Acyltransferase mutants with high acetylation activity and use thereof

Abstract

The application discloses an acyltransferase mutant with high acetylation activity and application thereof, and belongs to the technical field of enzyme engineering. The acyltransferase mutant provided by the application contains one or more mutation sites in the 62th and 64th positions on the basis of the acyltransferase mutant with the amino acid sequence shown in SEQ ID NO. 1. The acyltransferase mutant obtained by the application has higher acyltransferase activity and catalytic efficiency than the original enzyme before mutation, and can improve the yield of N-acetyl-trans-4-hydroxyproline. Therefore, the acyltransferase mutant of the application has important significance in the efficient biosynthesis of N-acetyl-trans-4-hydroxyproline, and has wide application prospects in the fields of biological materials, medicines or cosmetics.

Description

TECHNICAL FIELD

[0001] The present application relates to an acyltransferase mutant with high acetylation activity and its application, belonging to the technical field of enzyme engineering. BACKGROUND

[0002] N-acetyl-trans-4-hydroxyproline is widely used in the fields of cosmetic care and biological medicine, and has high market demand and economic value. Currently, N-acetyl-trans-4-hydroxyproline on the market is mainly synthesized by chemical reaction of hydroxyproline with acetylating reagents. This process is complex, has tedious separation steps and low efficiency, resulting in high production cost. In addition, chemical synthesis has a negative impact on the environment, limiting its popularization and application in industry.

[0003] In recent years, enzyme-catalyzed synthesis of N-acetyl-trans-4-hydroxyproline has shown good development momentum. Acyltransferase MsAcT from Mycobacterium smegmatis has become one of the most concerned acyltransferases due to its high activity, high stability and wide range of esterification reaction substrates. Patent CN117586979A discloses an acyltransferase mutant, which uses hydroxyproline as a substrate and utilizes a genetically engineered strain expressing the acyltransferase mutant in the technology for catalysis. The yield of N-acetyl-trans-4-hydroxyproline reaches 312.5 mg / L or more. Among them, the genetically engineered strain expressing mutant K97A / F154A can synthesize N-acetyl-trans-4-hydroxyproline with a yield of 488 mg / L.

[0004] However, the yield of N-acetyl-trans-4-hydroxyproline is still low. The research on the synthesis of N-acetyl-trans-4-hydroxyproline using MsAcT for catalysis is still in the primary stage, and the catalytic efficiency of the enzyme needs to be further improved. In addition, MsAcT has mixed hydrolytic enzyme and acyltransferase activity, and its high hydrolytic enzyme activity hinders the accumulation of the product. Therefore, further exploration and development of MsAcT to improve its catalytic efficiency and acyltransferase activity (AT:H) is the key to improving the yield of N-acetyl-trans-4-hydroxyproline, and has extremely high economic and practical value. SUMMARY

[0005] In order to further tap the potential of acyltransferase (MsAcT) and improve the enzyme activity of acyltransferase, the present application provides an acyltransferase mutant. Compared with the mutant before mutation, the conversion rate of the mutant for hydroxyproline is greatly improved, further improving the yield of N-acetyl-trans-4-hydroxyproline.

[0006] A first object of the present application is to provide an acyltransferase mutant with high acetylation activity, which contains one or more mutation sites at positions 62 and 64 based on an acyltransferase mutant with an amino acid sequence as shown in SEQ ID NO. 1.

[0007] SEQ ID NO. 1:

[0008] MAKRILCFGDCLTWGWVPVEDGAPTERFAPDVRWTGVLAQQLGADFEVIEEGLSARTTNIDDPTDPRLNGASYLPSCLATHLPLDLVIIMLGTNDTKAYFRRTPLDIALGMSVLVTQVLTSAGGVGTTYPAPKVLVVSPPPLAPMPHPWFQLIFEGGEQKTTELARVYSALASFMKVPFFDAGSVISTDGVDGIHFTEANNRDLGVALAEQVRSLL

[0009] In an embodiment of the present application, a nucleotide sequence encoding the acyltransferase mutant with an amino acid sequence as shown in SEQ ID NO. 1 is as shown in SEQ ID NO. 2.

[0010] SEQ ID NO. 2:

[0011] ATGGCCAAACGCATCTTATGTTTCGGAGACtgcCTGACTTGGGGATGGGTTCCCGTGGAAGACGGTGCTCCTACTGAACGCTTTGCGCCAGATGTACGCTGGACCGGCGTATTAGCTCAGCAGTTAGGCGCTGACTTTGAAGTCATTGAGGAAGGTTTGTCTGCCCGTACGACAAACATCGATGATCCCACCGACCCGCGTTTGAATGGAGCGAGCTATCTTCCCTCGTGCCTGGCAACTCACTTACCGCTGGATCTTGTTATTATCATGTTGGGCACAAACGACACCAAGGCGTATTTCCGCCGCACACCCCTTGATATTGCACTTGGGATGTCGGTGCTTGTCACACAGGTCTTGACATCCGCGGGGGGAGTAGGCACGACATATCCCGCACCGAAAGTATTAGTCGTCTCGCCTCCTCCCTTGGCACCCATGCCTCACCCCTGGTTTCAACTGATTTTTGAGGGCGGAGAACAAAAGACAACGGAACTTGCCCGTGTGTATTCAGCGCTGGCTTCGTTTATGAAAGTCCCGTTCTTTGATGCCGGTAGCGTGATCAGTACAGACGGCGTAGATGGTATCCACTTTACGGAGGCCAATAACCGTGATCTGGGGGTGGCACTGGCGGAGCAAGTTCGCTCACTTTTA

[0012] wherein, in the amino acid sequence of SEQ ID NO. 1, the amino acid residues at the 62nd and 64th amino acid sites are within the active pocket of the acyltransferase MsAcT.

[0013] In an embodiment of the present application, the acyltransferase mutant is obtained by mutating the aspartic acid at the 62nd amino acid site of the acyltransferase mutant having the amino acid sequence of SEQ ID NO. 1 to lysine, and is named as D62K.

[0014] In an embodiment of the present application, the acyltransferase mutant is obtained by mutating the aspartic acid at the 62nd amino acid site of the acyltransferase mutant having the amino acid sequence of SEQ ID NO. 1 to arginine, and is named as D62R.

[0015] ​In one embodiment of the present invention, the threonine at position 64 of the acyltransferase mutant having the amino acid sequence shown in SEQ ID NO. 1 is mutated to asparagine, and is named: T64N.

[0016] The present invention also provides a gene encoding the mutant or a recombinant vector carrying the gene.

[0017] The present invention also provides a recombinant cell expressing the mutant or carrying the gene or the recombinant vector.

[0018] In one embodiment of the present invention, the recombinant cell uses bacteria or fungi as expression hosts.

[0019] In one embodiment of the present invention, the host of the recombinant cell is Escherichia coli C43 (DE3).

[0020] The present invention also provides a recombinant enzyme catalyst containing the above-mentioned acyltransferase mutant sequence, wherein the catalyst is in any one of the following forms:

[0021] (1) culturing a recombinant expression transformant, isolating a transformant cell expressing a recombinase containing the acyltransferase mutant sequence, and obtaining the recombinase catalyst;

[0022] (2) culturing the recombinant expression transformant, isolating the transformant cells expressing the recombinant enzyme containing the acyltransferase mutant sequence, disrupting the transformant cells expressing the recombinant enzyme to obtain a cell disrupted liquid, and obtaining the recombinant enzyme catalyst;

[0023] (3) culturing the recombinant expression transformant, isolating the transformant cells expressing the recombinant enzyme containing the acyltransferase mutant sequence, disrupting the transformant cells expressing the recombinant enzyme to obtain a cell disruption solution, freeze-drying the cell disruption solution to obtain a freeze-dried enzyme powder, and obtaining the recombinant enzyme catalyst.

[0024] The present invention also provides a method for increasing acyltransferase activity (AT:H); the AT:H is the ratio of acyltransferase activity to hydrolysis activity;

[0025] The method comprises mutating the aspartic acid at position 62 of the acyltransferase mutant of the amino acid sequence shown in SEQ ID NO. 1 to lysine or arginine;

[0026] Alternatively, the threonine at position 64 of the acyltransferase mutant having the amino acid sequence shown in SEQ ID NO. 1 is mutated to asparagine.

[0027] The application also provides a method for improving the yield or conversion rate of N-acetyl-trans-4-hydroxyproline, which comprises adding the acyltransferase mutant, the recombinant cell or the recombinant enzyme catalyst into a reaction system containing hydroxyproline and an acyl donor to prepare N-acetyl-trans-4-hydroxyproline.

[0028] In an embodiment of the application, the acyl donor is vinyl acetate.

[0029] In an embodiment of the application, the reaction system further contains a phosphate buffer.

[0030] In an embodiment of the application, the final concentration of hydroxyproline in the system is 100-350 mM, and the final concentration of the acyl donor in the system is 1-20% v / v.

[0031] In an embodiment of the application, the method comprises adding MsAcT mutant with a final concentration of 0.4 mg / mL into 100 mM phosphate buffer (pH 7.0) containing 250 mM hydroxyproline and 10% (v / v) vinyl acetate, and reacting at 45°C for 12 h.

[0032] The application also provides the use of the acyltransferase mutant, the recombinant cell or the recombinant enzyme catalyst in catalyzing the preparation of N-acetyl-trans-4-hydroxyproline from hydroxyproline.

[0033] In an embodiment of the application, the use comprises adding the acyltransferase mutant, the recombinant cell or the recombinant enzyme catalyst into a reaction system containing hydroxyproline and an acyl donor to prepare N-acetyl-trans-4-hydroxyproline.

[0034] In an embodiment of the application, the acyl donor is vinyl acetate.

[0035] In an embodiment of the application, the reaction system further contains a phosphate buffer.

[0036] In an embodiment of the application, the final concentration of hydroxyproline in the system is 100-350 mM, and the final concentration of the acyl donor in the system is 1-20% v / v.

[0037] In an embodiment of the application, the use comprises adding MsAcT mutant with a final concentration of 0.4 mg / mL into 100 mM phosphate buffer (pH 7.0) containing 250 mM hydroxyproline and 10% (v / v) vinyl acetate, and reacting at 45°C for 12 h.

[0038] The application also provides application of the mutant, the gene, the recombinant carrier, the recombinant cell, the recombinant enzyme catalyst or the method in the field of biomaterials, medicines or cosmetics.

[0039] Beneficial effects:

[0040] The application is based on structural information combined with simulation calculation, adopts PCR method for site-directed saturation mutation, and finally obtains MsAcT mutants with high acyltransferase activity. The acyltransferase mutants obtained by the application have higher acyltransferase activity and catalytic efficiency than the original enzyme before mutation, and can improve production efficiency. Among them, the mutant D62K has the best effect, and the enzyme activity is as high as 5.9; when catalyzing the substrate, the yield of N-acetyl-trans-4-hydroxyproline is 7.27 g / L, which is increased by 160% compared with that before mutation.

[0041] Therefore, the acyltransferase mutants of the application have important significance in the efficient biosynthesis of N-acetyl-trans-4-hydroxyproline, and have wide application prospects in the field of biomaterials, medicines or cosmetics. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 is a figure according to the energy screening result of alanine scanning combined with simulation mutation;

[0043] Figure 2 is a figure according to the energy screening site result of simulation mutation;

[0044] Figure 3 is an agarose gel electrophoresis figure. DETAILED DESCRIPTION

[0045] The technical solutions of the application will be described below in combination with the embodiments of the application. Obviously, the described embodiments are only a part of the embodiments of the application, rather than all the embodiments of the application. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the application. The materials and reagents used in the embodiments are commercially available if not otherwise specified.

[0046] The culture medium and buffer involved in the embodiments are as follows:

[0047] LB solid culture medium (1L): 10g of tryptone, 5g of yeast powder, 10g of NaCl and 17g of agar powder.

[0048] LB liquid culture medium: 10g of tryptone, 5g of yeast powder and 10g of NaCl are dissolved in deionized water and then diluted to 1000mL.

[0049] Binding Buffer: Dissolve 17.54 g of NaCl and 6.00 g of NaH2PO4 in deionized water and dilute to 1000 mL. Adjust the pH to 8 with NaOH solution.

[0050] Elution buffer with different imidazole concentrations: NaCl 29.22 g, Tris 2.42 g, imidazole 0.68 g / 27.23 g / 68.32 g (final concentration 2 / 400 / 1000 mM) were dissolved in deionized water and the volume was adjusted to 1000 mL. The pH was adjusted to 8 with hydrochloric acid.

[0051] The p-nitrophenyl acetate involved in the examples was purchased from Sangon Biotech (Shanghai) Co., Ltd. with the product number 830-03-5; hydroxyproline was purchased from Sangon Biotech (Shanghai) Co., Ltd. with the product number 51-35-4; all competent cells used were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0052] Example 1: Construction of a highly active acyltransferase mutant

[0053] 1. Site-directed mutagenesis strategy

[0054] The active pocket of acyltransferase MsAcT (S11C) (amino acid sequence SEQ ID NO. 1) was analyzed using Discovery studio software. Alanine scanning was performed on all amino acid residues in the nucleotide sequence of ... Figure 1 ), and further simulate saturation mutation prediction to screen the optimal mutation method ( Figure 2 ), and finally determined 9 mutation modes, including D62E, D62H, D62K, D62R, D62W, T64H, T64F, T64Y and T64N.

[0055] 2. Construction of mutant recombinant plasmid

[0056] (1) Chemically synthesizing a gene encoding an acyltransferase mutant MsAcT(S11C) having an amino acid sequence as shown in SEQ ID NO. 1 (the nucleotide sequence of the gene is shown in SEQ ID NO. 2), wherein MsAcT(S11C) is obtained by mutating the serine at position 11 of the wild-type acyltransferase MsAcT to cysteine;

[0057] (2) Construction of recombinant vector: using the primer of Table 1, using the commercial plasmid pET22b as the skeleton, the acyltransferase Misact (S11C) sequence is connected to the pET-22b (+) vector as the target gene template, linear amplification is carried out by PCR technology respectively, after linear amplification, using One Step Seamless Cloning Mix homologous recombination enzyme, incubating at 50°C for 30 min, the target gene MsAcT (S11C) is connected to the pET22b vector, and the recombinant vector pET-22b (+)-MsAcT (S11C) is obtained;

[0058] Table 1 primer sequence

[0059]

[0060] (3) Using the nucleotide sequence of the recombinant plasmid pET-22b (+)-MsAcT (S11C) as a template, a primer containing a mutation site is designed (Table 2):

[0061] MsAcT(S11C / D62E) mutant (or referred to as D62E mutant) is obtained by mutating aspartic acid at position 62 of the acyltransferase mutant MsAcT(S11C) having the amino acid sequence shown in SEQ ID NO. 1 to glutamic acid; MsAcT(S11C / D62H) mutant (or referred to as D62H mutant) is obtained by mutating aspartic acid at position 62 of the acyltransferase mutant MsAcT(S11C) having the amino acid sequence shown in SEQ ID NO. 1 to histidine; MsAcT(S11C / D62K) mutant (or referred to as D62K mutant) is obtained by mutating aspartic acid at position 62 of the acyltransferase mutant MsAcT(S11C) having the amino acid sequence shown in SEQ ID NO. 1 to lysine; MsAcT(S11C / D62W) mutant (or referred to as D62W mutant) is obtained by mutating aspartic acid at position 62 of the acyltransferase mutant MsAcT(S11C) having the amino acid sequence shown in SEQ ID NO. 1 to tryptophan; MsAcT(S11C / D62R) mutant (or referred to as D62R mutant) is obtained by mutating aspartic acid at position 62 of the acyltransferase mutant MsAcT(S11C) having the amino acid sequence shown in SEQ ID NO. 1 to arginine; MsAcT(S11C / T64H) mutant (or referred to as T64H mutant) is obtained by mutating threonine at position 64 of the acyltransferase mutant MsAcT(S11C) having the amino acid sequence shown in SEQ ID NO. 1 to histidine; MsAcT(S11C / T64F) mutant (or referred to as T64F mutant) is obtained by mutating threonine at position 64 of the acyltransferase mutant MsAcT(S11C) having the amino acid sequence shown in SEQ ID NO. 1 to phenylalanine; MsAcT(S11C / T64Y) mutant (or referred to as T64Y mutant) is obtained by mutating threonine at position 64 of the acyltransferase mutant MsAcT(S11C) having the amino acid sequence shown in SEQ ID NO. 1 to tyrosine; MsAcT(S11C / T64N) mutant (or referred to as T64N mutant) is obtained by mutating threonine at position 64 of the acyltransferase mutant MsAcT(S11C) having the amino acid sequence shown in SEQ ID NO. 1 to asparagine.

[0062] The PCR technique was used to carry out site-directed mutation, and the recombinant plasmids pET-22b(+)-MsAcT(S11C / D62E), pET-22b(+)-MsAcT(S11C / D62H), pET-22b(+)-MsAcT(S11C / D62K), pET-22b(+)-MsAcT(S11C / D62W), pET-22b(+)-MsAcT(S11C / D62R), pET-22b(+)-MsAcT(S11C / T64H), pET-22b(+)-MsAcT(S11C / T64F), pET-22b(+)-MsAcT(S11C / T64Y), and pET-22b(+)-MsAcT(S11C / T64N) were obtained.

[0063] The PCR reaction system is shown in Table 3, and the PCR reaction conditions are as follows: 98 ℃ for 30 s; 98 ℃ for 10 s, 58 ℃ for 5 s, 72 ℃ for 3 min, for a total of 30 cycles; 72 ℃ for 2 min; and 4 ℃ preservation.

[0064] The PCR products were detected by 1% agarose gel electrophoresis. Figure 3 The PCR products were transformed into E. coli JM109 competent cells, positive transformants were selected, plasmids were extracted, and sequencing verification was performed, and finally seven mutants were successfully constructed, which were E. coli-JM09-MsAcT(S11C / D62E), E. coli-JM09-MsAcT(S11C / D62K), E. coli-JM09-MsAcT(S11C / D62R), E. coli-JM09-MsAcT(S11C / T64H), E. coli-JM09-MsAcT(S11C / T64F), E. coli-JM09-MsAcT(S11C / T64Y), and E. coli-JM09-MsAcT(S11C / T64N).

[0065] Table 2 primer

[0066]

[0067] Table 3 PCR reaction system

[0068]

[0069] Example 2: Expression analysis and purification of acyltransferase mutant proteins

[0070] 1. Induced expression of acyltransferase mutant proteins

[0071] The successfully sequencing verified recombinant plasmids pET-22b(+)-MsAcT(S11C), pET-22b(+)-MsAcT(S11C / D62E), pET-22b(+)-MsAcT(S11C / D62K), pET-22b(+)-MsAcT(S11C / D62R), pET-22b(+)-MsAcT(S11C / T64H), pET-22b(+)-MsAcT(S11C / T64F), pET-22b(+)-MsAcT(S11C / T64Y), pET-22b(+)-MsAcT(S11C / T64N) were transformed into E. coli C43(DE3) competent cells which can highly express hydrophobic proteins, i.e. to obtain recombinant E. coli containing the above mutant plasmids.

[0072] The successfully constructed recombinant E. coli was inoculated into 4 mL LB liquid medium containing ampicillin (100 μg / mL) for activation, and cultured at 37°C, 200 rpm for 12 h; then inoculated into 50 mL LB liquid medium at 1% (v / v) inoculation amount, and cultured at 37°C, 220 rpm for 3-4 h to OD 600 = 0.6-0.8; 250 μM IPTG (isopropyl-β-D-thiogalactoside) was added, and low-temperature induction was carried out at 16°C, 220 rpm for 16 h. The fermentation broth was centrifuged at 4°C, 5000 rpm for 5 min, and the bacterial cells were collected; the bacterial cells were resuspended with 20 mL Binding Buffer; the cells were broken by an ultrasonic cell disruptor under the condition of ice water bath for 20 min (power 45% ultrasonic for 2 s, intermittent for 2 s); centrifuged at 4°C, 10000 rpm for 40 min, and the supernatant was used as crude enzyme. 15 μL of the crude enzyme was mixed with 5 μL of 5 SDS PAGE loading buffer, and then subjected to SDS-PAGE analysis at 100°C metal bath for 10 min.

[0073] 2. Protein purification

[0074] The crude enzyme obtained in step 1 was filtered through a 0.22 μm filter membrane and then loaded onto a pre-equilibrated nickel column; first, non-specifically bound proteins were eluted with 10 column volumes of elution buffer (2 mM imidazole); then, the target protein was eluted with 3 column volumes of elution buffer (400 mM imidazole); finally, the remaining impurities were washed with 10 volumes of elution buffer (1000 mM imidazole); the collected protein purification liquid was dialyzed at 4°C for 12 h to remove imidazole (dialysis buffer: 100 mmol / L pH 7.0 phosphate buffer), to obtain the purified enzyme liquid, which was stored in a 4°C refrigerator.

[0075] Finally, seven purified enzyme liquids were obtained, which were a purified enzyme liquid containing MsAcT (S11C), a purified enzyme liquid containing MsAcT (S11C / D62E), a purified enzyme liquid containing MsAcT (S11C / D62K), a purified enzyme liquid containing MsAcT (S11C / D62R), a purified enzyme liquid containing MsAcT (S11C / T64H), a purified enzyme liquid containing MsAcT (S11C / T64F), a purified enzyme liquid containing MsAcT (S11C / T64Y), and a purified enzyme liquid containing MsAcT (S11C / T64N).

[0076] Example 3: Acyltransferase activity (AT:H) determination of acyltransferase mutants

[0077] The hydrolytic and acyltransferase activities of MsAcT mutants were determined using the p-nitrophenyl acyltransferase (pNP-AcT) method. The hydrolytic activity was evaluated by monitoring the release of p-nitrophenol from p-nitrophenyl ester at 405 nm. The acyltransferase activity was determined by measuring the increase in the release of p-nitrophenol (pNP) after the addition of methanol. The acetylation activity of the mutants was reflected by the ratio of acyltransferase activity to hydrolytic activity (AT:H).

[0078] The experiment was carried out at 25°C, and the reaction scale was 1 mL.

[0079] Hydrolytic enzyme activity experiment group: p-nitrophenyl acetate (final concentration of 1 mM), benzyl alcohol (final concentration of 10 mM), the purified enzyme liquid containing MsAcT (S11C), the purified enzyme liquid containing MsAcT (S11C / D62E), the purified enzyme liquid containing MsAcT (S11C / D62K), the purified enzyme liquid containing MsAcT (S11C / D62R), the purified enzyme liquid containing MsAcT (S11C / T64H), the purified enzyme liquid containing MsAcT (S11C / T64F), the purified enzyme liquid containing MsAcT (S11C / T64Y), or the purified enzyme liquid containing MsAcT (S11C / T64N) (final concentration of 10 mg / L) obtained in Example 2, 200 mM potassium phosphate buffer (pH 7.5), and DMSO with a final concentration of 0.1% (v / v) were sequentially added to each well.

[0080] Acylation enzyme activity experiment group: based on the hydrolytic enzyme activity experiment group, benzyl alcohol with a final concentration of 10 mM was added.

[0081] Control group: Add p-nitrophenyl acetate (final concentration of 1 mM), benzyl alcohol (final concentration of 10 mM), 200 mM potassium phosphate buffer (pH 7.5) and 0.1% (v / v) DMSO (final concentration) into each well in sequence.

[0082] After 15 min of reaction of the above three groups, 200 μL was taken and added to a 96-well plate, and the absorbance A of each well of the above three groups was detected at a wavelength of 405 nm using a microplate reader. 405 The amount of generated p-nitrophenol (pNP) was calculated.

[0083] The calculation formula of acyltransferase activity (AT:H) is as follows:

[0084] AT:H = (n2-n0) / (n1-n0)

[0085] Wherein, n0, n1, n2 represent the amount of generated pNP (i.e. absorbance A 405 ) in the Control group, the hydrolytic enzyme activity experimental group and the acyltransferase activity experimental group, respectively.

[0086] The results of the acyltransferase activity determination of the acyltransferase mutants showed that, compared with the original enzyme, the AT:H of four mutants was significantly increased, and they were further applied (Table 4).

[0087] Table 4 Determination of acyltransferase activity (AT:H) of acyltransferase mutants

[0088] Mutant Control MsAcT (S11C) D62E D62K D62R T64N T64Y T64F T64H AT:H 1.03 2.71 2.21 3.94 2.96 3.76 2.19 2.77 2.58

[0089] Example 4: Catalytic synthesis of N-acetyl-trans-4-hydroxyproline by acyltransferase mutants

[0090] The reaction scale was 1 mL.

[0091] Experimental group: Add hydroxyproline with a final concentration of 250 mM, 10% (v / v) vinyl acetate into 100 mM phosphate buffer (pH 7.0), and then add purified enzyme solution containing MsAcT (S11C), purified enzyme solution containing MsAcT (S11C / D62E), purified enzyme solution containing MsAcT (S11C / D62K), purified enzyme solution containing MsAcT (S11C / D62R), purified enzyme solution containing MsAcT (S11C / T64H), purified enzyme solution containing MsAcT (S11C / T64F), purified enzyme solution containing MsAcT (S11C / T64Y) or purified enzyme solution containing MsAcT (S11C / T64N) with a final concentration of 0.4 mg / mL.

[0092] Control: 250 mM final concentration of trans-4-hydroxy-L-proline, 10% (v / v) vinyl acetate was added to 100 mM phosphate buffer (pH 7.0).

[0093] The solution was sampled after 12 h of reaction at 45℃. After adding 4 volumes of methanol to the sample and mixing, the mixture was left to stand at -20℃ for 4 h, then centrifuged at 4℃ at 12000 rpm for 5 min, and the supernatant was aspirated for high performance liquid chromatography detection.

[0094] The HPLC detection conditions for N-acetyl-trans-4-hydroxyproline were as follows: using an Agilent high performance liquid chromatograph, liquid separation was performed by Diamond C-18, 4.6 x 250 mm column: flow rate 0.8 mL / min, mobile phase A was 2% methanol aqueous solution, mobile phase B was 2% methanol and 0.1% phosphoric acid aqueous solution. Column temperature 25℃, detection wavelength 205 nm. Single sample was run for 20 min.

[0095] The results of N-acetyl-trans-4-hydroxyproline production determination of the acyltransferase mutants showed that, compared with the original enzyme MsAcT (S11C), the N-acetyl-trans-4-hydroxyproline production of three mutants was significantly increased, which were T64N, D62R and D62K mutants.

[0096] Table 5 N-acetyl-trans-4-hydroxyproline production determination of acyltransferase mutants

[0097]

[0098] The sequences involved in the present application are as follows:

[0099] SEQ ID NO. 1:

[0100] MAKRILCFGDCLTWGWVPVEDGAPTERFAPDVRWTGVLAQQLGADFEVIEEGLSARTTNIDDPTDPRLNGASYLPSCLATHLPLDLVIIMLGTNDTKAYFRRTPLDIALGMSVLVTQVLTSAGGVGTTYPAPKVLVVSPPPLAPMPHPWFQLIFEGGEQKTTELARVYSALASFMKVPFFDAGSVISTDGVDGIHFTEANNRDLGVALAEQVRSLL

[0101] SEQ ID NO. 2:

[0102] ATGGCCAAACGCATCTTATGTTTCGGAGACtgcCTGACTTGGGGATGGGTTCCCGTGGAAGACGGTGCTCCTACTGAACGCTTTGCGCCAGATGTACGCTGGACCGGCGTATTAGCTCAGCAGTTAGGCGCTGACTTTGAAGTCATTGAGGAAGGTTTGTCTGCCCGTACGACAAACATCGATGATCCCACCGACCCGCGTTTGAATGGAGCGAGCTATCTTCCCTCGTGCCTGGCAACTCACTTACCGCTGGATCTTGTTATTATCATGTTGGGCACAAACGACACCAAGGCGTATTTCCGCCGCACACCCCTTGATATTGCACTTGGGATGTCGGTGCTTGTCACACAGGTCTTGACATCCGCGGGGGGAGTAGGCACGACATATCCCGCACCGAAAGTATTAGTCGTCTCGCCTCCTCCCTTGGCACCCATGCCTCACCCCTGGTTTCAACTGATTTTTGAGGGCGGAGAACAAAAGACAACGGAACTTGCCCGTGTGTATTCAGCGCTGGCTTCGTTTATGAAAGTCCCGTTCTTTGATGCCGGTAGCGTGATCAGTACAGACGGCGTAGATGGTATCCACTTTACGGAGGCCAATAACCGTGATCTGGGGGTGGCACTGGCGGAGCAAGTTCGCTCACTTTTA

[0103] Although the present application has been disclosed in its preferred embodiments with reference to the accompanying drawings, it is to be understood that the application is not limited to those precise embodiments, and that various changes and modifications can be effected therein by one skilled in the art without departing from the spirit or scope of the application. Therefore, the scope of the present application should be determined by the appended claims.

Claims

1. An acyltransferase mutant having high acetylation activity, characterized in that, the aspartic acid at position 62 of the acyltransferase mutant with amino acid sequence as shown in SEQ ID NO. 1 is mutated to lysine; or the aspartic acid at position 62 of the acyltransferase mutant with amino acid sequence as shown in SEQ ID NO. 1 is mutated to arginine; or the threonine at position 64 of the acyltransferase mutant with amino acid sequence as shown in SEQ ID NO. 1 is mutated to asparagine.

2. A gene encoding the mutant of claim 1 or a recombinant vector carrying the gene.

3. A recombinant cell expressing the mutant of claim 1 or carrying the gene of claim 2 or the recombinant vector.

4. The recombinant cell of claim 3, wherein, The recombinant cell is a bacterial or fungal expression host.

5. A recombinant enzyme catalyst comprising the acylase mutant sequence of claim 1, wherein, The catalyst is in any of the following forms: (1) culturing the recombinant expression transformant, isolating the transformant cells expressing the recombinant enzyme containing the sequence of the acyltransferase mutant of claim 1 to obtain the recombinant enzyme catalyst; (2) culturing the recombinant expression transformant, isolating the transformant cells expressing the recombinant enzyme containing the sequence of the acyltransferase mutant of claim 1, crushing the transformant cells expressing the recombinant enzyme to obtain a cell crushing solution, and obtaining the recombinant enzyme catalyst; (3) culturing the recombinant expression transformant, isolating the transformant cells expressing the recombinant enzyme containing the sequence of the acyltransferase mutant of claim 1, crushing the transformant cells expressing the recombinant enzyme to obtain a cell crushing solution, freeze-drying the cell crushing solution to obtain a freeze-dried enzyme powder, and obtaining the recombinant enzyme catalyst.

6. A method of increasing acyltransferase AT:H, characterized by, the aspartic acid at position 62 of the acyltransferase mutant with amino acid sequence as shown in SEQ ID NO. 1 is mutated to lysine or arginine; or the threonine at position 64 of the acyltransferase mutant with amino acid sequence as shown in SEQ ID NO. 1 is mutated to asparagine; wherein the AT:H is the ratio of acyltransferase activity to hydrolysis activity.

7. Use of the acyltransferase mutant of claim 1, the gene or recombinant vector of claim 2, the recombinant cell of claim 3 or 4, or the recombinant enzyme catalyst of claim 5 in catalyzing the preparation of N-acetyl-trans-4-hydroxyproline from hydroxyproline.

8. Use according to claim 7, characterized in that, The use is adding the acyltransferase mutant of claim 1, the recombinant cell of claim 3 or 4, or the recombinant enzyme catalyst of claim 5 to a reaction system containing hydroxyproline and an acyl donor to prepare N-acetyl-trans-4-hydroxyproline.

9. Use according to claim 8, characterized in that, The acyl donor is vinyl acetate; the reaction system further contains a phosphate buffer; the final concentration of hydroxyproline in the system is 100-350 mM, and the final concentration of the acyl donor in the system is 1-20% v / v.

10. A method for improving the yield or conversion of N-acetyl-trans-4-hydroxyproline, characterized in that, The method is that the acyltransferase mutant of claim 1 or the recombinant cell of claim 3 or 4 or the recombinant enzyme catalyst of claim 5 is added to a reaction system containing hydroxyproline and an acyl donor to carry out a reaction, thereby preparing N-acetyl-trans-4-hydroxyproline.

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