Sheep-derived acylase mutant and application thereof

Abstract

The invention belongs to the technical field of biology, and discloses a sheep-derived acylase mutant and application thereof. Compared with wild-type acylase, the sheep-derived acylase mutant disclosed by the invention has the advantages that the catalytic activity is improved, and the sheep-derived acylase mutant can efficiently and stably act on acylation reaction to generate octanoyl amino acid. Therefore, the sheep-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 particularly relates to a sheep-derived acylase mutant and application thereof. Background Art

[0002] The development of high-quality, multifunctional and environmentally friendly surfactants has become the main direction of the surfactant industry, and amino acid surfactants have been the focus of development and synthesis in recent years. Amino acid surfactants have many advantages, such as a wide range of biomass raw materials, low toxicity and side effects, mild performance, low irritation and good biodegradability, and it also has good emulsification, wetting, solubilization, dispersion, foaming and other properties. Therefore, it has attracted much attention and has been gradually applied to many fields such as washing, personal care and food industry.

[0003] Amino acid surfactants can usually be classified into acidic, neutral, and alkaline according to the number of amino and carboxyl groups; they can also be divided into anionic, cationic, amphoteric, and nonionic according to the different charges of the hydrophilic group. Among them, N-acyl amino acid surfactants are still the most important amino acid surfactants, and their synthesis methods are divided into direct and indirect methods. Direct synthesis methods include enzyme-catalyzed synthesis and dehydration condensation, and indirect synthesis methods include fatty acid chloride acylation, fatty nitrile hydrolysis acylation, fatty acid anhydride acylation, amide carbonylation reaction, etc. Among them, dehydration condensation and indirect synthesis methods are often subject to the large amount of waste generated by chemical synthesis, the danger of synthetic raw materials, high-precision equipment, and high energy consumption generated by the process, which does not conform to the concept of green synthesis; and although enzyme catalysis has the advantage of green environmental protection, it often has the disadvantages of low catalytic efficiency and expensive enzyme preparations. Therefore, it is necessary to develop more efficient and convenient enzyme preparations. If you want to develop enzymes with high catalytic efficiency, you must introduce the concept of synthetic biology and develop high-catalytic enzyme preparations suitable for industrial production through enzyme evolution methods such as computational design and random mutation.

[0004] Octanoyl amino acid surfactants are a type of surfactant with good biocompatibility and biodegradability. Due to their mild effects, they are often used in personal care products such as shampoo, shower gel, and facial cleansers. 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 group and an amino acid, which can be obtained by acylation of octanoic acid and amino acid. Acylase is a kind of enzyme that catalyzes acylation reaction, and its main function is to transfer acyl groups (such as fatty acids) to other molecules. It can play a key role in various biochemical processes in the body, including fatty acid metabolism, biosynthesis and degradation. It is widely present in animals, plants, fungi and bacteria. Among them, industrially produced acylase is often obtained from pig kidneys. In addition, studies have also confirmed that acylases from herbivores and mammals have certain acylation capabilities, but they have not yet been applied to the synthesis of surfactants of octanoyl amino acids. In order to explore the catalytic ability of acylases from more sources and provide a new synthetic route for the industrial production of octanoyl amino acids, the present invention has conducted synthetic tests and optimized transformations on sheep-derived acylases with high homology to human and pig sources and acylation activity to obtain sheep-derived acylases with high catalytic activity and promote the synthesis of octanoyl amino acids. Summary of the invention

[0006] The present invention is based on the sheep acylase synthesis gene pAcy obtained in previous experiments, and is modified to obtain a mutant with improved activity. Thus, the present invention is proposed.

[0007] The invention provides a sheep-derived acylase mutant, which is a mutant having one, two, three, four, five, six or more amino acid residue substitution mutations selected from the following sites on the basis of the acylase whose amino acid sequence is 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 and K284H.

[0008] Specifically, there are mutants with mutations in the amino acid residues at the following two positions: R41Q, Q88V; R41Q, T201N; R41Q, A183M; Q88V, T201N; Q88V, K284E; A183M, K284E.

[0009] Preferably, there are mutants with amino acid residue mutations in the following three positions: R41Q, Q88V, T201N; R41Q, Q88V, A183M; R41Q, T201N, K284E; R41Q, A183M, K284E; Q88V, T201N, A183M; Q88V, T201N, K284E; T201N, A183M, K284E.

[0010] There are mutants with amino acid residue mutations at the following four positions: R41Q, Q88V, T201N, A183M; R41Q, Q88V, T201N, K284E; R41Q, T201N, A183M, K284E;

[0011] Or mutants with amino acid residue mutations at the following five positions: 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; R41 Q, Q88V, T201N, K284E, Q291A; R41Q, Q88V, T201N, K284E, A306S; R41Q, Q88V, T201N, K284E, A306L; R 41Q, Q88V, T201N, K284E, A306M; R41Q, Q88V, T201N, K284E, A306V; R41Q, Q88V, T201N, K284E, A306E;

[0012] Or mutants with amino acid residue mutations at the following six positions: 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 there are mutants with amino acid residue mutations at the following seven positions:

[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 invention provides a gene encoding the acylase mutant.

[0016] The present invention provides a recombinant expression vector containing the coding gene, specifically in the form of a pET vector framework.

[0017] The present invention provides a genetically engineered bacterium containing the coding gene or the recombinant expression vector, such as Escherichia coli.

[0018] The present invention provides the acylase mutant or the use of its encoding nucleic acid in the preparation of octanoyl amino acid compounds, specifically in catalyzing the conversion of octanoic acid into octanoyl amino acids.

[0019] The present invention provides a method for preparing an octanoyl amino acid compound, which comprises using the genetically engineered bacteria as a whole-cell catalyst, octanoic acid and amino acids as substrates, and synthesizing the octanoyl amino acid compound;

[0020] 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.

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

[0022] The reaction conditions include using a wet bacterial cell dosage of 5-30 g / L, an octanoic acid concentration of 1.44-72 g / L, a 60%-100% saturated amino acid solution, and a pH 6.0-8.0 phosphate buffer as a reaction medium to form a reaction system, and performing a biocatalytic reaction for 20-28 hours at a temperature of 35-65° C. and a shaking table speed of 100-800 rpm to obtain an octanoyl amino acid compound.

[0023] The present invention provides a sheep-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 octanoic acid and amino acids are used as substrates to synthesize an octanoyl amino acid surfactant, thereby greatly improving production efficiency and having high production application value. DETAILED DESCRIPTION

[0024] 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 so-called mutation site in the following examples refers to the difference position in the amino acid sequence of the mutant acylase derived from sheep containing the amino acid sequence with SEQ ID NO: 1.

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

[0026] The gene was synthesized according to the reported sheep acylase (Ovis aries aminoacylase 1 (ACY1), transcript variant X1, mRNA, Ovis aries (sheep), XM_004018411.6) and named pAcy. Its amino acid sequence is shown in SEQ ID NO: 1, and its nucleic acid sequence of the cDNA coding region is shown in SEQ ID NO: 2.

[0027] 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.

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

[0029] 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.

[0030] 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.

[0031] 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 a transformant (1). A plasmid was prepared from the above-mentioned bacteria using the Tiangen plasmid mini-extraction kit to obtain a recombinant plasmid pET-28a (+) -Acy. DNA sequencing confirmed that the nucleotide sequence of the coding region of the plasmid was consistent with sequence 2. The transformant (1) produced an acylase (1) as a mutant acylase derived from sheep.

[0032] 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.

[0033] The wet cells obtained above were used to determine the octanoyl amino acid synthesis activity. The wet cell dosage of the synthesis reaction system was 5-30 g / L, the octanoic acid concentration was 1.44-72 g / L, 60%-100% saturated amino acid solution, pH 6.0-8.0 phosphate buffer was used as the reaction medium to form the reaction system, and the biocatalytic reaction was carried out for 20-28 hours at a temperature of 35-65° C. and a shaking speed of 100-800 rpm to obtain an octanoyl amino acid compound.

[0034] [Example 2] Obtaining mutant acylase (2)

[0035] By introducing random mutations into candidate transformation sites, saturation mutations are performed, low conversion rate sites are eliminated, and advantageous mutation points with high conversion rates are retained. These sites are then combined and superimposed, and new mutation sites are introduced during the superposition process, and multiple rounds of evolutionary screening are performed.

[0036] 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.

[0037] The PCR product was analyzed by 1% agarose gel electrophoresis, and the presence of an amplified product of about 6.5 kb was confirmed.

[0038] 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.

[0039] Escherichia coli BL21(DE3) was transformed to obtain a transformant (2). The transformant (2) produced mutant (2) which was a dominant mutant acylase derived from sheep.

[0040] The transformant (2) produced acylase (2) having the mutations described in the table based on Example 1, and its culture and activity determination scheme were the same as Example 1.

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

[0042] In order to obtain a transformant (3) expressing a mutant acylase having an amino acid residue substitution as shown in Table 1, appropriate primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (3). The transformant (3) produced an acylase (3) having the mutation described in the table.

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

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

[0045] In order to obtain a transformant (4) expressing a mutant acylase having an amino acid residue substitution as shown in Table 1, appropriate primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (4). The transformant (4) produced an acylase (4) having the mutation described in the table.

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

[0047] Table 1

[0048]

[0049] 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. From Table 1, it can be found that sites 41, 201, and 284 can be used as advantageous sites for improving the conversion rate, and saturation mutations can be performed on them to observe whether there is a possibility of mutations with higher conversion rates.

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

[0051] In order to obtain a transformant (5) expressing a mutant acylase having an amino acid residue substitution as shown in Table 2, appropriate primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (5). The transformant (5) produced an acylase (5) having the mutations described in the table.

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

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

[0054] In order to obtain a transformant (6) expressing a mutant acylase having an amino acid residue substitution as shown in Table 2, appropriate primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (6). The transformant (6) produced an acylase (6) having the mutation described in the table.

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

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

[0057] In order to obtain a transformant (7) expressing a mutant acylase having an amino acid residue substitution as shown in Table 2, appropriate primers were designed and the same operation as in Example 2 was performed to obtain a plasmid to obtain a transformant (7). The transformant (7) produced an acylase (7) having the mutation described in the table.

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

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

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

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

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

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

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

[0065] [Example 10] Obtaining mutant acylase (10)

[0066] In order to obtain a transformant (10) expressing a mutant acylase having an amino acid residue substitution as shown in Table 2, appropriate primers were designed and a plasmid was obtained by the same operation as in Example 2 to obtain a transformant (10).

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

[0068] Table 2

[0069]

[0070] It can be found from Table 2 that the conversion rate of Q88 mutation to V and L is greatly improved, and the M, S, A, and G mutations are improved. These mutations can be used as advantageous sites for improving the conversion rate.

[0071] [Example 11] Obtaining mutant acylase (11)

[0072] In order to obtain a transformant (11) expressing a mutant acylase having an amino acid residue substitution as shown in Table 3, appropriate primers were designed and a plasmid was obtained by the same operation as in Example 2 to obtain a transformant (11).

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

[0074] [Example 12] Obtaining mutant acylase (12)

[0075] In order to obtain a transformant (12) expressing a mutant acylase having an amino acid residue substitution as shown in Table 3, appropriate primers were designed and a plasmid was obtained by the same operation as in Example 2 to obtain a transformant (12).

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

[0077] [Example 13] Obtaining mutant acylase (13)

[0078] In order to obtain a transformant (13) expressing a mutant acylase having an amino acid residue substitution as shown in Table 3, appropriate primers were designed and a plasmid was obtained by the same operation as in Example 2 to obtain a transformant (13).

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

[0080] [Example 14] Obtaining mutant acylase (14)

[0081] In order to obtain a transformant (14) expressing a mutant acylase having an amino acid residue substitution as shown in Table 3, appropriate primers were designed and a plasmid was obtained by the same operation as in Example 2 to obtain a transformant (14).

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

[0083] Table 3

[0084]

[0085] It can be found from Table 3 that the conversion rates of A183 site mutations to M, S, N, and L are improved to varying degrees, among which A183M has the most significant improvement and can be used as a dominant mutation for improving conversion rate.

[0086] [Example 15] Obtaining mutant acylase (15)

[0087] In order to obtain a transformant (15) expressing a mutant acylase having an amino acid residue substitution as shown in Table 4, appropriate primers were designed and a plasmid was obtained by the same operation as in Example 2 to obtain a transformant (15).

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

[0089] [Example 16] Obtaining mutant acylase (16)

[0090] In order to obtain a transformant (16) expressing a mutant acylase having an amino acid residue substitution as shown in Table 4, appropriate primers were designed and a plasmid was obtained by the same operation as in Example 2 to obtain a transformant (16).

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

[0092] [Example 17] Obtaining mutant acylase (17)

[0093] In order to obtain a transformant (17) expressing a mutant acylase having an amino acid residue substitution as shown in Table 4, appropriate primers were designed and a plasmid was obtained by the same operation as in Example 2 to obtain a transformant (17).

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

[0095] Table 4

[0096]

[0097] It can be found from Table 4 that mutation of T201 site to V, I, and A can improve the conversion rate, among which T201V and T201I have slight improvements, while T201A has the greatest improvement. This site can be introduced as a mutation to improve the conversion rate.

[0098] [Example 18] Obtaining mutant acylase (18)

[0099] In order to obtain a 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 by the same operation as in Example 2 to obtain a transformant (18).

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

[0101] [Example 19] Obtaining mutant acylase (19)

[0102] In order to obtain a 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 by the same operation as in Example 2 to obtain a transformant (19).

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

[0104] [Example 20] Obtaining mutant acylase (20)

[0105] In order to obtain a 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 by the same operation as in Example 2 to obtain a transformant (20).

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

[0107] [Example 21] Obtaining mutant acylase (21)

[0108] In order to obtain a 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 by the same operation as in Example 2 to obtain a transformant (21).

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

[0110] [Example 22] Obtaining mutant acylase (22)

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

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

[0113] Table 5

[0114]

[0115] It can be found from Table 5 that the conversion rate is improved when K284 is mutated to D, E, L, Y, and E, and this site can be used as a dominant site for improving the conversion rate. [Example 23] Obtaining mutant acylase (23)

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

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

[0118] [Example 24] Obtaining mutant acylase (24)

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

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

[0121] [Example 25] Obtaining mutant acylase (25)

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

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

[0124] [Example 26] Obtaining mutant acylase (26)

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

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

[0127] [Example 27] Obtaining mutant acylase (27)

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

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

[0130] [Example 28] Obtaining mutant acylase (28)

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

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

[0133] Table 6

[0134]

[0135] It can be found from Table 6 that the conversion rate of the two-point combination is improved compared with the single-point combination, and it can be used as the mother of a new round of evolution to continue superimposing mutation research.

[0136] [Example 29] Obtaining mutant acylase (29)

[0137] In order to obtain a transformant (29) expressing a mutant acylase having amino acid residue substitutions as shown in Table 7, a vector obtained by plasmid extraction from transformant (23) was used as a cloning parent, appropriate primers were designed, and the plasmid was obtained by the same operation as in Example 2 to obtain a transformant (29).

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

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

[0140] In order to obtain a transformant (30) expressing a mutant acylase having amino acid residue substitutions as shown in Table 7, a vector obtained by plasmid extraction from transformant (23) was used as a cloning parent, appropriate primers were designed, and the plasmid was obtained by the same operation as in Example 2 to obtain a transformant (30).

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

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

[0143] In order to obtain a transformant (31) expressing a mutant acylase having amino acid residue substitutions as shown in Table 7, a vector obtained by plasmid extraction from transformant (23) was used as a cloning parent, appropriate primers were designed, and the plasmid was obtained by the same operation as in Example 2 to obtain a transformant (31).

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

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

[0146] In order to obtain a transformant (32) expressing a mutant acylase having amino acid residue substitutions as shown in Table 7, a vector obtained by plasmid extraction from transformant (23) was used as a cloning parent, appropriate primers were designed, and the plasmid was obtained by the same operation as in Example 2 to obtain a transformant (32).

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

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

[0149] In order to obtain a transformant (33) expressing a mutant acylase having amino acid residue substitutions as shown in Table 7, a vector obtained by plasmid extraction from transformant (23) was used as a cloning parent, appropriate primers were designed, and the plasmid was obtained by the same operation as in Example 2 to obtain a transformant (33).

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

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

[0152] In order to obtain a transformant (34) expressing a mutant acylase having amino acid residue substitutions as shown in Table 7, a vector obtained by plasmid extraction from transformant (23) was used as a cloning parent, appropriate primers were designed, and the plasmid was obtained by the same operation as in Example 2 to obtain a transformant (34).

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

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

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

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

[0157] Table 7

[0158]

[0159] It can be seen from Table 7 that these three-point mutation combinations are improved compared to one-point and two-point combinations, among which the combination of 41, 201, and 284 performs best. [Example 36] Obtaining mutant acylase (36)

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

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

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

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

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

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

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

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

[0168] Table 8

[0169]

[0170]

[0171] It can be found from Table 8 that 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 improves conversion rate the most and can be used as the mother version for a new round of evolution.

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

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

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

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

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

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

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

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

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

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

[0182] In order to obtain a transformant (37) expressing a mutant acylase having amino acid residue substitutions as shown in Table 9, a vector obtained by plasmid extraction from the transformant (40) was used as a cloning parent, appropriate primers were designed, and the plasmid was obtained by the same operation as in Example 2 to obtain a transformant (42).

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

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

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

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

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

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

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

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

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

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

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

[0194] In order to obtain a transformant (46) expressing a mutant acylase having amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from transformant (37) was used as a cloning parent, appropriate primers were designed, and the plasmid was obtained by the same operation as in Example 2 to obtain a transformant (46).

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

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

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

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

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

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

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

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

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

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

[0205] Table 9

[0206]

[0207]

[0208] It can be found from Table 9 that by superimposing the advantageous sites obtained from the above evolution, the conversion rate of the five-point combination mutation obtained is improved compared with the four-point combination.

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

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

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

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

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

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

[0215] [Example 52] Obtaining mutant acylase (52)

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

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

[0218] Table 10

[0219]

[0220] It can be found from Table 10 that the introduction of site combination helps to improve the transformation rate, and a mutant with a six-site combination is obtained.

[0221] [Example 53] Obtaining mutant acylase (53)

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

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

[0224] [Example 54] Obtaining mutant acylase (54)

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

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

[0227] [Example 55] Obtaining mutant acylase (55)

[0228] In order to obtain a transformant (55) expressing a mutant acylase having amino acid residue substitutions as shown in Table 11, a vector obtained by plasmid extraction from the transformant (52) was used as a cloning parent, appropriate primers were designed, and the plasmid was obtained by the same operation as in Example 2 to obtain a transformant (55).

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

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

[0231] In order to obtain a transformant (56) expressing a mutant acylase having amino acid residue substitutions as shown in the table, a vector obtained by plasmid extraction from the transformant (52) was used as a cloning parent, appropriate primers were designed, and the plasmid was obtained by the same operation as in Example 2 to obtain a transformant (56).

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

[0233] Table 11

[0234]

[0235]

[0236] It can be found from Table 11 that compared with the six-point combination, the seven-point combination still has different degrees of improvement in conversion rate by introducing different mutation forms, which proves that the mutation site superposition scheme still has room for improvement.

Claims

1. A sheep-derived acylase mutant, characterized in that: It is a mutant having one, two, three, four, five, six or more amino acid residue substitution mutations selected from the following positions based on the amino acid sequence of the acylase 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.

2. The acylase mutant according to claim 1, characterized in that There are mutants with mutations in the amino acid residues at the following two positions: R41Q, Q88V; R41Q, T201N; R41Q, A183M; Q88V, T201N; Q88V, K284E; A183M, K284E.

3. The acylase mutant according to claim 1, characterized in that There are mutants with amino acid residue mutations at the following three positions: R41Q, Q88V, T201N; R41Q, Q88V, A183M; R41Q, T201N, K284E; R41Q, A183M, K284E; Q88V, T201N, A183M; Q88V, T201N, K284E; T201N, A183M, K284E.

4. The acylase mutant according to claim 1, characterized in that There are mutants with amino acid residue mutations at the following four positions: R41Q, Q88V, T201N, A183M; R41Q, Q88V, T201N, K284E; R41Q, T201N, A183M, K284E; Or there are mutants with amino acid residue mutations at the following five positions: 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, T201 N, K284E, Q291A; R41Q, Q88V, T201N, K284E, A306S; R41Q, Q88V, T201N, K284E, A306L; R41Q, Q8 8V, T201N, K284E, A306M; R41Q, Q88V, T201N, K284E, A306V; R41Q, Q88V, T201N, K284E, A306E; Or there are mutants with amino acid residue mutations at the following six positions: R41Q, Q88V, T201N, K284E, E231K, A306S; R41Q, Q88V, T201N, R41Q, Q88V, T201N, K284E, E23 1K, Q291M; R41Q, Q88V, T201N, R41Q, Q88V, T201N, K284E, Q291E, A306S; R41Q, Q88V, T201N; Or there are mutants with amino acid residue mutations at the following seven positions: 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.

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 preparing an octanoyl amino acid compound, in particular, in catalyzing the conversion of octanoic acid into octanoyl amino acid.

9. A method for preparing an octanoyl amino acid compound, characterized in that: The method comprises using the genetically engineered bacteria as claimed in claim 7 as a whole-cell catalyst and using octanoic acid and amino acids as substrates to synthesize octanoyl amino acid compounds; 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, characterized in that: The amino acid is selected from glycine, arginine, glutamic acid, lysine, and alanine; The reaction conditions include using a wet bacterial cell dosage of 5-30 g / L, an octanoic acid concentration of 1.44-72 g / L, a 60%-100% saturated amino acid solution, and a pH 6.0-8.0 phosphate buffer as a reaction medium to form a reaction system, performing a biocatalytic reaction for 20-28 hours at a temperature of 35-65° C. and a shaking table speed of 100-800 rpm to obtain an octanoyl amino acid compound.

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