Horse-derived acylase mutant and application thereof in synthesis of lauroyl glycine

Abstract

The invention belongs to the technical field of biology, and discloses a horse-derived acylase mutant and application thereof in synthesis of lauroyl glycine. 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 production enzyme, 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 in synthesizing lauroylglycine. Background Art

[0002] Amino acid surfactants not only have a wide range of biomass raw materials, few toxic side effects, mild performance, low irritation, and foaming properties, but also often have the advantages of good surface activity, health and safety, and strong antibacterial ability. Therefore, they have attracted much attention and are gradually used in many fields such as washing, personal care, and food industry.

[0003] N-acyl amino acid surfactants are the main amino acid surfactants. Common N-acyl amino acid surfactants include sodium lauroyl glycinate, sodium lauroyl sarcosinate, sodium lauroyl glutamate, potassium lauroyl glutamate, sodium cocoyl glutamate, sodium cocoyl glycinate, potassium cocoyl glycinate, etc. Its synthetic raw materials come from various acidic, basic or neutral amino acids such as aspartic acid, glutamic acid, arginine, alanine, glycine, leucine, proline, serine and protein hydrolysates. Hydrophobic ends such as fatty acids or alkyl chlorides can be connected to the amino group, a-COOH or side chain group of amino acids. If fatty acids or alkyl halides react with amino groups, corresponding N-acyl or N-alkyl amino acid derivatives are produced respectively; if fatty amines or fatty alcohols condense with carboxyl groups, N-alkyl or O-alkyl ester amino acid derivatives are obtained respectively; different reaction modes give different varieties of products, so amino acid surfactants have chemical structure diversity and diversity of physical, chemical and biological properties.

[0004] The synthesis methods of N-acylamino acid surfactants are divided into direct and indirect methods. Indirect synthesis methods include fatty acid chloride acylation, fatty nitrile hydrolysis acylation, fatty acid anhydride acylation, amide carbonylation reaction, etc. Among them, the Schotten-Baumann condensation method, in which fatty acid chlorides replace fatty acids and react with amino acids in alkaline solution, is currently the most widely used synthesis method in laboratories or industries. It has the advantages of relatively low equipment requirements, low-cost and easy-to-obtain raw materials, mild reaction conditions, and easy to handle by-products. However, since it involves the use of phosgene to convert free fatty acids into corresponding acyl chlorides during the production process, 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 atomic economy. Direct synthesis of fatty acid raw materials includes enzyme-catalyzed synthesis and dehydration condensation. Among them, lipase-catalyzed synthesis is subject to factors such as low conversion rate, long reaction time and expensive enzyme preparations; while dehydration condensation is subject to factors such as harsh reaction conditions, high equipment requirements and high energy consumption, and needs to be improved. Therefore, there is an urgent need to develop a novel synthesis scheme for N-acyl amino acid surfactants / a series of novel N-acyl amino acid surfactants to provide a more convenient and efficient new route for laboratory / industrial production of N-acyl amino acid surfactants.

[0005] Aminoacylase I (ACY1) has thus entered the public eye. ACY1 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. Its physiological function is believed to be to rescue metabolic amino acids and inhibit the growth of cancer cells. It is usually used in the industry to split amino acids. Because it can also catalyze the production of N-acyl amino acids in non-aqueous phases, it plays an important role in biosynthesis and metabolism. The mechanism of using it to synthesize lauroylglycine is as follows:

[0006]

[0007] However, although ACY1 has catalytic advantages for the above synthetic reactions, it is often limited by the difficulty of reversible reactions to proceed unidirectionally, resulting in product hydrolysis, low enzyme catalytic efficiency, low enzyme stability, inability to continue reaction or difficulty in recovery, resulting in low synthetic yields. Therefore, there are still few studies on this type of reaction on the market. At present, one of the main sources of industrial ACY1 is extracted from pig kidneys. Although it has been found that ACY1 from sheep, humans, dogs, etc. has a certain catalytic effect on acylation reactions, there are still only a few related research reports, which inevitably leads to the disadvantages of single source of ACY1 and low richness. Therefore, by blasting the ACY1 sequences from humans, pigs, etc., and checking the sequence similarities one by one, the horse ACY1 with higher similarity was selected for research. It is hoped that on the basis of the acylation function of ACY1 from humans and pigs, mutation sites can be introduced to make it have a certain catalytic efficiency, providing a new selection direction for the ACY1 catalytic synthesis pathway of lauroylglycine. Summary of the invention

[0008] The present invention is based on the maleylase synthetic gene pAcy obtained in previous experiments, and is modified to obtain a mutant with improved activity. Thus, the present invention is proposed.

[0009] The present invention first provides a horse acylase mutant, which is a mutant having one, two, three, four, five, six or more amino acid residue mutations selected from the following sites based on the wild-type acylase with an amino acid sequence as shown in SEQ ID NO.1: T24Y, V35E, E40K, Q50G, A102C, T181N, S224G, S292A, Q160E, T14Y, K284L, V306E, S320A, K232D, A166S, A130S, T368H, L395S.

[0010] Specifically, there are mutants in which the amino acid residues are mutated at the following two positions: E40K, A102C; E40K, T181N; E40K, S224G; E40K, S292A; A102C, T181N; A102C, S224G; A102C, S292A; T181N, S224G; T181N, S292A; S224G, S292A.

[0011] In a specific embodiment, there are mutants with mutations in amino acid residues at the following three positions: E40K, T181N, S224G; E40K, T181N, S292A; A102C, T181N, S224G; A102C, T181N, S292A; T181N, S224G, S292A; E40K, T181N, S292A; E40K, T181N, S224G; S292A, E40K, T181N.

[0012] In a further embodiment, there are mutants with amino acid residue mutations at the following four positions: E40K, T181N, S224G, S292A; E40K, T181N; S224G, S292A;

[0013] Or mutants with amino acid residue mutations at the following five positions: E40K, T181N, S224G, S292A, T14Y

[0014] Or mutants with amino acid residue mutations at the following six positions: E40K, T181N, S224G, S292A, T14Y, K284L; E40K, T181N, S224G, S292A, T14Y, V306E; E40K, T181N, S224G, S292A, Q160E, K284L; E40K, T181N, S224G, S292A, Q160E, S320A;

[0015] Or mutants with amino acid residue mutations at the following seven positions: E40K, T181N, S224G, S292A, T14Y, Q160E, S320A; E40K, T181N, S224G, S292A, Q160E, K232D, S320A; E40K, T181N, S224G, S292A, K232D, V 306E, S320A; E40K, T181N, S224G, S292A, Q160E, K284L, V306E; E40K, T181N, S224 G, S292A, Q160E, K284L, S320A; E40K, T181N, S224G, S292A, K284L, V306E, S320A;

[0016] Or mutants with eight amino acid residue mutations: E40K, T181N, S224G, S292A, T14Y, Q160E, K232D, K284L; E40K, T181N, S224G, S292A, T14Y, Q160E, K232D, V306E; E40K, T181N, S224G, S292A, T14Y, Q160E, K232D, V306E 284L, V306E; E40K, T181N, S224G, S292A, T14Y, Q160E, K284L, S320A; E40K, T181N, S224G, S 292A, Q160E, K284L, V306E, S320A; E40K, T181N, S224G, S292A, T14Y, Q160E, K284L, V306E,

[0017] Or mutants with mutations in the following nine amino acid residues: E40K, T181N, S224G, S292A, T14Y, Q160E, A166S, V306E, S320A; E40K, T181N, S224G, S292A, T14Y, Q160E, A166S, V306E, S320A;

[0018] Or mutants with amino acid residue mutations at the following ten positions: E40K, T181N, S224G, S292A, T14Y, Q160E, A166S, K284L, V306E, S320A;

[0019] Or mutants with amino acid residue mutations at the following eleven positions: E40K, T181N, S224G, S292A, T14Y, A130S, Q160E, A166S, K284L, V306E, S320A;

[0020] Or mutants with amino acid residue mutations at the following twelve positions: E40K, T181N, S224G, S292A, T14Y, A130S, Q160E, A166S, K284L, V306E, S320A, T368H; E40K, T181N, S224G, S292A, T14Y, Q40G, A130S, Q160E, A166S, K284L, V306E, T368H; E40K, T181N, S224G, S292A, T14Y, V35E, Q160E, A166S, K284L, V306E, S320A, L395S; E40K, T181N, S224G, S292A, V35E, A130S, Q160E, A166S, K284L, V306E, T368H, L395S.

[0021] The present invention provides a gene encoding the acylase mutant.

[0022] The present invention also provides a recombinant expression vector containing the coding gene, specifically in the form of a pET vector framework.

[0023] The present invention provides a genetically engineered bacterium containing the coding gene or the recombinant expression vector, such as Escherichia coli.

[0024] The present invention further provides the use of the acylase mutant or its encoding nucleic acid in the preparation of lauroylglycine, specifically in catalyzing the conversion of lauric acid into lauroylglycine.

[0025] The present invention also provides a method for preparing lauroyl glycine, which comprises using the genetically engineered bacteria as a whole-cell catalyst, and using lauric acid and glycine as substrates to synthesize lauroyl glycine;

[0026] The method of obtaining the whole-cell catalyst specifically comprises the following steps: centrifuging the fermented bacterial solution, discarding the supernatant, and collecting the precipitate to obtain wet bacterial cells;

[0027] The reaction conditions are: the dosage 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%; the reaction conditions are: the biocatalytic reaction is carried out under the conditions of temperature 35-65°C, pH 5.5-8.5, and stirring speed 100-300 rpm to obtain lauroyl glycine.

[0028] The present invention provides a horse-derived acylase mutant, whose catalytic activity is significantly improved compared with that of a wild-type acylase. The wet bacterial cells obtained by fermentation and culturing a recombinant bacterium containing a mutant coding gene of the acylase are used as a biocatalyst, and lauric acid and glycine are used as substrates to synthesize a lauroylglycine surfactant, thereby greatly improving production efficiency and having high production application value. DETAILED DESCRIPTION

[0029] The embodiments are described in more detail by the following examples, but the present invention is not limited by the following examples. It should be noted that the mutation site in the following examples refers to the position of the difference in amino acid sequence from the mutant acylase derived from horse containing the amino acid sequence with SEQ ID NO: 1.

[0030] Comparative Example 1

[0031] Based on the maleylase sequence obtained in previous experiments, the gene pAcy was synthesized, the amino acid sequence of which is shown in Sequence 1, and the nucleic acid sequence of its cDNA coding region is shown in Sequence 2.

[0032] 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.

[0033] The PCR product was analyzed by 1% agarose gel electrophoresis, and the presence of an amplified product of about 1.2 kb was confirmed.

[0034] 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.

[0035] 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.

[0036] 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 then 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) as a mutant acylase derived from horse.

[0037] 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.

[0038] The wet cells obtained above were used to test the lauroylglycine synthesis activity. Synthesis reaction system (10 mL): 360 mg of lauric acid, 10 ml of saturated glycine solution, 0.2 g of wet cells. Reaction time 14 h.

[0039] Reaction conditions: temperature 35-65°C, pH 5.5-8.5.

[0040] 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.

[0041] Comparative Example 2

[0042] Candidate modification sites are introduced through analysis of sequence conservation and random mutation. The mutation risk of low-conservation sites is unknown, and the sites cannot be superimposed one by one. Therefore, it is necessary to first perform single-point conversion rate screening, eliminate low-conversion rate sites, retain high-conversion rate advantage mutation sites, and then combine and superimpose, and introduce new mutation sites during the superposition process, and perform multiple rounds of evolutionary screening.

[0043] After artificially synthesizing the recombinant plasmid pET-28a(+)-Acy, the plasmid was used as a template and primer pair sequence 5 was used to amplify the fragment by PCR to introduce the desired mutation site into the transformant (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.

[0044] The PCR product was analyzed by 1% agarose gel electrophoresis, and the presence of an amplified product of about 6.5 kb was confirmed.

[0045] 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.

[0046] Escherichia coli BL21(DE3) was transformed to obtain a transformant (2). The transformant (2) produced the mutant (2) which was a dominant mutant acylase derived from horse.

[0047] The transformant (2) produced the acylase (2) having the mutations described in the table based on Comparative Example 1, and its culture and activity measurement scheme were the same as those of Comparative Example 1.

[0048] [Example 1] Obtaining mutant acylase (3)

[0049] In order to obtain a transformant (3) expressing a mutant acylase having an amino acid residue substitution as shown in the table, appropriate primers were designed, and the plasmid was obtained in the same manner as in Comparative Example 2 to obtain a transformant (3). The transformant (3) produced an acylase (3) having the mutation described in the table.

[0050] The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1. The results are shown in Table 1.

[0051] [Example 2] Obtaining mutant acylase (4)

[0052] In order to obtain a transformant (4) expressing a mutant acylase having an amino acid residue substitution as shown in the table, appropriate primers were designed and the plasmid was obtained in the same manner as in Comparative Example 2 to obtain a transformant (4). The transformant (4) produced an acylase (4) having the mutation described in the table.

[0053] The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1. The results are shown in Table 1.

[0054] [Example 3] Obtaining mutant acylase (5)

[0055] In order to obtain a transformant (5) expressing a mutant acylase having an amino acid residue substitution as shown in the table, appropriate primers were designed, and the plasmid was obtained in the same manner as in Comparative Example 2 to obtain a transformant (5). The transformant (5) produced an acylase (5) having the mutation described in the table.

[0056] The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1. The results are shown in Table 1.

[0057] [Example 4] Obtaining mutant acylase (6)

[0058] In order to obtain a transformant (6) expressing a mutant acylase having an amino acid residue substitution as shown in the table, appropriate primers were designed, and the plasmid was obtained in the same manner as in Comparative Example 2 to obtain a transformant (6). The transformant (6) produced an acylase (6) having the mutation described in the table.

[0059] The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1. The results are shown in Table 1.

[0060] [Example 5] Obtaining mutant acylase (7)

[0061] In order to obtain a transformant (7) expressing a mutant acylase having an amino acid residue substitution as shown in the table, appropriate primers were designed, and the plasmid was obtained in the same manner as in Comparative Example 2 to obtain a transformant (7). The transformant (7) produced an acylase (7) having the mutation described in the table.

[0062] The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1. The results are shown in Table 1.

[0063] Table 1

[0064]

[0065] For superimposed point mutations, the relative value of the synthetic conversion rate relative to the wild type is defined as >1.5 as a significant improvement, 1.2-1.5 as an improvement, 1-1.2 as a slight improvement, 0.8-1 as a slight decrease, 0.5-0.8 as a decrease, and <0.5 as a sharp decrease. It can be found from Table 1 that the conversion rate of the amino acid 24 of Comparative Example 2 mutated to Y is sharply reduced, proving that not all low-conservative sites can be used for the modification site of the conversion rate improvement. This point is not conducive to evolution and is eliminated; mutations T181N and S224G have improvements, and S292A, E40K, and A102C have slight improvements. These mutations can be used as advantageous sites for improving the conversion rate.

[0066] [Example 6] Obtaining mutant acylase (8)

[0067] In order to obtain a transformant (8) expressing a mutant acylase having an amino acid residue substitution as shown in the table, a vector obtained by plasmid extraction from the transformant (1) was used as a cloning parent, and appropriate primers were designed. The plasmid was obtained by the same operation as in Comparative Example 2 to obtain a transformant (8). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 2.

[0068] [Example 7] Obtaining mutant acylase (9)

[0069] In order to obtain a transformant (9) expressing a mutant acylase having an amino acid residue substitution as shown in the table, a vector obtained by plasmid extraction from the transformant (1) was used as a cloning parent, and appropriate primers were designed. The plasmid was obtained by the same operation as in Comparative Example 2 to obtain a transformant (9). The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1, and the results are shown in Table 2.

[0070] The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1. The results are shown in Table 2.

[0071] [Example 8] Obtaining mutant acylase (10)

[0072] In order to obtain a transformant (10) expressing a mutant acylase having an amino acid residue substitution as shown in the table, a vector obtained by plasmid extraction from the transformant (1) was used as a cloning parent, and appropriate primers were designed. The plasmid was obtained by the same operation as in Comparative Example 2 to obtain a transformant (10). The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1, and the results are shown in Table 2.

[0073] The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1. The results are shown in Table 2.

[0074] [Example 9] Obtaining mutant acylase (11)

[0075] In order to obtain a transformant (11) expressing a mutant acylase having an amino acid residue substitution as shown in the table, a vector obtained by plasmid extraction from the transformant (1) was used as a cloning parent, and appropriate primers were designed. The plasmid was obtained by the same operation as in Comparative Example 2 to obtain a transformant (11). The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1, and the results are shown in Table 2.

[0076] The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1. The results are shown in Table 2.

[0077] [Example 10] Obtaining mutant acylase (11)

[0078] In order to obtain a transformant (11) expressing a mutant acylase having an amino acid residue substitution as shown in the table, a vector obtained by plasmid extraction from the transformant (1) was used as a cloning parent, and appropriate primers were designed. The plasmid was obtained by the same operation as in Comparative Example 2 to obtain a transformant (11). The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1, and the results are shown in Table 2.

[0079] The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1. The results are shown in Table 2.

[0080] [Example 11] Obtaining mutant acylase (12)

[0081] In order to obtain a transformant (12) expressing a mutant acylase having an amino acid residue substitution as shown in the table, a vector obtained by plasmid extraction from the transformant (1) was used as a cloning parent, and appropriate primers were designed. The plasmid was obtained by the same operation as in Comparative Example 2 to obtain a transformant (12). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 2.

[0082] The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1. The results are shown in Table 2.

[0083] [Example 11] Obtaining mutant acylase (13)

[0084] In order to obtain a transformant (13) expressing a mutant acylase having an amino acid residue substitution as shown in the table, a vector obtained by plasmid extraction from the transformant (1) was used as a cloning parent, and appropriate primers were designed. The plasmid was obtained by the same operation as in Comparative Example 2 to obtain a transformant (13). The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1, and the results are shown in Table 2.

[0085] The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1. The results are shown in Table 2.

[0086] [Example 12] Obtaining mutant acylase (14)

[0087] In order to obtain a transformant (14) expressing a mutant acylase having an amino acid residue substitution as shown in the table, a vector obtained by plasmid extraction from the transformant (1) was used as a cloning parent, and appropriate primers were designed. The plasmid was obtained by the same operation as in Comparative Example 2 to obtain a transformant (14). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 2.

[0088] The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1. The results are shown in Table 2.

[0089] [Example 13] Obtaining mutant acylase (15)

[0090] In order to obtain a transformant (15) expressing a mutant acylase having an amino acid residue substitution as shown in the table, a vector obtained by plasmid extraction from the transformant (1) was used as a cloning parent, and appropriate primers were designed. The plasmid was obtained by the same operation as in Comparative Example 2 to obtain a transformant (15). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 2.

[0091] The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1. The results are shown in Table 2.

[0092] [Example 14] Obtaining mutant acylase (16)

[0093] In order to obtain a transformant (16) expressing a mutant acylase having an amino acid residue substitution as shown in the table, a vector obtained by plasmid extraction from the transformant (1) was used as a cloning parent, and appropriate primers were designed. The plasmid was obtained by the same operation as in Comparative Example 2 to obtain a transformant (16). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 2.

[0094] The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1. The results are shown in Table 2.

[0095] [Example 15] Obtaining mutant acylase (17)

[0096] In order to obtain a transformant (17) expressing a mutant acylase having amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from the transformant (1) was used as a cloning parent, and appropriate primers were designed. The plasmid was obtained by the same operation as in Comparative Example 2 to obtain a transformant (17). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 2.

[0097] The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1. The results are shown in Table 2.

[0098] Table 2

[0099]

[0100]

[0101] It can be found from Table 2 that the related mutations at sites 40 and 102 have a slight improvement, the combination of sites 181, 224 and 292 is more helpful in improving the conversion rate, and the combination of sites 181 and 224 has a significant improvement. These mutations can be used as advantageous sites for improving the conversion rate.

[0102] [Example 16] Obtaining mutant acylase (18)

[0103] In order to obtain a transformant (18) expressing a mutant acylase having amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from transformant (5) was used as a cloning parent, and appropriate primers were designed. The plasmid was obtained by the same operation as in Comparative Example 2 to obtain a transformant (18). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 3.

[0104] The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1. The results are shown in Table 3.

[0105] [Example 17] Obtaining mutant acylase (19)

[0106] In order to obtain a transformant (19) expressing a mutant acylase having amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from transformant (5) was used as a cloning parent, appropriate primers were designed, and a plasmid was obtained by the same operation as in Comparative Example 2 to obtain a transformant (19). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 3.

[0107] The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1. The results are shown in Table 3.

[0108] [Example 18] Obtaining mutant acylase (20)

[0109] In order to obtain a transformant (20) expressing a mutant acylase having an amino acid residue substitution as shown in the table, a vector obtained by plasmid extraction from the transformant (5) was used as a cloning parent, and appropriate primers were designed. The plasmid was obtained by the same operation as in Comparative Example 2 to obtain a transformant (20). The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1, and the results are shown in Table 3.

[0110] The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1. The results are shown in Table 3.

[0111] [Example 19] Obtaining mutant acylase (21)

[0112] In order to obtain a transformant (21) expressing a mutant acylase having amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from transformant (5) was used as a cloning parent, and appropriate primers were designed. The plasmid was obtained by the same operation as in Comparative Example 2 to obtain a transformant (21). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 3.

[0113] The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1. The results are shown in Table 3.

[0114] [Example 20] Obtaining mutant acylase (22)

[0115] In order to obtain a transformant (22) expressing a mutant acylase having amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from transformant (5) was used as a cloning parent, appropriate primers were designed, and a plasmid was obtained by the same operation as in Comparative Example 2 to obtain a transformant (22). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 3.

[0116] The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1. The results are shown in Table 3.

[0117] Table 3

[0118]

[0119]

[0120] For the superimposed point mutations, the relative value of the synthetic conversion rate relative to the parent is defined as a significant improvement of >1.5, 1.2-1.5 as an improvement, and 1-1.2 as a slight improvement. It can be found from Table 3 that the combination of 40, 181, and 292 has a significant improvement, and the combination of 224 with 181, 292 and 40, 181 also helps to improve the conversion rate, so these four sites are further superimposed.

[0121] [Example 21] Obtaining mutant acylase (23)

[0122] In order to obtain a transformant (23) expressing a mutant acylase having amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from the transformant (19) was used as a cloning parent, and appropriate primers were designed. The plasmid was obtained by the same operation as in Comparative Example 2 to obtain a transformant (23). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 4.

[0123] The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1. The results are shown in Table 4.

[0124] Table 4

[0125]

[0126] It can be found from Table 4 that the four-site combination of 40, 181, 224, and 292 has an improved conversion rate compared to the three-site combination, and can be used as the mother plant for a new round of evolutionary superposition to screen the sites.

[0127] [Example 22] Obtaining mutant acylase (24)

[0128] In order to obtain a transformant (24) expressing a mutant acylase having amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from the transformant (23) was used as a cloning parent, and appropriate primers were designed. The plasmid was obtained by the same operation as in Comparative Example 2 to obtain a transformant (24). The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1, and the results are shown in Table 5.

[0129] The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1. The results are shown in Table 5.

[0130] [Example 23] Obtaining mutant acylase (25)

[0131] In order to obtain a transformant (25) expressing a mutant acylase having amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from the transformant (23) was used as a cloning parent, and appropriate primers were designed. The plasmid was obtained by the same operation as in Comparative Example 2 to obtain a transformant (25). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 5.

[0132] The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1. The results are shown in Table 5.

[0133] [Example 24] Obtaining mutant acylase (26)

[0134] In order to obtain a transformant (26) expressing a mutant acylase having amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from the transformant (23) was used as a cloning parent, and appropriate primers were designed. The plasmid was obtained by the same operation as in Comparative Example 2 to obtain a transformant (26). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 5.

[0135] The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1. The results are shown in Table 5.

[0136] [Example 25] Obtaining mutant acylase (27)

[0137] In order to obtain a transformant (27) expressing a mutant acylase having amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from the transformant (23) was used as a cloning parent, and appropriate primers were designed. The plasmid was obtained by the same operation as in Comparative Example 2 to obtain a transformant (27). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 5.

[0138] The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1. The results are shown in Table 5.

[0139] [Example 26] Obtaining mutant acylase (28)

[0140] In order to obtain a transformant (28) expressing a mutant acylase having amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from the transformant (23) was used as a cloning parent, and appropriate primers were designed. The plasmid was obtained by the same operation as in Comparative Example 2 to obtain a transformant (28). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 5.

[0141] The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1. The results are shown in Table 5.

[0142] Table 5

[0143]

[0144] It can be found from Table 5 that the introduction of sites 160 and 320 has a certain improvement on the conversion rate of the previous four-site combination, and these mutations can be used as advantageous sites for improving the conversion rate.

[0145] [Example 27] Obtaining mutant acylase (29)

[0146] In order to obtain a transformant (29) expressing a mutant acylase having amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from the transformant (23) was used as a cloning parent, and appropriate primers were designed. The plasmid was obtained by the same operation as in Comparative Example 2 to obtain a transformant (29). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 6.

[0147] The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1. The results are shown in Table 6.

[0148] [Example 28] Obtaining mutant acylase (30)

[0149] In order to obtain a transformant (30) expressing a mutant acylase having amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from transformant (23) was used as a cloning parent, appropriate primers were designed, and a plasmid was obtained by the same operation as in Comparative Example 2 to obtain a transformant (30). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 6.

[0150] The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1. The results are shown in Table 6.

[0151] [Example 29] Obtaining mutant acylase (31)

[0152] In order to obtain a transformant (31) expressing a mutant acylase having amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from transformant (23) was used as a cloning parent, appropriate primers were designed, and a plasmid was obtained by the same operation as in Comparative Example 2 to obtain a transformant (31). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 6.

[0153] The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1. The results are shown in Table 6.

[0154] [Example 30] Obtaining mutant acylase (32)

[0155] In order to obtain a transformant (32) expressing a mutant acylase having amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from transformant (23) was used as a cloning parent, and appropriate primers were designed. The plasmid was obtained by the same operation as in Comparative Example 2 to obtain a transformant (32). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 6.

[0156] The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1. The results are shown in Table 6.

[0157] [Example 31] Obtaining mutant acylase (33)

[0158] In order to obtain a transformant (33) expressing a mutant acylase having amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from the transformant (23) was used as a cloning parent, and appropriate primers were designed. The plasmid was obtained by the same operation as in Comparative Example 2 to obtain a transformant (33). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 6.

[0159] The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1. The results are shown in Table 6.

[0160] [Example 32] Obtaining mutant acylase (34)

[0161] In order to obtain a transformant (34) expressing a mutant acylase having amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from transformant (23) was used as a cloning parent, and appropriate primers were designed. The plasmid was obtained by the same operation as in Comparative Example 2 to obtain a transformant (34). The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1, and the results are shown in Table 6.

[0162] The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1. The results are shown in Table 6.

[0163] Table 6

[0164]

[0165]

[0166] It can be found from Table 6 that the introduction of sites 160, 232, 284, 306, and 320 all help to improve the conversion rate to a certain extent, and these mutations can be used as advantageous sites for improving the conversion rate.

[0167] [Example 33] Obtaining mutant acylase (35)

[0168] In order to obtain a transformant (35) expressing a mutant acylase having amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from the transformant (24) was used as a cloning parent, and appropriate primers were designed. The plasmid was obtained by the same operation as in Comparative Example 2 to obtain a transformant (35). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 7.

[0169] The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1. The results are shown in Table 7.

[0170] [Example 34] Obtaining mutant acylase (36)

[0171] In order to obtain a transformant (36) expressing a mutant acylase having amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from the transformant (24) was used as a cloning parent, and appropriate primers were designed. The plasmid was obtained by the same operation as in Comparative Example 2 to obtain a transformant (36). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 7.

[0172] The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1. The results are shown in Table 7.

[0173] [Example 35] Obtaining mutant acylase (37)

[0174] In order to obtain a transformant (37) expressing a mutant acylase having amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from transformant (24) was used as a cloning parent, appropriate primers were designed, and plasmids were obtained by the same operation as in Comparative Example 2 to obtain a transformant (37). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 7.

[0175] The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1. The results are shown in Table 7.

[0176] [Example 36] Obtaining mutant acylase (38)

[0177] In order to obtain a transformant (38) expressing a mutant acylase having amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from the transformant (24) was used as a cloning parent, and appropriate primers were designed. The plasmid was obtained by the same operation as in Comparative Example 2 to obtain a transformant (38). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 7.

[0178] The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1. The results are shown in Table 7.

[0179] [Example 37] Obtaining mutant acylase (39)

[0180] In order to obtain a transformant (39) expressing a mutant acylase having amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from the transformant (24) was used as a cloning parent, and appropriate primers were designed. The plasmid was obtained by the same operation as in Comparative Example 2 to obtain a transformant (39). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 7.

[0181] The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1. The results are shown in Table 7.

[0182] Table 7

[0183]

[0184]

[0185] It can be found from Table 7 that the introduction of 14 sites is helpful to improve the conversion rate to a certain extent. The site selection combination of 160, 232, 284, 306, and 320 sites can also improve the conversion rate. These mutation combinations can be used as advantageous solutions to improve the conversion rate.

[0186] [Example 38] Obtaining mutant acylase (40)

[0187] In order to obtain a transformant (40) expressing a mutant acylase having amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from the transformant (38) was used as a cloning parent, and appropriate primers were designed. The plasmid was obtained by the same operation as in Comparative Example 2 to obtain a transformant (40). The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1, and the results are shown in Table 8.

[0188] The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1. The results are shown in Table 8.

[0189] [Example 39] Obtaining mutant acylase (41)

[0190] In order to obtain a transformant (41) expressing a mutant acylase having amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from the transformant (38) was used as a cloning parent, and appropriate primers were designed. The plasmid was obtained by the same operation as in Comparative Example 2 to obtain a transformant (41). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 8.

[0191] The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1. The results are shown in Table 8.

[0192] Table 8

[0193]

[0194] It can be found from Table 8 that the substitution of site 166 slightly improved the conversion rate, and site 306 also helped to improve the conversion rate. On the basis of the eight-site combination, the advantageous sites can still be replaced or superimposed to improve the enzyme activity. These mutation combinations can be used as an advantageous solution to improve the conversion rate. [Example 40] Obtaining mutant acylase (42)

[0195] In order to obtain a transformant (42) expressing a mutant acylase having amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from the transformant (40) was used as a cloning parent, and appropriate primers were designed. The plasmid was obtained by the same operation as in Comparative Example 2 to obtain a transformant (42). The synthesis of lauroylglycine was evaluated according to the culture and activity assay protocol described in Comparative Example 1, and the results are shown in Table 9.

[0196] The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1. The results are shown in Table 9.

[0197] Table 9

[0198]

[0199] It can be found from Table 9 that by superimposing the advantageous sites obtained from the above evolution, the conversion rate of the obtained ten-site combination mutation is improved compared with the nine-site combination.

[0200] [Example 41] Obtaining mutant acylase (43)

[0201] In order to obtain a transformant (43) expressing a mutant acylase having amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from the transformant (42) was used as a cloning parent, and appropriate primers were designed. The plasmid was obtained by the same operation as in Comparative Example 2 to obtain a transformant (43). The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1, and the results are shown in Table 10.

[0202] The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1. The results are shown in Table 10.

[0203] Table 10

[0204]

[0205]

[0206] It can be found from Table 10 that the introduction of site 130 is helpful to improve the conversion rate, and mutants with eleven combinations are obtained.

[0207] [Example 41] Obtaining mutant acylase (43)

[0208] In order to obtain a transformant (43) expressing a mutant acylase having amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from the transformant (42) was used as a cloning parent, and appropriate primers were designed. The plasmid was obtained by the same operation as in Comparative Example 2 to obtain a transformant (43). The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1, and the results are shown in Table 11.

[0209] The synthesis of lauroylglycine was evaluated according to the culture and activity measurement protocol described in Comparative Example 1. The results are shown in Table 11.

[0210] Table 11

[0211]

[0212]

[0213] It can be found from Table 11 that the introduction of V35E, Q50G, T368H, and L395S sites by different combinations of substitutions is helpful to improve the conversion rate, and mutants with a twelve-site combination are obtained, proving that the mutation site superposition scheme still has room for improvement.

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 an amino acid sequence as shown in SEQ ID NO.1: T24Y, V35E, E40K, Q50G, A102C, T181N, S224G, S292A, Q160E, T14Y, K284L, V306E, S320A, K232D, A166S, A130S, T368H, L395S.

2. The acylase mutant according to claim 1, characterized in that There are mutants with mutations in amino acid residues at the following two positions: E40K, A102C; E40K, T181N; E40K, S224G; E40K, S292A; A102C, T181N; A102C, S224G; A102C, S292A; T181N, S224G; T181N, S292A; S224G, S292A.

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: E40K, T181N, S224G; E40K, T181N, S292A; A102C, T181N, S224G; A102C, T181N, S292A; T181N, S224G, S292A; E40K, T181N, S292A; E40K, T181N, S224G; S292A, E40K, T181N.

4. The acylase mutant according to claim 1, characterized in that There are mutants with amino acid residue mutations at the following four positions: E40K, T181N, S224G, S292A; E40K, T181N; S224G, S292A; Or mutants with amino acid residue mutations at the following five positions: E40K, T181N, S224G, S292A, T14Y Or mutants with amino acid residue mutations at the following six positions: E40K, T181N, S224G, S292A, T14Y, K284L; E40K, T181N, S224G, S292A, T14Y, V306E; E40K, T181N, S224G, S292A, Q160E, K284L; E40K, T181N, S224G, S292A, Q160E, S320A; Or mutants with amino acid residue mutations at the following seven positions: E40K, T181N, S224G, S292A, T14Y, Q160E, S320A; E40K, T181N, S224G, S292A, Q160E, K232D, S320A; E40K, T181N, S224G, S292A, K232D, V306E, S320A; E40K, T181N, S224G, S292A, Q160E, K284L, V306E; E40K, T181N, S224G, S292A, Q160E, K284L, S320A; E40K, T181N, S224G, S292A, K284L, V306E, S320A; Or mutants with eight amino acid residue mutations: E40K, T181N, S224G, S292A, T14Y, Q160E, K232D, K284L; E40K, T181N, S224G, S292A, T14Y, Q160E, K232D, V306E; E40K, T181N, S224G, S292A, T14Y, Q160E, K284L, V306E; E40K, T181N, S224G, S292A, T14Y, Q160E, K284L, S320A; E40K, T181N, S224G, S292A, Q160E, K284L, V306E, S320A; E40K, T181N, S224G, S292A, T14Y, Q160E, K284L, V306E, Or mutants with amino acid residue mutations at the following nine positions: E40K, T181N, S224G, S292A, T14Y, Q160E, A166S, V306E, S320A; E40K, T181N, S224G, S292A, T14Y, Q160E, A166S, V306E, S320A; Or mutants with amino acid residue mutations at the following ten positions: E40K, T181N, S224G, S292A, T14Y, Q160E, A166S, K284L, V306E, S320A; Or mutants with amino acid residue mutations at the following eleven positions: E40K, T181N, S224G, S292A, T14Y, A130S, Q160E, A166S, K284L, V306E, S320A; Or mutants with amino acid residue mutations at the following twelve positions: E40K, T181N, S224G, S292A, T14Y, A130S, Q160E, A166S, K284L, V306E, S320A, T368H; E40K, T181N, S224G, S292A, T14Y, Q40G, A130S, Q160E, A166S, K284L, V306E, T368H; E40K, T181N, S224G, S292A, T14Y, V35E, Q160E, A166S, K284L, V306E, S320A, L395S; E40K, T181N, S224G, S292A, V35E, A130S, Q160E, A166S, K284L, V306E, T368H, L395S.

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

6. A recombinant expression vector containing the coding gene as claimed in claim 5, specifically in a pET vector framework.

7. The genetically engineered bacteria containing the coding gene as claimed in claim 3 or the recombinant expression vector as claimed in claim 6 is, for example, Escherichia coli.

8. Use of the acylase mutant according to any one of claims 1 to 4, or a nucleic acid encoding the mutant, in the preparation of lauroylglycine, in particular in catalyzing the conversion of lauric acid into lauroylglycine.

9. A method for preparing lauroyl glycine, characterized in that: The method comprises using the genetically engineered bacteria as claimed in claim 7 as a whole-cell catalyst and lauric acid and glycine as substrates to synthesize lauroylglycine; The method of obtaining the whole-cell catalyst specifically includes the following steps: centrifuging the fermented bacterial solution, discarding the supernatant, and collecting the precipitate to obtain the wet bacterial cells.

10. The preparation method according to claim 9, wherein the reaction conditions include a wet cell dosage of 5-30 g / L, a lauric acid concentration of 1.44-72 g / L, a 60%-100% saturated glycine solution; a temperature of 35-65° C., a pH of 5.5-8.5, and a stirring speed of 100-300 rpm to carry out a biocatalytic reaction to obtain lauroyl glycine.

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