A caprine acylase mutant and its use

Abstract

The application belongs to the technical field of biology, and discloses a goat-derived acylase mutant and application thereof. The goat-derived acylase mutant provided by the application has improved catalytic activity compared with wild-type acylase, can be used in acylation reaction in a high-efficiency and stable manner, and generates octanoyl amino acid. Therefore, the goat-derived acylase mutant provided by the application can be used as an amino acid surfactant production enzyme, and can be widely applied in different fields, and has great application potential in personal care products and the food industry.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biotechnology, and particularly relates to a mutant of goat acylase and application thereof. BACKGROUND

[0002] Developing high-quality multifunctional environmentally friendly surfactants has become the main direction of the surfactant industry, among which amino acid surfactants are the focus of development and synthesis in recent years. Amino acid surfactants have many advantages, such as wide source of biomass raw materials, small toxic and side effects, mild performance, small irritation and good biodegradability, and they also have good emulsifying, wetting, solubilizing, dispersing, foaming and other properties, so they are concerned and gradually applied in many fields such as washing, personal care and food industry.

[0003] Amino acid surfactants can be classified as acidic, neutral and basic according to the number of amino and carboxyl groups, or as anionic, cationic, amphoteric and non-ionic according to the different charge of the hydrophilic group. Among them, N-acyl amino acid surfactants are still the most important amino acid surfactants, and their synthesis methods include direct and indirect methods. Direct synthesis methods include enzyme catalysis and dehydration condensation, and indirect synthesis methods include fatty acyl chloride acylation, fatty nitrile hydrolysis acylation, fatty acid anhydride acylation, amide carbonylation and the like. Among them, dehydration condensation and indirect synthesis method are often subject to a large amount of waste produced by chemical synthesis, the danger of synthesis raw materials, high-precision equipment and large energy consumption in the process, which does not meet the concept of green synthesis; while enzyme catalysis has the advantages of green environmental protection, but often has the disadvantages of low catalytic efficiency and expensive enzyme preparation, so it is necessary to develop more efficient and convenient enzyme preparations, and to develop high-catalytic efficiency enzymes, which requires the introduction of the concept of synthetic biology, through enzyme evolution means such as computational design and random mutation, to develop high-catalytic enzyme preparations suitable for industrial production.

[0004] Octanoyl amino acid surfactants are a kind of surfactants with good biocompatibility and biodegradability, and they are often used in personal care products such as shampoo, shower gel and facial cleanser due to their mild effect. In addition, they can also be used as emulsifiers and stabilizers in the food industry and medical drugs.

[0005] The molecular structure of octanoyl amino acid contains an octanoyl and an amino acid, which can be obtained by acylation of octanoic acid with amino acid. Acylase is a kind of enzyme catalyzing acylation reaction, and mainly functions to transfer acyl (such as fatty acid) to other molecules, which can play a key role in various biochemical processes in organisms, including fatty acid metabolism, biosynthesis and degradation, etc. Acylase is widely present in animals, plants, fungi and bacteria, and the acylase for industrial production is often obtained from pig kidney. In addition, it has been confirmed by research that acylase from herbivorous animals and mammals has certain acylation capacity, but has not been applied to the synthesis of octanoyl amino acid surfactants. In order to explore more acylase catalytic capacity from different sources and provide a new synthesis route for industrial production of octanoyl amino acid, the present application tests and optimizes the synthesis of sheep acylase with high homology and acylation activity to human and pig sources, so as to obtain sheep acylase with high catalytic activity and promote the synthesis of octanoyl amino acid. SUMMARY

[0006] According to the sheep acylase synthesis gene pAcy obtained in the previous experiment, the present application obtains a mutant with improved activity. Thus, the present application is proposed.

[0007] The present application provides a sheep acylase mutant, which is a mutant with one, two, three, four, five, six or more amino acid residue substitution mutations selected from the following sites based on the acylase with the amino acid sequence shown in SEQ ID NO. 1: R41Q, T201N, K284R, Q88M, Q88L, Q88V, Q88S, Q88A, Q88G, A183M, A183S, A183N, A183L, T201V, T201I, T201A, K284D, K284E, K284L, K284Y, K284H.

[0008] Specifically, the mutant has two site amino acid residue mutations: R41Q, Q88V; R41Q, T201N; R41Q, A183M; Q88V, T201N; Q88V, K284E; A183M, K284E.

[0009] Preferably, the mutant has three site amino acid residue mutations: R41Q, Q88V, T201N; R41Q, Q88V, A183M; R41Q, T201N, K284E; R41Q, A183M, K284E; Q88V, T201N, A183M; Q88V, T201N, K284E; T201N, A183M, K284E.

[0010] Mutants with the following four-site amino acid residue mutations exist: R41Q, Q88V, T201N, A183M; R41Q, Q88V, T201N, K284E; R41Q, T201N, A183M, K284E;

[0011] Or mutants with the following five-site amino acid residue mutations exist: R41Q, Q88V, T201N, K284E, E231K; R41Q, Q88V, T201N, K284E, E231D; R41Q, Q88V, T201N, K284E, Q291M; R41Q, Q88V, T201N, K284E, Q291G; R41Q, Q88V, T201N, K284E, Q291E; R41Q, Q88V, T201N, K284E, Q291A; R41Q, Q88V, T201N, K284E, A306S; R41Q, Q88V, T201N, K284E, A306L; R41Q, Q88V, T201N, K284E, A306M; R41Q, Q88V, T201N, K284E, A306V; R41Q, Q88V, T201N, K284E, A306E;

[0012] Or mutants with the following six-site amino acid residue mutations exist: R41Q, Q88V, T201N, K284E, E231K, A306S; R41Q, Q88V, T201N, R41Q, Q88V, T201N, K284E, E231K, Q291M; R41Q, Q88V, T201N, R41Q, Q88V, T201N, K284E, Q291E, A306S; R41Q, Q88V, T201N;

[0013] Or mutants with the following seven-site amino acid residue mutations exist:

[0014] R41Q, Q88V, T201N, K284E, A183M, E231K, Q291E; R41Q, Q88V, T201N, K284E, A183M, E231K, Q291M; R41Q, Q88V, T201N, K284E, A183M, S239A, Q291M; R41Q, Q88V, T201N, K284E, E231K, S239A, Q291E.

[0015] The present application provides the coding gene of the acylase mutant.

[0016] The present application provides the recombinant expression vector containing the coding gene, specifically in the pET vector framework.

[0017] The present application provides a genetically engineered bacterium containing the coding gene or the recombinant expression vector, for example, Escherichia coli.

[0018] The present application provides an application of the acylase mutant or the coding nucleic acid thereof in preparing octanoyl amino acid compounds, in particular, in catalyzing the conversion of octanoic acid into octanoyl amino acid.

[0019] The present application provides a preparation method of octanoyl amino acid compounds, which comprises using the genetically engineered bacterium as a whole cell catalyst, and using octanoic acid and amino acid as substrates to synthesize octanoyl amino acid compounds.

[0020] The obtaining of the whole cell catalyst specifically comprises the following steps: centrifuging the fermentation culture bacterium liquid, discarding the supernatant, and collecting the precipitate to obtain wet bacterium.

[0021] Specifically, the amino acid is selected from glycine, arginine, glutamic acid, lysine and alanine.

[0022] The reaction condition comprises a reaction system composed of 5-30 g / L of wet bacterium, 1.44-72 g / L of octanoic acid, 60%-100% saturated amino acid solution, pH 6.0-8.0 phosphate buffer as a reaction medium, and a biological catalytic reaction is carried out under the condition of 35-65℃ of temperature, 100-800 rpm of shaking bed speed for 20-28 h to obtain octanoyl amino acid compounds.

[0023] The present application provides a sheep-derived acylase mutant, which has significantly improved catalytic activity compared with wild-type acylase. The wet bacterium obtained by fermentation culture of the recombinant bacterium containing the mutant coding gene of the acylase is used as a biological catalyst, and octanoyl amino acid surfactants can be synthesized by using octanoic acid and amino acid as substrates, so that the production efficiency can be greatly improved, and the mutant has high production application value. DETAILED DESCRIPTION

[0024] The embodiments are described in more detail by the following examples, but the present application is not limited by any of the following examples. It should be noted that the so-called mutation site in the following examples refers to the difference position in the amino acid sequence of the mutant acylase containing the amino acid sequence with sequence number 1 and derived from sheep.

[0025] [Example 1] Obtaining of wild-type acylase (1)

[0026] According to the reported gene synthesis of Ovis aries aminoacylase 1 (ACY1), transcript variant X1, mRNA, Ovis aries (sheep), XM_004018411.6), named pAcy, the amino acid sequence of which is shown in SEQ ID NO: 1, and the nucleic acid sequence of the cDNA coding region thereof is shown in SEQ ID NO: 2.

[0027] After the wild-type acylase cDNA coding region was artificially synthesized, the fragment was amplified by PCR using primer pair SEQ ID NO: 3 and SEQ ID NO: 4 and introducing Nde I and Xho I endonuclease sites on both sides of the fragment, with the gene as the template. The PCR reaction system: Takara Max DNA Polymerase 25 μL, ddH2O 20 μl, SEQ ID NO: 3 primer 1.5 μl, SEQ ID NO: 4 primer 1.5 μl, SEQ ID NO: 2 template 2 μl. The PCR reaction program: after 98°C pre-denaturation for 2 min, 98°C denaturation for 10 s, 58°C annealing for 15 s, 72°C extension for 10 s, a total of 25 cycles, and finally 72°C terminal extension for 5 min.

[0028] The PCR product was analyzed by 1% agarose gel electrophoresis, and it was confirmed that there was an amplified product of about 1.2 kb.

[0029] Only the DNA fragment of about 1.2 kb was cut from the above agarose gel, and the DNA in the gel strip was purified and recovered using the ordinary agarose gel DNA recovery kit of Tiangeng, and the purified product was double-digested with restriction enzymes Nde I and Xho I. After confirming that the band size was about 1.2 kb by 1% agarose gel electrophoresis, the gene digested fragment was again purified and recovered.

[0030] Similarly, the E. coli expression vector pET-28a(+) was cut with restriction enzymes Nde I and Xho I, agarose gel electrophoresis was performed, and only a DNA fragment of about 5.3 kb was cut from the agarose gel, and the vector digested fragment was obtained after purification and recovery.

[0031] Under the action of T4 DNA ligase, the about 1.2 kb and about 5.3 kb digested fragments obtained above were ligated, and E. coli BL21(DE3) was transformed to obtain transformant (1). The plasmid was prepared from the above bacterial body using the plasmid extraction kit of Tiangeng, and the recombinant plasmid pET-28a(+)-Acy was obtained, and DNA sequencing confirmed that the coding region nucleotide sequence of the plasmid was consistent with SEQ ID NO: 2. Transformant (1) produced acylase (1) as a mutant acylase derived from sheep.

[0032] The recombinant E. coli BL21(DE3) / pAcy-pET-28a(+) obtained above was inoculated into LB liquid medium containing 50 μg / ml kanamycin resistance, and cultured at 37°C, 200 rpm for 12 h, then inoculated into fresh LB liquid medium containing 50 μg / ml kanamycin resistance at 1% (v / v) inoculation amount, and cultured at 37°C, 200 rpm until the OD600 of the bacteria reached 0.6-0.8, then 0.1 mM IPTG was added, and the culture was induced at 20°C, 200 rpm for 15 h. The culture was centrifuged at 4°C, 8000 rpm for 20 min, the supernatant was discarded, and the precipitate was collected to obtain wet bacteria containing recombinant E. coli BL21(DE3) / pAcy-pET-28a(+) expressing the recombinant plasmid. The bacteria can be directly used as a biocatalyst.

[0033] The wet bacteria obtained above were used to determine the synthesis activity of octanoyl amino acids. The reaction system was composed of 5-30 g / L wet bacteria, 1.44-72 g / L octanoic acid, 60%-100% saturated amino acid solution, and pH 6.0-8.0 phosphate buffer as the reaction medium, and the biocatalytic reaction was carried out at 35-65°C, 100-800 rpm for 20-28 h to obtain octanoyl amino acid compounds.

[0034] [Example 2] Obtaining of Mutant Acylase (2)

[0035] The candidate modification sites introduced by random mutation were subjected to saturation mutation, the low conversion rate sites were eliminated, the high conversion rate advantage mutation sites were retained, then the sites were combined and superimposed, and new mutation sites were introduced in the superimposition process, and multiple rounds of evolution screening were carried out.

[0036] After the recombinant plasmid pET-28a(+)-Acy was artificially synthesized, the plasmid was used as a template, and the primer pair sequence 5 was used to amplify the fragment by PCR to introduce the mutation sites required by the transformant (2). The PCR reaction system: Takara Max DNA Polymerase 25 μL, ddH2O 20 μl, sequence 5 primer F 1.5 μl, primer R 1.5 μl, pET-28a(+)-Acy template 2 μl. The PCR reaction program: after pre-denaturation at 98°C for 2 min, 98°C denaturation for 10 s, 58°C annealing for 15 s, 72°C extension for 40 s, a total of 25 cycles, and finally 72°C final extension for 5 min.

[0037] The PCR product was analyzed by 1% agarose gel electrophoresis, and it was confirmed that there was an amplification product of about 6.5 kb.

[0038] From the agarose gel, only the DNA fragment of about 6.5 kb was cut out, and the DNA in the gel strip was purified and recovered using a common agarose gel DNA recovery kit from Tiangen.

[0039] E. coli BL21 (DE3) was transformed to obtain transformant (2). Transformant (2) produced mutant (2) as a dominant mutant acylase derived from sheep.

[0040] Transformant (2) produced acylase (2) with mutations as shown in the table on the basis of Example 1, and the culture and activity determination scheme were the same as in Example 1.

[0041] [Example 3] Obtaining of mutant acylase (3)

[0042] In order to obtain transformant (3) expressing mutant acylase with amino acid residue substitution as shown in Table 1, appropriate primers were designed, and plasmids were obtained by the same operation as in Example 2 to obtain transformant (3). Transformant (3) produced acylase (3) with mutations as shown in the table

[0043] The synthesis of octanoyl amino acids was evaluated according to the culture and activity determination scheme described in Example 1, and the results are shown in Table 1.

[0044] [Example 4] Obtaining of mutant acylase (4)

[0045] In order to obtain transformant (4) expressing mutant acylase with amino acid residue substitution as shown in Table 1, appropriate primers were designed, and plasmids were obtained by the same operation as in Example 2 to obtain transformant (4). Transformant (4) produced acylase (4) with mutations as shown in the table

[0046] The synthesis of octanoyl amino acids was evaluated according to the culture and activity determination scheme described in Example 1, and the results are shown in Table 1.

[0047] Table 1

[0048]

[0049] For the superimposed point mutations, the relative value of the synthesis conversion rate relative to the wild type >1.5 was defined as significant improvement, 1.2-1.5 as improvement, 1-1.2 as slight improvement, 0.8-1 as slight decrease, 0.5-0.8 as decrease, and <0.5 as severe decrease. As can be seen from Table 1, positions 41, 201 and 284 can be used as dominant positions for improving the conversion rate, and saturation mutation can be performed to observe whether there is a mutation with higher conversion rate.

[0050] [Example 5] Obtaining of mutant acylase (5)

[0051] To obtain transformant (5) expressing mutant acylase having amino acid residue substitution as shown in Table 2, appropriate primers were designed, and plasmid was obtained by the same procedure as in Example 2 to obtain transformant (5). Transformant (5) produced acylase (5) having mutation as shown in the table

[0052] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 2.

[0053] [Example 6] Obtaining of mutant acylase (6)

[0054] To obtain transformant (6) expressing mutant acylase having amino acid residue substitution as shown in Table 2, appropriate primers were designed, and plasmid was obtained by the same procedure as in Example 2 to obtain transformant (6). Transformant (6) produced acylase (6) having mutation as shown in the table

[0055] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 2.

[0056] [Example 7] Obtaining of mutant acylase (7)

[0057] To obtain transformant (7) expressing mutant acylase having amino acid residue substitution as shown in Table 2, appropriate primers were designed, and plasmid was obtained by the same procedure as in Example 2 to obtain transformant (7). Transformant (7) produced acylase (7) having mutation as shown in the table

[0058] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 2.

[0059] [Example 8] Obtaining of mutant acylase (8)

[0060] To obtain transformant (8) expressing mutant acylase having amino acid residue substitution as shown in Table 2, appropriate primers were designed, and plasmid was obtained by the same procedure as in Example 2 to obtain transformant (8).

[0061] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 2.

[0062] [Example 9] Obtaining of mutant acylase (9)

[0063] To obtain transformant (9) expressing mutant acylase having amino acid residue substitution as shown in Table 2, appropriate primers were designed, and plasmid was obtained by the same procedure as in Example 2 to obtain transformant (9).

[0064] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 2.

[0065] [Example 10] Obtaining of Mutant Acylases (10)

[0066] In order to obtain transformant (10) expressing mutant acylases having amino acid residue substitutions as shown in Table 2, appropriate primers were designed, and plasmids were obtained using the same procedures as in Example 2 to obtain transformant (10).

[0067] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 2.

[0068] Table 2

[0069]

[0070] As can be seen from Table 2, there is a large increase in conversion rate when Q88 is mutated to V and L, and there is a slight increase when M, S, A, and G are mutated. These mutations can be used as advantageous sites for increasing conversion rate.

[0071] [Example 11] Obtaining of Mutant Acylases (11)

[0072] In order to obtain transformant (11) expressing mutant acylases having amino acid residue substitutions as shown in Table 3, appropriate primers were designed, and plasmids were obtained using the same procedures as in Example 2 to obtain transformant (11).

[0073] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 3.

[0074] [Example 12] Obtaining of Mutant Acylases (12)

[0075] In order to obtain transformant (12) expressing mutant acylases having amino acid residue substitutions as shown in Table 3, appropriate primers were designed, and plasmids were obtained using the same procedures as in Example 2 to obtain transformant (12).

[0076] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 3.

[0077] [Example 13] Obtaining of Mutant Acylases (13)

[0078] In order to obtain transformant (13) expressing mutant acylases having amino acid residue substitutions as shown in Table 3, appropriate primers were designed, and plasmids were obtained using the same procedures as in Example 2 to obtain transformant (13).

[0079] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 3.

[0080] [Example 14] Obtaining of Mutant Acylases (14)

[0081] In order to obtain transformant (14) expressing mutant acylases having amino acid residue substitutions as shown in Table 3, appropriate primers were designed, and plasmids were obtained using the same procedures as in Example 2 to obtain transformant (14).

[0082] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 3.

[0083] Table 3

[0084]

[0085] As can be seen from Table 3, the conversion rate was improved to varying degrees when the A183 site was mutated to M, S, N, or L, with the most prominent improvement being A183M, which can be used as an advantageous mutation for improving conversion rate.

[0086] [Example 15] Obtaining of Mutant Acylases (15)

[0087] In order to obtain transformant (15) expressing mutant acylases having amino acid residue substitutions as shown in Table 4, appropriate primers were designed, and plasmids were obtained using the same procedures as in Example 2 to obtain transformant (15).

[0088] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 4.

[0089] [Example 16] Obtaining of Mutant Acylases (16)

[0090] In order to obtain transformant (16) expressing mutant acylases having amino acid residue substitutions as shown in Table 4, appropriate primers were designed, and plasmids were obtained using the same procedures as in Example 2 to obtain transformant (16).

[0091] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 4.

[0092] [Example 17] Obtaining of Mutant Acylases (17)

[0093] In order to obtain transformant (17) expressing mutant acylases having amino acid residue substitutions as shown in Table 4, appropriate primers were designed, and plasmids were obtained using the same procedures as in Example 2 to obtain transformant (17).

[0094] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 4.

[0095] Table 4

[0096]

[0097] As can be seen from Table 4, mutation of site T201 to V, I, or A can increase the conversion rate, with T201V and T201I increasing slightly, and T201A increasing the most. This site can be used as a mutation for increasing the conversion rate.

[0098] [Example 18] Obtaining of Mutant Acylase (18)

[0099] In order to obtain transformant (18) expressing a mutant acylase having an amino acid residue substitution as shown in Table 5, appropriate primers were designed, and a plasmid was obtained using the same procedures as in Example 2, to obtain transformant (18).

[0100] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 5.

[0101] [Example 19] Obtaining of Mutant Acylase (19)

[0102] In order to obtain transformant (19) expressing a mutant acylase having an amino acid residue substitution as shown in Table 5, appropriate primers were designed, and a plasmid was obtained using the same procedures as in Example 2, to obtain transformant (19).

[0103] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 5.

[0104] [Example 20] Obtaining of Mutant Acylase (20)

[0105] In order to obtain transformant (20) expressing a mutant acylase having an amino acid residue substitution as shown in Table 5, appropriate primers were designed, and a plasmid was obtained using the same procedures as in Example 2, to obtain transformant (20).

[0106] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 5.

[0107] [Example 21] Obtaining of Mutant Acylase (21)

[0108] In order to obtain transformant (21) expressing a mutant acylase having an amino acid residue substitution as shown in Table 5, appropriate primers were designed, and a plasmid was obtained using the same procedures as in Example 2, to obtain transformant (21).

[0109] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 5.

[0110] [Example 22] Obtaining of Mutant Acylase (22)

[0111] In order to obtain transformant (22) expressing mutant acylase having amino acid residue substitution as shown in Table 5, appropriate primers were designed, and a plasmid was obtained using the same procedure as in Example 2, to obtain transformant (22).

[0112] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 5.

[0113] Table 5

[0114]

[0115] As can be seen from Table 5, there is an increase in conversion rate when K284 is mutated to D, E, L, Y, or E, and this site can be an advantageous site for increasing conversion rate.

[0116] In order to obtain transformant (23) expressing mutant acylase having amino acid residue substitution as shown in Table 6, the vector obtained by extracting the plasmid from transformant (19) was used as a cloning parent, appropriate primers were designed, and a plasmid was obtained using the same procedure as in Example 2, to obtain transformant (23).

[0117] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 6.

[0118] [Example 24] Obtaining of Mutant Acylase (24)

[0119] In order to obtain transformant (24) expressing mutant acylase having amino acid residue substitution as shown in Table 6, the vector obtained by extracting the plasmid from transformant (23) was used as a cloning parent, appropriate primers were designed, and a plasmid was obtained using the same procedure as in Example 2, to obtain transformant (24).

[0120] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 6.

[0121] [Example 25] Obtaining of Mutant Acylase (25)

[0122] In order to obtain transformant (25) expressing mutant acylase having amino acid residue substitution as shown in Table 6, the vector obtained by extracting the plasmid from transformant (23) was used as a cloning parent, appropriate primers were designed, and a plasmid was obtained using the same procedure as in Example 2, to obtain transformant (25).

[0123] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 6.

[0124] [Example 26] Obtaining of Mutant Acylase (26)

[0125] In order to obtain transformant (26) expressing mutant acylase having amino acid residue substitution as shown in Table 6, the vector obtained by plasmid extraction from transformant (23) was used as the cloning parent, appropriate primers were designed, and a plasmid was obtained using the same procedures as in Example 2 to obtain transformant (26).

[0126] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 6.

[0127] [Example 27] Obtaining of Mutant Acylase (27)

[0128] In order to obtain transformant (27) expressing mutant acylase having amino acid residue substitution as shown in Table 6, the vector obtained by plasmid extraction from transformant (23) was used as the cloning parent, appropriate primers were designed, and a plasmid was obtained using the same procedures as in Example 2 to obtain transformant (27).

[0129] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 6.

[0130] [Example 28] Obtaining of Mutant Acylase (28)

[0131] In order to obtain transformant (28) expressing mutant acylase having amino acid residue substitution as shown in Table 6, the vector obtained by plasmid extraction from transformant (23) was used as the cloning parent, appropriate primers were designed, and a plasmid was obtained using the same procedures as in Example 2 to obtain transformant (28).

[0132] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 6.

[0133] Table 6

[0134]

[0135] As can be seen from Table 6, the conversion rate is improved in the two-point combination compared to the single point, which can be used as the parent for further mutation research.

[0136] [Example 29] Obtaining of Mutant Acylase (29)

[0137] To obtain transformant (29) expressing mutant acylase having amino acid residue substitution as shown in Table 7, the 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 procedure as in Example 2 to obtain transformant (29).

[0138] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 7.

[0139] [Example 30] Obtaining of mutant acylase (30)

[0140] To obtain transformant (30) expressing mutant acylase having amino acid residue substitution as shown in Table 7, the 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 procedure as in Example 2 to obtain transformant (30).

[0141] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 7.

[0142] [Example 31] Obtaining of mutant acylase (31)

[0143] To obtain transformant (31) expressing mutant acylase having amino acid residue substitution as shown in Table 7, the 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 procedure as in Example 2 to obtain transformant (31).

[0144] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 7.

[0145] [Example 32] Obtaining of mutant acylase (32)

[0146] To obtain transformant (32) expressing mutant acylase having amino acid residue substitution as shown in Table 7, the 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 procedure as in Example 2 to obtain transformant (32).

[0147] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 7.

[0148] [Example 33] Obtaining of mutant acylase (33)

[0149] To obtain transformant (33) expressing mutant acylase having amino acid residue substitution as shown in Table 7, the 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 procedure as in Example 2 to obtain transformant (33).

[0150] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 7.

[0151] [Example 34] Obtaining of mutant acylase (34)

[0152] To obtain transformant (34) expressing mutant acylase having amino acid residue substitution as shown in Table 7, the 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 procedure as in Example 2 to obtain transformant (34).

[0153] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 7.

[0154] [Example 35] Obtaining of mutant acylase (35)

[0155] To obtain transformant (35) expressing mutant acylase having amino acid residue substitution as shown in Table 7, the vector obtained by plasmid extraction from transformant (24) was used as a cloning parent, appropriate primers were designed, and a plasmid was obtained by the same procedure as in Example 2 to obtain transformant (35).

[0156] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 7.

[0157] Table 7

[0158]

[0159] As can be seen from Table 7, these three-point mutation combinations have an improvement compared to one-point and two-point, and the combination of 41, 201, and 284 shows the best performance.[Example 36] Obtaining of mutant acylase (36)

[0160] To obtain transformant (36) expressing mutant acylase having amino acid residue substitution as shown in Table 8, the vector obtained by plasmid extraction from transformant (35) was used as a cloning parent, appropriate primers were designed, and a plasmid was obtained by the same procedure as in Example 2 to obtain transformant (36).

[0161] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 8.

[0162] [Example 37] Obtaining of mutant acylase (37)

[0163] In order to obtain transformant (37) expressing mutant acylase having amino acid residue substitution as shown in Table 8, the vector obtained by plasmid extraction from transformant (35) was used as a cloning parent, appropriate primers were designed, and a plasmid was obtained by the same procedure as in Example 2 to obtain transformant (37).

[0164] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 8.

[0165] [Example 38] Obtaining of mutant acylase (38)

[0166] In order to obtain transformant (38) expressing mutant acylase having amino acid residue substitution as shown in Table 8, the vector obtained by plasmid extraction from transformant (35) was used as a cloning parent, appropriate primers were designed, and a plasmid was obtained by the same procedure as in Example 2 to obtain transformant (38).

[0167] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 8.

[0168] Table 8

[0169]

[0170]

[0171] As can be seen from Table 8, these four-point mutation combinations can be used as advantageous solutions for improving conversion rate, among which the combination of 41, 88, 201 and 284 can improve conversion rate the most and can be used as a parent for a new round of evolution.

[0172] [Example 39] Obtaining of mutant acylase (39)

[0173] In order to obtain transformant (39) expressing mutant acylase having amino acid residue substitution as shown in Table 9, the vector obtained by plasmid extraction from transformant (37) was used as a cloning parent, appropriate primers were designed, and a plasmid was obtained by the same procedure as in Example 2 to obtain transformant (39).

[0174] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 9.

[0175] [Example 40] Obtaining of mutant acylase (40)

[0176] To obtain transformant (40) expressing mutant acylase having amino acid residue substitution as shown in Table 9, the vector obtained by plasmid extraction from transformant (37) was used as a cloning parent, appropriate primers were designed, and a plasmid was obtained by the same procedure as in Example 2 to obtain transformant (40).

[0177] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 9.

[0178] [Example 41] Obtaining of mutant acylase (41)

[0179] To obtain transformant (41) expressing mutant acylase having amino acid residue substitution as shown in Table 9, the vector obtained by plasmid extraction from transformant (37) was used as a cloning parent, appropriate primers were designed, and a plasmid was obtained by the same procedure as in Example 2 to obtain transformant (41).

[0180] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 9.

[0181] [Example 42] Obtaining of mutant acylase (42)

[0182] To obtain transformant (42) expressing mutant acylase having amino acid residue substitution as shown in Table 9, the vector obtained by plasmid extraction from transformant (40) was used as a cloning parent, appropriate primers were designed, and a plasmid was obtained by the same procedure as in Example 2 to obtain transformant (42).

[0183] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 9.

[0184] [Example 43] Obtaining of mutant acylase (43)

[0185] To obtain transformant (43) expressing mutant acylase having amino acid residue substitution as shown in Table 9, the vector obtained by plasmid extraction from transformant (37) was used as a cloning parent, appropriate primers were designed, and a plasmid was obtained by the same procedure as in Example 2 to obtain transformant (43).

[0186] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 9.

[0187] [Example 44] Obtaining of mutant acylase (44)

[0188] To obtain transformant (44) expressing mutant acylase having amino acid residue substitution as shown in Table 9, the vector obtained by extracting plasmid from transformant (37) was used as a cloning parent, appropriate primers were designed, and a plasmid was obtained by the same procedure as in Example 2 to obtain transformant (44).

[0189] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 9.

[0190] [Example 45] Obtaining of mutant acylase (45)

[0191] To obtain transformant (45) expressing mutant acylase having amino acid residue substitution as shown in Table 9, the vector obtained by extracting plasmid from transformant (37) was used as a cloning parent, appropriate primers were designed, and a plasmid was obtained by the same procedure as in Example 2 to obtain transformant (45).

[0192] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 9.

[0193] [Example 46] Obtaining of mutant acylase (46)

[0194] To obtain transformant (46) expressing mutant acylase having amino acid residue substitution as shown in Table 9, the vector obtained by extracting plasmid from transformant (37) was used as a cloning parent, appropriate primers were designed, and a plasmid was obtained by the same procedure as in Example 2 to obtain transformant (46).

[0195] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 9.

[0196] [Example 47] Obtaining of mutant acylase (47)

[0197] To obtain transformant (47) expressing mutant acylase having amino acid residue substitution as shown in Table 9, the vector obtained by extracting plasmid from transformant (37) was used as a cloning parent, appropriate primers were designed, and a plasmid was obtained by the same procedure as in Example 2 to obtain transformant (47).

[0198] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 9.

[0199] [Example 48] Obtaining of mutant acylase (48)

[0200] To obtain transformant (48) expressing mutant acylase having amino acid residue substitution as shown in Table 9, the vector obtained by extracting plasmid from transformant (37) was used as a cloning parent, appropriate primers were designed, and plasmid was obtained by the same procedure as in Example 2 to obtain transformant (48).

[0201] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 9.

[0202] [Example 49] Obtaining of mutant acylase (49)

[0203] To obtain transformant (49) expressing mutant acylase having amino acid residue substitution as shown in Table 9, the vector obtained by extracting plasmid from transformant (37) was used as a cloning parent, appropriate primers were designed, and plasmid was obtained by the same procedure as in Example 2 to obtain transformant (49).

[0204] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 9.

[0205] Table 9

[0206]

[0207]

[0208] As can be seen from Table 9, the conversion rate was improved by stacking the advantageous sites obtained by the above evolution into a five-point combination mutation compared to the four-point combination.

[0209] [Example 50] Obtaining of mutant acylase (50)

[0210] To obtain transformant (50) expressing mutant acylase having amino acid residue substitution as shown in Table 10, the vector obtained by extracting plasmid from transformant (37) was used as a cloning parent, appropriate primers were designed, and plasmid was obtained by the same procedure as in Example 2 to obtain transformant (50).

[0211] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 10.

[0212] [Example 51] Obtaining of mutant acylase (51)

[0213] To obtain transformant (51) expressing mutant acylase having amino acid residue substitution as shown in Table 10, the vector obtained by extracting plasmid from transformant (37) was used as a cloning parent, appropriate primers were designed, and plasmid was obtained by the same procedure as in Example 2 to obtain transformant (51).

[0214] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 10.

[0215] [Example 52] Obtaining of Mutant Acylases (52)

[0216] In order to obtain transformant (52) expressing mutant acylases having amino acid residue substitutions as shown in Table 10, the vector obtained by plasmid extraction from transformant (37) was used as the cloning parent, appropriate primers were designed, and a plasmid was obtained using the same procedures as in Example 2 to obtain transformant (52).

[0217] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 10.

[0218] Table 10

[0219]

[0220] As can be seen from Table 10, the introduction of site combinations was helpful in improving conversion, and six-point mutant combinations were obtained.

[0221] [Example 53] Obtaining of Mutant Acylases (53)

[0222] In order to obtain transformant (53) expressing mutant acylases having amino acid residue substitutions as shown in Table 11, the vector obtained by plasmid extraction from transformant (52) was used as the cloning parent, appropriate primers were designed, and a plasmid was obtained using the same procedures as in Example 2 to obtain transformant (53).

[0223] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 11.

[0224] [Example 54] Obtaining of Mutant Acylases (54)

[0225] In order to obtain transformant (54) expressing mutant acylases having amino acid residue substitutions as shown in Table 11, the vector obtained by plasmid extraction from transformant (52) was used as the cloning parent, appropriate primers were designed, and a plasmid was obtained using the same procedures as in Example 2 to obtain transformant (54).

[0226] The synthesis of octanoyl amino acids was evaluated according to the culture and activity assay protocol described in Example 1, and the results are shown in Table 11.

[0227] [Example 55] Obtaining of Mutant Acylases (55)

[0228] To obtain transformant (55) expressing mutant acylase with amino acid residue substitution as shown in Table 11, use the vector obtained by plasmid extraction from transformant (52) as the cloning parent, design appropriate primers, and obtain plasmid by the same operation as in Example 2 to obtain transformant (55).

[0229] The synthesis of octanoyl amino acids was evaluated according to the culture and activity determination protocol described in Example 1, and the results are shown in Table 11.

[0230] [Example 56] Obtaining of mutant acylase (56)

[0231] To obtain transformant (56) expressing mutant acylase with amino acid residue substitution as shown in Table 11, use the vector obtained by plasmid extraction from transformant (52) as the cloning parent, design appropriate primers, and obtain plasmid by the same operation as in Example 2 to obtain transformant (56).

[0232] The synthesis of octanoyl amino acids was evaluated according to the culture and activity determination protocol described in Example 1, and the results are shown in Table 11.

[0233] Table 11

[0234]

[0235]

[0236] As can be seen from Table 11, compared with six-point combination, the conversion rate is still improved to different degrees by introducing different mutations, proving that the mutation site stacking scheme still has room for improvement.

Claims

1. A mutant of a goat-derived acylase, characterized in that, which is a mutant of acylase whose amino acid sequence is shown as SEQ ID NO. 1, in which only the following amino acid residue substitution mutations exist: (1) R41Q; (2) R41Q and Q88V; (3) R41Q and T201N; (4) R41Q and A183M; (5) R41Q, Q88V and T201N; (6) R41Q, Q88V and A183M; (7) R41Q, T201N and K284E; (8) R41Q, A183M and K284E; (9) R41Q, Q88V, T201N and A183M; (10) R41Q, Q88V, T201N and K284E; (11) R41Q, T201N, A183M and K284E; (12) R41Q, Q88V, T201N, K284E and E231K; (13) R41Q, Q88V, T201N, K284E and E231D; (14) R41Q, Q88V, T201N, K284E and Q291M; (15) R41Q, Q88V, T201N, K284E and Q291G; (16) R41Q, Q88V, T201N, K284E and Q291E; (17) R41Q, Q88V, T201N, K284E and Q291A; (18) R41Q, Q88V, T201N, K284E and A306S; (19) R41Q, Q88V, T201N, K284E and A306L; (20) R41Q, Q88V, T201N, K284E and A306M; (21) R41Q, Q88V, T201N, K284E and A306V; (22) R41Q, Q88V, T201N, K284E and A306E; (23) R41Q, Q88V, T201N, K284E, E231K and A306S; (24) R41Q, Q88V, T201N, K284E, E231K and Q291M; (25) R41Q, Q88V, T201N, K284E, Q291E and A306S; (26) R41Q, Q88V, T201N, K284E, A183M, E231K and Q291E; (27) R41Q, Q88V, T201N, K284E, A183M, E231K and Q291M; (28) R41Q, Q88V, T201N, K284E, A183M, S239A and Q291M; or (29) R41Q, Q88V, T201N, K284E, E231K, S239A and Q291E.

2. A coding gene of the acylase mutant according to claim 1.

3. A recombinant expression vector containing the coding gene according to claim 2. which is pET-28a(+) vector.

4. The recombinant expression vector of claim 3, wherein, ​ 5. A genetically engineered bacterium comprising the coding gene according to claim 2, or the recombinant expression vector according to claim 3 or 4.

6. The genetically engineered bacteria as described in claim 5, characterized in that, It is Escherichia coli.

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