having the mutations described in the table.

[0104] In order to obtain a transformant (28) expressing a mutant acylase having an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (28). The transformant (28) produced an acylase (28) having the mutations described in the table.

[0105] In order to obtain a transformant (29) expressing a mutant acylase having an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain 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] From the above table, it can be found that 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] In order to obtain a transformant (30) expressing a mutant acylase having an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (30). The transformant (30) produced an acylase (30) having the mutation described in the table.

[0111] In order to obtain a transformant (31) expressing a mutant acylase having an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (31). The transformant (31) produced an acylase (31) having the mutation described in the table.

[0112] In order to obtain a transformant (32) expressing a mutant acylase having an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (32). The transformant (32) produced an acylase (32) having the mutation described in the table.

[0113] In order to obtain a transformant (33) expressing a mutant acylase having an amino acid residue substitution as shown in the table, a plasmid was obtained in the same manner as in Example 2, except that the primer of sequence number 36 was used instead of the primer of sequence number 5, thereby obtaining a transformant (33). The transformant (33) produced an acylase (33) having the mutation described in the table.

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

[0115]

[0116]

[0117] From the above table, it can be found that the conversion rate is significantly improved when the amino acid at position 205 is mutated to Q, and there is an improvement when it is mutated to S, L, and 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] In order to obtain a transformant (34) expressing a mutant acylase having an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (34). The transformant (34) produced an acylase (34) having the mutation described in the table.

[0120] In order to obtain a transformant (35) expressing a mutant acylase having an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (35). The transformant (35) produced an 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] It can be found from the above table that 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 a dominant site for improving the conversion rate.

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

[0125] In order to obtain a transformant (36) expressing a mutant acylase having an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (36). The transformant (36) produced an acylase (36) having the mutation described in the table.

[0126] In order to obtain a transformant (37) expressing a mutant acylase having an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (37). The transformant (37) produced an acylase (37) having the mutation described in the table.

[0127] In order to obtain a transformant (38) expressing a mutant acylase having an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (38). The transformant (38) produced an acylase (38) having the mutation described in the table.

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

[0129]

[0130] From the table above, it can be found that 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 the conversion rate.

[0131] Example 11. Obtaining acylase mutants (39) to (45) and activity determination

[0132] In order to obtain a transformant (39) expressing a mutant acylase having an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (39). The transformant (39) produced an acylase (39) having the mutation described in the table.

[0133] In order to obtain a transformant (40) expressing a mutant acylase having an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (40). The transformant (40) produced an acylase (40) having the mutation described in the table.

[0134] In order to obtain a transformant (41) expressing a mutant acylase having an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (41). The transformant (41) produced an acylase (41) having the mutation described in the table.

[0135] In order to obtain a transformant (42) expressing a mutant acylase having an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (42). The transformant (42) produced an acylase (42) having the mutation described in the table.

[0136] In order to obtain a transformant (43) expressing a mutant acylase having an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (43). The transformant (43) produced an acylase (43) having the mutation described in the table.

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

[0138] In order to obtain a transformant (45) expressing a mutant acylase having an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (45). The transformant (45) produced an 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] From the above table, it can be found that the conversion rate is significantly improved when the amino acid at position 288 is mutated into R, M, and V, and there is an improvement when it is mutated into F, Q, and T, and there is a slight improvement 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 activity determination

[0143] In order to obtain a transformant (46) expressing a mutant acylase having an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (46). The transformant (46) produced an acylase (46) having the mutation described in the table.

[0144] In order to obtain a transformant (47) expressing a mutant acylase having an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (47). The transformant (47) produced an acylase (47) having the mutation described in the table.

[0145] In order to obtain a transformant (48) expressing a mutant acylase having an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (48). The transformant (48) produced an acylase (48) having the mutation described in the table.

[0146] In order to obtain a transformant (49) expressing a mutant acylase having an amino acid residue substitution as shown in the table, a plasmid was obtained in the same manner as in Example 2, except that the primer of sequence number 52 was used instead of the primer of sequence number 5, thereby obtaining a transformant (49). The transformant (49) produced an acylase (49) having the mutations described in the table.

[0147] In order to obtain a transformant (50) expressing a mutant acylase having an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (50). The transformant (50) produced an acylase (50) having the mutation described in the table.

[0148] In order to obtain a transformant (51) expressing a mutant acylase having an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (51). The transformant (51) produced an acylase (51) having the mutation 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 to G or N, and there is an improvement when it is mutated to 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 activity determination

[0153] In order to obtain a transformant (52) expressing a mutant acylase having an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (52). The transformant (52) produced an acylase (52) having the mutation described in the table.

[0154] In order to obtain a transformant (53) expressing a mutant acylase having an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (53). The transformant (53) produced an 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] It can be found from the above table that 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 a dominant site for improving the conversion rate.

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

[0159] In order to obtain a transformant (54) expressing a mutant acylase having an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (54). The transformant (54) produced an acylase (54) having the mutation described in the table.

[0160] In order to obtain a transformant (55) expressing a mutant acylase having an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (55). The transformant (55) produced an acylase (55) having the mutation described in the table.

[0161] In order to obtain a transformant (56) expressing a mutant acylase having an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (56). The transformant (56) produced an acylase (56) having the mutation described in the table.

[0162] In order to obtain a transformant (57) expressing a mutant acylase having an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (57). The transformant (57) produced an acylase (57) having the mutations described in the table.

[0163] In order to obtain a transformant (58) expressing a mutant acylase having an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (58). The transformant (58) produced an acylase (58) having the mutation described in the table.

[0164] In order to obtain a transformant (59) expressing a mutant acylase having an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (59). The transformant (59) produced an acylase (59) having the mutation described in the table.

[0165] In order to obtain a transformant (60) expressing a mutant acylase having an amino acid residue substitution as shown in the table, corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (60). The transformant (60) produced an acylase (60) having the mutation described in the table.

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

[0167]

[0168] From the above table, it can be found that the conversion rate is significantly improved when the amino acid at position 328 is mutated into M, V, I, and L, and there is an improvement when it is mutated into Q, and there is a slight improvement in activity when it is mutated into D and 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 activity determination

[0170] In order to obtain a transformant (61) expressing a mutant acylase having an amino acid residue substitution as shown in the table, the vector obtained by plasmid extraction of the transformant (8) was used to replace the original cloning plasmid parent, and the corresponding primers were designed and the plasmid was obtained in the same manner as in Example 2 to obtain a transformant (61). The transformant (61) produced an acylase (61) having the mutations described in the table.

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

[0172] In order to obtain a transformant (62) expressing a mutant acylase having an amino acid residue substitution as shown in the table, the vector obtained by plasmid extraction from the transformant (8) was used to replace the original cloning plasmid parent, and the corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (62). The transformant (62) produced an 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 superimposed multi-point mutations, the relative value of the synthetic conversion rate relative to the parent transformant is defined as a significant improvement when it is >1.3, an improvement when it is 1.1-1.3, and a slight improvement when it is 1-1.1. From the above table, it can be found that the conversion rate is significantly improved when the amino acids at positions 44 and 65 are mutated to W and Y, respectively. The combination of these two points can improve the conversion rate compared to a single point.

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

[0177] In order to obtain a transformant (63) expressing a mutant acylase having an amino acid residue substitution as shown in the table, the same operation as in Example 2 was performed except that the vector obtained by plasmid extraction from the transformant (7) was used to replace the original cloning plasmid parent and the primer of sequence number 10 was used instead of the primer of sequence number 5 to obtain a plasmid to obtain a transformant (63). The transformant (63) produced an acylase (63) having the mutation described in the table.

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

[0179]

[0180] From the above table, it can be found that the conversion rate is slightly improved when the amino acids at positions 44 and 65 are mutated to L and Y respectively, indicating that the combined mutation of overlapping points is not limited to a certain mutation form, and the combination of the two points can improve the conversion rate compared with a single point.

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

[0182] In order to obtain a transformant (64) expressing a mutant acylase having an amino acid residue substitution as shown in the table, the vector obtained by plasmid extraction of the transformant (3) was used to replace the original cloning plasmid parent, and the corresponding primers were designed and the plasmid was obtained in the same manner as in Example 2 to obtain a transformant (64). The transformant (64) produced an acylase (64) having the mutations described in the table.

[0183] In order to obtain a transformant (65) expressing a mutant acylase having an amino acid residue substitution as shown in the table, the vector obtained by plasmid extraction of the transformant (3) was used to replace the original cloning plasmid parent, and the corresponding primers were designed and the plasmid was obtained in the same manner as in Example 2 to obtain a transformant (65). The transformant (65) produced an acylase (65) having the mutations described in the table.

[0184] In order to obtain a transformant (66) expressing a mutant acylase having an amino acid residue substitution as shown in the table, the vector obtained by plasmid extraction of the transformant (3) was used to replace the original cloning plasmid parent, and the corresponding primers were designed and the plasmid was obtained in the same manner as in Example 2 to obtain a transformant (66). The transformant (66) produced an acylase (66) having the mutations described in the table.

[0185] In order to obtain a transformant (67) expressing a mutant acylase having an amino acid residue substitution as shown in the table, the vector obtained by plasmid extraction of the transformant (3) was used to replace the original cloning plasmid parent, and the corresponding primers were designed and the plasmid was obtained in the same manner as in Example 2 to obtain a transformant (67). The transformant (67) produced an 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] From the above table, it can be found that the conversion rate is significantly improved when the amino acids at positions 44 and 115 are mutated into E, E, or E, M, respectively, indicating that the overlapping point combination is not limited to a specific site, and the two-site combination can improve the conversion rate compared with the single-site mutation.

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

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

[0191] In order to obtain a transformant (68) expressing a mutant acylase having an amino acid residue substitution as shown in the table, the vector obtained by plasmid extraction of the transformant (62) was used to replace the original cloning plasmid parent, and the corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (68). The transformant (68) produced an acylase (68) having the mutations described in the table.

[0192] In order to obtain a transformant (69) expressing a mutant acylase having an amino acid residue substitution as shown in the table, the vector obtained by plasmid extraction from the transformant (62) was used to replace the original cloning plasmid parent, and the corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (69). The transformant (69) produced an acylase (69) having the mutations described in the table.

[0193] In order to obtain a transformant (70) expressing a mutant acylase having an amino acid residue substitution as shown in the table, the vector obtained by plasmid extraction from the transformant (62) was used to replace the original cloning plasmid parent, and the corresponding primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (70). The transformant (70) produced an 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] From the above table, it can be found that 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 activity determination

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

[0199] In order to obtain a transformant (72) expressing a mutant acylase having an amino acid residue substitution as shown in the table, the vector obtained by plasmid extraction from the transformant (20) was used to replace the original cloning plasmid parent, and the corresponding primers were designed to amplify the corresponding fragments. The plasmid was obtained by the same operation as above to obtain a transformant (72). The transformant (72) produced an 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] From the above table, it can be found that the conversion rate is significantly improved when the amino acids at positions 173, 205, and 259 are mutated to M, S, E or M, S, A, respectively. This three-point combination can improve the conversion rate compared to single-point mutation.

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

[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] In order to obtain a transformant (73) expressing a mutant acylase having an amino acid residue substitution as shown in the table, the vector obtained by plasmid extraction from the transformant (67) was used to replace the original cloning plasmid parent, and the corresponding primers were designed to amplify the corresponding fragments, and the plasmid was obtained by the same operation as in Example 19 to obtain a transformant (73). The transformant (73) produced an acylase (73) having the mutations described in the table.

[0206] In order to obtain a transformant (74) expressing a mutant acylase having an amino acid residue substitution as shown in the table, the vector obtained by plasmid extraction from the transformant (67) was used to replace the original cloning plasmid parent, and the corresponding primers were designed to amplify the corresponding fragments. The plasmid was obtained by the same operation as in Example 19 to obtain a transformant (74). The transformant (74) produced an acylase (74) having the mutations described in the table.

[0207] In order to obtain a transformant (75) expressing a mutant acylase having an amino acid residue substitution as shown in the table, the vector obtained by plasmid extraction from the transformant (67) was used to replace the original cloning plasmid parent, and the corresponding primers were designed to amplify the corresponding fragments. The plasmid was obtained by the same operation as in Example 19 to obtain a transformant (75). The transformant (75) produced an acylase (75) having the mutations described in the table.

[0208] In order to obtain a transformant (76) expressing a mutant acylase having an amino acid residue substitution as shown in the table, the vector obtained by plasmid extraction of the transformant (67) was used to replace the original cloning plasmid parent, and the corresponding primers were designed to amplify the corresponding fragments. The plasmid was obtained by the same operation as in Example 19 to obtain a transformant (76). The transformant (76) produced an 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 the amino acids at positions 44, 115, 328, and 252 are mutated to W, E, A, and Q, or the amino acids at positions 44, 65, 151, and 173 are mutated to W, Y, A, and M, or the amino acids at positions 44, 65, 151, and 307 are mutated to W, Y, A, and G, or the 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 the two-point mutation.

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

[0213] It can be seen from acylase mutants 73 to 76 that the combined mutation of 4 sites is helpful to improve the conversion rate, so more site mutations are superimposed.

[0214] In order to obtain a transformant (77) expressing a mutant acylase having an amino acid residue substitution as shown in the table, the vector obtained by plasmid extraction from the transformant (73) was used to replace the original cloning plasmid parent, and the corresponding primers were designed to amplify the corresponding fragments. The plasmid was obtained by the same operation as in Example 19 to obtain a transformant (77). The transformant (77) produced an acylase (77) having the mutations described in the table.

[0215] In order to obtain a transformant (78) expressing a mutant acylase having an amino acid residue substitution as shown in the table, the vector obtained by plasmid extraction from the transformant (73) was used to replace the original cloning plasmid parent, and the corresponding primers were designed to amplify the corresponding fragments. The plasmid was obtained by the same operation as in Example 19 to obtain a transformant (78). The transformant (78) produced an acylase (78) having the mutations described in the table.

[0216] In order to obtain a transformant (79) expressing a mutant acylase having an amino acid residue substitution as shown in the table, the vector obtained by plasmid extraction from the transformant (73) was used to replace the original cloning plasmid parent, and the corresponding primers were designed to amplify the corresponding fragments, and the plasmid was obtained by the same operation as in Example 19 to obtain a transformant (79). The transformant (79) produced an acylase (79) having the mutations described in the table.

[0217] In order to obtain a transformant (80) expressing a mutant acylase having an amino acid residue substitution as shown in the table, the vector obtained by plasmid extraction from the transformant (73) was used to replace the original cloning plasmid parent, and the corresponding primers were designed to amplify the corresponding fragments, and the plasmid was obtained by the same operation as in Example 19 to obtain a transformant (80). The transformant (80) produced an 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 the 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 the amino acids at positions 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 the four-point mutation.

Claims

1. A horse acylase mutant, characterized in that: It is a mutant having one, two, three, four, five, six or more amino acid residue mutations selected from the following positions based on the wild-type acylase with the amino acid sequence as shown in SEQ ID NO.1: 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.

2. The acylase mutant according to claim 1, characterized in that There are mutants with mutations in the amino acid residues at the following two positions: Q44W, W65E; Q44W, W65Y; Q44L, W65Y; Q44E, K115R; Q44E, K115M; Q44E, K115L; Q44E, K115E.

3. The acylase mutant according to claim 1, characterized in that There are mutants with mutations in amino acid residues at the following three positions: Q44W, W65Y, G151A; Q44W, W65Y, G151S; Q44W, W65Y, S328M; L173M, G205S, G259E; L173M, G205S, G259A.

4. The acylase mutant according to claim 1, characterized in that There are mutants with amino acid residue mutations at the following four positions: Q44W, W65Y, G151A, L173M; Q44W, W65Y, G151A, Q307G; Q44W, K115E, S328M, W323Y; Q44W, K115E, S328A, N252Q; Or there are mutants with mutations in amino acid residues at the following five positions: Q44W, W65Y, G151A, L173M, G205S, G259E; Q44W, W65Y, G151A, L173M, G205S, G259A; Q44W, W65Y, G151A, L173M, G205S, G259L; Q44W, W65Y, G151A, L173M, G205S, E288R.

5. A nucleic acid encoding the acylase mutant according to any one of claims 1 to 4.

6. A vector containing the encoding nucleic acid as claimed in claim 5, such as an expression vector, more specifically, the starting vector is a pET vector.

7. The genetically engineered bacteria of the encoding nucleic acid according to claim 5 or the vector according to claim 6 are, for example, bacteria or fungi, and specifically, the starting bacteria are Escherichia coli, Bacillus subtilis or Pichia pastoris.

8. Use of the acylase mutant according to any one of claims 1 to 4, its encoding nucleic acid, or a vector or genetically engineered bacteria containing the encoding nucleic acid in biocatalytic synthesis of lauroylglycine.

9. A method for preparing lauroyl glycine, characterized in that: The method comprises using the acylase mutant according to any one of claims 1 to 4 as a biocatalyst, and using lauric acid and amino acids as substrates to form a reaction system to generate lauroylglycine; Specifically, the wet cell, crude enzyme solution or pure enzyme obtained by fermentation culture of the acylase mutant or the genetically engineered bacteria is used as a biocatalyst. Preferably, the genetically engineered bacteria is used as a whole cell catalyst in the form of wet cell.

10. The preparation method according to claim 9, characterized in that: In the reaction system, the dosage 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%; The reaction conditions are: 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; More specifically, the wet bacteria 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 kanamycin resistance, and cultured at 37°C and 200 rpm for 12 hours; then inoculated into a fresh LB liquid culture medium containing 50 μg / ml kanamycin resistance at a 1% (v / v) inoculation amount, and cultured at 37°C and 200 rpm until the OD600 of the bacteria reaches 0.6-0.8; IPTG with a final concentration of 0.1 mM is added, and the culture is induced at 20°C and 200 rpm for 15 hours; then the culture is centrifuged at 4°C and 8000 rpm for 20 minutes, the supernatant is discarded, and the precipitate is collected to obtain the wet bacteria.

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