CN115960862B - Cutinase mutant and application thereof in synthesis of 1, 4-diacetylpiperazine - Google Patents

Abstract

A cutinase mutant and application thereof in synthesizing 1, 4-diacetylpiperazine belong to the technical field of enzyme engineering. Aiming at the problems of poor recognition and low catalytic activity of piperazine nitrogen heterocyclic substrates of wild cutinase, the invention carries out rational mutation on natural cutinase to obtain two cutinase mutants with stronger recognition and binding capacities on piperazine. The obtained cutinase mutant is added into a solvent containing piperazine and an acetyl donor, so that piperazine can be directly acylated into 1, 4-diacetylpiperazine, and the yield of the 1, 4-diacetylpiperazine is improved. The invention provides a novel biocatalysis tool for the acylation reaction of piperazine nitrogen heterocyclic compounds.

Description

A cutinase mutant and its application in the synthesis of 1,4-diacetylpiperazine

Technical Field

The invention belongs to the technical field of enzyme engineering, and particularly relates to a cutinase mutant and application thereof in synthesizing 1,4-diacetylpiperazine.

Background technique

Piperazine is an important pharmaceutical intermediate. It can be used to synthesize dozens of drugs such as piperazine phosphate, guanidine piperazine, and enoxacin. It can also be used to produce wetting agents, emulsifiers, dispersants, antioxidants, preservatives, stabilizers, plastic processing, and rubber additives. Piperazine-modified drugs and functional reagents mainly introduce piperazine molecules through acylation reactions. Piperazine has two secondary amine structures. Through acetylation modification and deacetylation reactions, the two secondary amines can be connected to different functional groups, which can greatly enrich the types of drug structures and significantly affect the pharmacokinetic properties of such compounds. Therefore, the acetylation reaction of piperazine, especially the construction of 1,4-diacetylpiperazine compounds, is of great significance to the above-mentioned drugs and functional reagents. At present, the preparation of 1,4-diacetylpiperazine mainly uses traditional chemical methods. This process requires coupling reagents to activate carboxylic acids to form active intermediates such as acyl chlorides, anhydrides, and acyl azides. The active intermediates further undergo nucleophilic substitution with amines to form amides. However, this method has a very low conversion rate and requires the use of many corrosive and highly toxic reagents, resulting in poor atom economy. Under the requirements of green chemistry, a new method for synthesizing 1,4-diacetylpiperazine compounds is urgently needed.

In recent years, biocatalysis (enzyme catalysis) has been popularized as a sustainable technology, does not require any protection or deprotection steps, usually has high enantioselectivity, and can be carried out under mild conditions. The cutinase used in the present invention belongs to α/β hydrolase, has the characteristic glycine (Gly)-tyrosine (Tyr)-serine (Ser) glutamine (Gln)-glycine (Gly) amino acid sequence and serine (Ser)-histidine (His)-aspartic acid (Asp) catalytic triad of cutinase, and can efficiently catalyze dehydration/hydrolysis reaction. Relative to lipase, most cutinases do not have the "lid" in the lipase structure, so the catalytic key amino acid serine of cutinase is exposed to the solvent; in addition, the cutinase catalytic triad is generally located in the surface groove surrounded by hydrophobic amino acids, which is conducive to accommodating substrates. This brings a wider substrate range to cutinase, such as the polymerization and degradation of polyesters, and this characteristic also provides more possibilities for the biocatalytic preparation of 1,4-diacetylpiperazine.

However, the mildness of enzyme catalysis limits the application of enzymes in industrialization. In order to obtain more ideal catalytic activity or a wider range of reaction conditions, it is necessary to modify the existing enzymes based on the wild type through chemical modification, irrational and rational design of enzyme molecular structure, etc.

Summary of the invention

Aiming at the problem that the wild-type cutinase has poor recognition of piperazine nitrogen heterocyclic substrates and low catalytic activity, the present invention performs mutation on the basis of the original cutinase sequence (the original cutinase is derived from Aspergillus oryzae, and the amino acid sequence is shown in SEQ ID NO.5), thereby providing a cutinase mutant with higher catalytic efficiency.

The specific technical solutions of the present invention are as follows:

The first object of the present invention is to provide a cutinase mutant having improved 1,4-diacetylpiperazine productivity, wherein the cutinase mutant is any one of the following mutants:

Mutant cuitinaseM1, the amino acid sequence of which is shown in SEQ ID NO.1;

The mutant cuitinaseM2, the amino acid sequence is shown in SEQ ID NO.2.

The second object of the present invention is to provide the coding sequence of the above-mentioned Cutinase mutant:

The coding sequence of the mutant cuitinaseM1 is shown in SEQ ID NO.3;

The coding sequence of the mutant cuitinaseM2 is shown in SEQ ID NO.4.

The third object of the present invention is to provide a recombinant vector containing the above coding sequence.

The fourth object of the present invention is to provide a recombinant bacterium containing the above recombinant vector.

The fifth object of the present invention is to provide the use of the above-mentioned cutinase mutant, coding sequence, recombinant vector or recombinant bacteria in the synthesis of 1,4-diacetylpiperazine.

The sixth object of the present invention is to provide a method for synthesizing 1,4-diacetylpiperazine, wherein piperazine and an acetyl donor are dissolved in a solvent, the above-mentioned cutinase mutant is added, and 1,4-diacetylpiperazine is obtained by catalysis.

In one embodiment of the present invention, the piperazine concentration is 0.1-5 g/L; the acetyl donor is vinyl acetate; the molar ratio of vinyl acetate to piperazine is 2.2-3:1; and the cutinase concentration is 0.1-10 mg/L.

In one embodiment of the present invention, the solvent is any one or a mixture of two or more of N,N-dimethylformamide, acetonitrile, dimethyl sulfoxide, tetrahydrofuran, n-hexane, ethyl acetate and water.

In one embodiment of the present invention, the catalytic reaction temperature is 20-50° C., and the reaction time is 0.5-24 h.

The seventh object of the present invention is to provide a method for preparing the above-mentioned Cutinase mutant, comprising the following steps:

(1) constructing a plasmid vector containing a gene encoding the cutinase mutant;

(2) Introducing the mutant plasmid into Escherichia coli BL21;

(3) selecting positive clones for fermentation culture, collecting cells by centrifugation, and the supernatant of cell wall disruption is the crude enzyme solution of the cutinase mutant;

(4) Purify the crude enzyme solution using a nickel column to obtain the target protein, and then concentrate it using an ultrafiltration tube to obtain the purified enzyme.

In one embodiment of the present invention, the mutant plasmid in step (2) is pET-28a-cutinaseM1 or pET-28a-cutinaseM2.

In one embodiment of the present invention, the fermentation culture in step (3) refers to culturing the recombinant Escherichia coli at 37° C. to an OD ₆₀₀ between 0.6 and 0.8 using LB-kana liquid culture medium, and inducing to obtain a fermentation broth.

In one embodiment of the present invention, the induction condition is to add IPTG with a final concentration of 0.5 mM and induce at a temperature of 16° C. for 16 h.

In one embodiment of the present invention, the method for obtaining the cell wall-breaking supernatant in step (3) is to centrifuge the fermentation broth, discard the supernatant, resuspend the bacteria using PBS buffer, and repeat the centrifugation step twice to obtain a recombinant Escherichia coli suspension, use a high-pressure cell disruptor to disrupt the bacteria, and centrifuge under certain conditions, discard the precipitate, and obtain the cell wall-breaking supernatant.

In one embodiment of the present invention, the centrifugation condition is 4° C. and 8000 rpm for 5 min.

In one embodiment of the present invention, the protein purification process in step (4) requires the preparation of solvents required for protein purification, and the required solvent preparation method is as follows: accurately weigh _{8.95g Na2HPO4} · _12H2O , 8.766g NaCl, and _0.3404g imidazole in 500mL UP water to obtain BindingBuffer; accurately weigh 8.95g _Na2HPO4 ·12H2O, 8.766g NaCl, and _1.5318g imidazole in 500mL UP water to obtain WashingBuffer; accurately weigh 8.95g _Na2HPO4 · _12H2O , 8.766g _NaCl , and 17.02g imidazole in 500mL UP water to obtain ElutionBuffer.

In one embodiment of the present invention, the protein purification step in step (4) is: first use 5 column volumes of Binding Buffer to balance the column, repeatedly load the crude enzyme solution three times, use 5 column volumes of Washing Buffer to wash away impurities, and use 3 column volumes of Elution Buffer to collect the target protein.

In one embodiment of the present invention, the gas phase detection conditions are: the gas chromatograph uses an Agilent 8890GC system, the gas phase detection conditions are: 30m×250μm×250μm HP-INNOWAX chromatographic column, heater 220°C, air flow 400mL/min, hydrogen fuel gas flow 30mL/min, tail gas (N ₂ ) flow 25mL/min. The column oven temperature is up to 270°C, and the column oven temperature gradient is set as: initial temperature 80°C, 15°C/min heating to 200°C and then maintained for 5min, 20°C/min heating to 240°C and maintained for 5min.

Beneficial effects of the present invention:

The present invention obtains mutant cutinases, cuitinaseM1 and cuitinaseM2, with stronger recognition and binding ability to piperazine by rationally mutating cutinases derived from the microorganism Aspergillus oryzae. The mutant cutinases can effectively bind to piperazine and continuously catalyze two-step acetylation reactions, and can use low-activity acetic acid as an acetyl donor to directly acylate piperazine into 1,4-diacetylpiperazine, thereby providing a new biocatalytic tool for the acylation reaction of piperazine-type nitrogen heterocyclic compounds, and has the following advantages:

(1) Compared with the wild-type Aspergillus oryzae cutinase, the mutant cutinases cuitinase M1 and cuitinase M2 have stronger recognition and binding abilities for piperazine. They can efficiently remove water molecules through the serine (Ser)-histidine (His)-aspartic acid (Asp) catalytic triad. After catalyzing piperazine to 1-acetylpiperazine, they can further catalyze the second step acetylation reaction by combining with 1-acetylpiperazine to generate 1,4-diacetylpiperazine. The yields are increased by 6.1 and 7.4 times, respectively, compared with the wild-type cutinase.

(2) Compared with traditional chemical methods, this method is green and environmentally friendly, with less pollution to the environment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a gas phase detection result of piperazine catalyzed by the cutinase mutant cutinaseM1 in Example 3; wherein, in FIG. 1 , 7.117 min is the gas phase peak result of 1-acetylpiperazine, and 14.928 min is the gas phase peak result of 1,4-diacetylpiperazine;

FIG2 is the gas phase detection result of piperazine catalyzed by the cutinase mutant cutinaseM2 in Example 4; wherein, 7.117 min in FIG2 is the gas phase peak result of 1-acetylpiperazine, and 14.928 min is the gas phase peak result of 1,4-diacetylpiperazine.

Detailed ways

The present invention aims to provide a cutinase mutant and its application in the synthesis of 1,4-diacetylpiperazine. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of the present invention to implement and apply the technology of the present invention.

In order to enable those skilled in the art to better understand the present invention, the present invention is further described in detail below in conjunction with specific implementation methods.

The LB-kana liquid culture medium used in the present invention is prepared as follows: tryptone (10 g), yeast extract (5 g), sodium chloride (5 g), 1 mL of 1 mol/L sodium hydroxide aqueous solution to adjust the pH to 7.4, add kanamycin to a final concentration of 50 ug/mL, dilute to 1 L with deionized water, and steam sterilize under high pressure for 20 min.

The gas phase detection conditions involved in the present invention are: the gas chromatograph adopts the Agilent 8890GC system, the gas phase detection conditions are: 30m×250μm×250μm HP-INNOWAX chromatographic column, heater 220°C, air flow 400mL/min, hydrogen fuel gas flow 30mL/min, tail gas (N ₂ ) flow 25mL/min. The column oven temperature is up to 270°C, and the column oven temperature gradient is set as follows: initial temperature 80°C, 15°C/min heating to 200°C and then maintained for 5min, 20°C/min heating to 240°C and maintained for 5min.

Example 1: A cutinase mutant with improved 1,4-diacetylpiperazine yield and its coding sequence and related recombinant vector

This example provides a mutant of cutinase with stronger recognition and binding ability to piperazine, wherein the mutant is selected from any one of mutants (1) to (2):

(1) Mutant cuitinaseM1, the amino acid sequence of which is shown in SEQ ID NO.1:

MHLAIKSLFVSLLGASVLASPLPSNALVERNAPLNEFLSALLSHLPAIDGTIDAVSGVITDFDQLLADLTGARTTQNGYIGVCTDYTVLFARGTSEPGNVGVLVGPPLSEAFEQAVGAKALSFQGVNGYNADVAGILAGSDAAGSKSMASLASEVLSKCPDTKLVMSGYSQGCQIVHNAVEQLPAADASKISSVLLFGDPYAGKAFPNVDASRVHTVCHAGDTICNNSVVWLPPHLTYAVDVTNAVQFAVAAAN;

(2) Mutant cuitinaseM2, the amino acid sequence of which is shown in SEQ ID NO.2:

MHLAIKSLFVSLLGASVLASPLPSNALVERNAPLNEFLSALLSHLPAIDGTIDAVSGVITDFDQLLADLTGSRTTQNGYIGVCTDYTVLFARGTSEPGNVGVLVGPPLSEAFELAVGAKALSFQGVNGYNADVAGYLAGSDAAGSKSMASLASEVLSKCPDTKLVMSGYSQGCQIVHNAVEQLPAADASKISSVLLFGDPYAAKAFPNVDASRVHTVCHAGDTICNNSVVILPPHLTYAVDVTNAVQFAVAAAN.

The above mutants are obtained by rational mutation on the basis of wild-type cutinase. The mutation refers to: on the basis of the amino acid sequence of wild-type cutinase derived from Aspergillus oryzae (as shown in SEQ ID NO.5), tyrosine at position 136 is mutated to isoleucine, isoleucine at position 231 is mutated to tryptophan to obtain mutant cuitinaseM1, or on the basis of the amino acid sequence of wild-type cutinase derived from Aspergillus oryzae (as shown in SEQ ID NO.5), alanine at position 72 is mutated to serine, glutamine at position 114 is mutated to leucine, and glycine at position 203 is mutated to alanine to obtain mutant cuitinaseM2.

SEQ ID NO.5:

MHLAIKSLFVSLLGASVLASPLPSNALVERNAPLNEFLSALLSHLPAIDGTIDAVSGVITDFDQLLADLTGARTTQNGYIGVCTDYTVLFARGTSEPGNVGVLVGPPLSEAFEQAVGAKALSFQGVNGYNADVAGYLAGSDAAGSKSMASLASEVLSKCPDTKLVMSGYSQGCQIVHNAVEQLPAADASKISSVLLFGDPYAGKAFPNVDASRVHTVCHAGDTICNNSVVILPPHLTYAVDVTNAVQFAVAAAN

In this example, by mutating the wild-type cutinase, the recognition and binding ability of the wild-type cutinase to piperazine is improved, thereby improving the catalytic activity of the wild-type cutinase and enhancing the ability of the enzyme to synthesize 1,4-diacetylpiperazine.

This example also provides the coding sequence of the above-mentioned cutinase mutants. The coding sequence of the mutant cuitinaseM1 is shown in SEQ ID NO.3; the coding sequence of the mutant cuitinaseM2 is shown in SEQ ID NO.4.

SEQ ID NO.3:

ATGCACCTGGCCATCAAGAGCCTGTTCGTGAGCCTGCTGGGCGCCAGCGTGCTGGCCAGCCCCCTGCCCAGCAACGCCCTGGTGGAGAGGAACGCCCCCCTGAACGAGTTCCTGAGCGCCCTGCTGAGCCACCTGCCCGCCATCGACGGCACCATCGACGCCGTGAGCGGCGTGATCACCGACTTCGACCAGCTGCTGGCCGACCTGACCGGCGCCAGGACCACCCAGAACGGCTACATCGGCGTGTGCACCGACTACACCGTGCTGTTCGCCAGGGGCACCAGCGAGCCCGGCAACGTGGGCGTGCTGGTGGGCCCCCCCCTGAGCGAGGCCTTCGAGCAGGCCGTGGGCGCCAAGGCCCTGAGCTTCCAGGGCGTGAAC GGCTACAACGCCGACGTGGCCGGCATCCTGGCCGGCAGCGACGCCGCCGGCAGCAAGAGCATGGCCAGCCTGGCCAGCGAGGTGCTGAGCAAGTGCCCCGACACCAAGCTGGTGATGAGCGGCTACAGCCAGGGCTGCCAGATCGTGCACAACGCCGTGGAGCAGCTGCCCGCCGCCGACGCCAGCAAGATCAGCAGCGTGCTGCTGTTCGGCGACCCCTACGCCGGCAAGGCCTTCCCCAACGTGGACGCCAGCAGGGTGCACACCGTGTGCCACGCCGGCGACACCATCTGCAACAACAGCGTGGTGTGGCTGCCCCCCCACCTGACCTACGCCGTGGACGTGACCAACGCCGTGCAGTTCGCCGTGGCCGCCGCCAAC

SEQ ID NO.4:

ATGCACCTGGCCATCAAGAGCCTGTTCGTGAGCCTGCTGGGCGCCAGCGTGCTGGCCAGCCCCCTGCCCAGCAACGCCCTGGTGGAGAGGAACGCCCCCCTGAACGAGTTCCTGAGCGCCCTGCTGAGCCACCTGCCCGCCATCGACGGCACCATCGACGCCGTGAGCGGCGTGATCACCGACTTCGACCAGCTGCTGGCCGACCTGACCGGCAGCAGGACCACCCAGAACGGCTACATCGGCGTGTGCACCGACTACACCGTGCTGTTCGCCAGGGGCACCAGCGAGCCCGGCAACGTGGGCGTGCTGGTGGGCCCCCCCCTGAGCGAGGCCTTCGAGCTGGCCGTGGGCGCCAAGGCCCTGAGCTTCCAGGGCGTGAAC GGCTACAACGCCGACGTGGCCGGCTACCTGGCCGGCAGCGACGCCGCCGGCAGCAAGAGCATGGCCAGCCTGGCCAGCGAGGTGCTGAGCAAGTGCCCCGACACCAAGCTGGTGATGAGCGGCTACAGCCAGGGCTGCCAGATCGTGCACAACGCCGTGGAGCAGCTGCCCGCCGCCGACGCCAGCAAGATCAGCAGCGTGCTGCTGTTCGGCGACCCCTACGCCGCCAAGGCCTTCCCCAACGTGGACGCCAGCAGGGTGCACACCGTGTGCCACGCCGGCGACACCATCTGCAACAACAGCGTGGTGATCCTGCCCCCCCACCTGACCTACGCCGTGGACGTGACCAACGCCGTGCAGTTCGCCGTGGCCGCCGCCAAC

This embodiment also provides a recombinant vector comprising the mutant coding sequence. The recombinant vector preferably uses pET-28a as the original expression vector; the present invention has no particular limitation on the method for constructing the recombinant vector, and conventional methods in the art can be used.

This embodiment also provides a recombinant strain comprising the above recombinant vector. The recombinant bacteria preferably uses Escherichia coli as a host bacteria; the Escherichia coli is preferably E. coli BL21 (DE3). The present invention has no special restrictions on the construction method of the recombinant strain, and conventional methods in the art can be used.

Example 2: A method for preparing the cutinase mutant described in Example 1

method one:

Step 1: Introduce the cutinaseM1-modified plasmid pET-28a-cutinaseM1 into Escherichia coli BL21, culture in LB-kana liquid medium at 37°C until _OD600 is between 0.6 and 0.8, add IPTG with a final concentration of 0.5mM, and induce at 16°C for 16h; obtain the cells by centrifugation, the centrifugation conditions are: 4°C, 8000rpm for 5min; wash the cells by centrifugation with PBS, resuspend them, and obtain a recombinant Escherichia coli suspension.

Step 2: Use a high-pressure cell disruptor to disrupt the bacteria and centrifuge at 4°C and 13,500 rpm for 30 min. Discard the precipitate to obtain a crude enzyme solution.

Step 3: Use 5 column volumes of Binding Buffer to equilibrate the nickel column, repeatedly load the crude enzyme solution three times, use 5 column volumes of Washing Buffer to wash away the impurities, use 3 column volumes of Elution Buffer to collect the target protein, and finally obtain the purified enzyme cutinase M1, and measure the protein concentration using the Bradford method.

Method Two:

Step 1: Introduce the cutinaseM2-modified plasmid pET-28a-cutinaseM2 into Escherichia coli BL21, culture in LB-kana liquid medium at 37°C until _OD600 is between 0.6 and 0.8, add IPTG with a final concentration of 0.5mM, and induce at 16°C for 16h; obtain the cells by centrifugation, the centrifugation conditions are: 4°C, 8000rpm for 5min; wash the cells by centrifugation with PBS, resuspend them, and obtain a recombinant Escherichia coli suspension.

Step 2: Use a high-pressure cell disruptor to disrupt the bacteria and centrifuge at 4°C and 13,500 rpm for 30 min. Discard the precipitate to obtain a crude enzyme solution.

Step 3: Use 5 column volumes of Binding Buffer to equilibrate the nickel column, repeatedly load the crude enzyme solution three times, use 5 column volumes of Washing Buffer to wash away the impurities, use 3 column volumes of Elution Buffer to collect the target protein, and finally obtain the purified enzyme cutinase M2, and measure the protein concentration using the Bradford method.

The _solvent preparation method required in the above method is as follows: accurately weigh _8.95gNa2HPO4 · _12H2O , 8.766gNaCl, and _0.3404g imidazole in 500mLUP water to obtain BindingBuffer; accurately weigh _8.95gNa2HPO4 · _12H2O , 8.766gNaCl, and _1.5318g imidazole in 500mLUP water to obtain WashingBuffer; accurately weigh _8.95gNa2HPO4 · _12H2O , 8.766gNaCl, and 17.02g imidazole in 500mLUP water to obtain ElutionBuffer.

Example 3: Application of cutinase mutant cutinaseM1 in the synthesis of 1,4-diacetylpiperazine

Purified enzyme cutinaseM1 was obtained according to method 1 in Example 2, and the protein concentration was measured by Bradford method to be 5.5 mg/mL. Substrate piperazine (11.6 μmol, 2 g/L) and vinyl acetate (34.8 μmol) were added to N, N-dimethylformamide, and 45.5 μL of cutinaseM1 recombinant cutinase solution was added, and the final enzyme concentration was 0.5 mg/mL; the final volume of the catalytic system was 500 μL; the catalytic reaction was carried out at 30°C for 12 hours, and the yield of 1-acetylpiperazine was 53.47% and the yield of 1,4-diacetylpiperazine was 11.86% (see Figure 1 for gas phase detection results). 1-Acetylpiperazine is a synthetic precursor of 1,4-diacetylpiperazine. Piperazine first undergoes the first step of acetylation to generate 1-acetylpiperazine, and then undergoes the second step of acetylation to generate 1,4-diacetylpiperazine.

The yield calculation formula is as follows:

1-Acetylpiperazine yield = (1-Acetylpiperazine yield / 1-Acetylpiperazine amount of substance) / amount of substrate piperazine × 100%

1,4-diacetylpiperazine yield = (1,4-diacetylpiperazine yield / 1,4-diacetylpiperazine amount of substance) / amount of substrate piperazine × 100%

Example 4: Application of cutinase mutant cutinase M2 in the synthesis of 1,4-diacetylpiperazine

Purified enzyme cutinase M2 was obtained according to method 2 in Example 2, and the protein concentration was measured by Bradford method to be 8.4 mg/mL. Substrate piperazine (11.6 μmol, 2 g/L) and vinyl acetate (34.8 μmol) were added to N, N-dimethylformamide, and 30.0 μL of cutinase M2 recombinant cutinase solution was added, and the final enzyme concentration was 0.5 mg/mL; the final volume of the catalytic system was 500 μL; the catalytic reaction was carried out at 30° C. for 12 h, and the yield of 1-acetylpiperazine was 51.42% and the yield of 1,4-diacetylpiperazine was 14.26% as determined by Agilent 8890GC gas phase instrument (see Figure 2 for gas phase detection results).

Comparative Example 1:

Prepare wild-type cutinase by referring to the method described in Example 2:

Step 1: The wild-type Aspergillus oryzae cutinase-modified plasmid pET-28a-Aspergillus oryzae was introduced into Escherichia coli BL21, cultured in LB-kana liquid medium at 37°C until OD ₆₀₀ was between 0.6 and 0.8, added IPTG with a final concentration of 0.5 mM, and induced at 16°C for 16 hours; the centrifugation conditions were: centrifugation at 4°C and 8000 rpm for 5 minutes; the bacteria were washed by centrifugation with PBS, and resuspended to obtain a recombinant Escherichia coli suspension.

Step 2: Use a high-pressure cell disruptor to disrupt the bacteria and centrifuge at 4°C and 13,500 rpm for 30 min. Discard the precipitate to obtain a crude enzyme solution.

Step 3: Use 5 column volumes of Binding Buffer to equilibrate the nickel column, repeatedly load the crude enzyme solution three times, use 5 column volumes of Washing Buffer to wash away the impurities, and use 3 column volumes of Elution Buffer to collect the target protein. Finally, the wild-type Aspergillus oryzae cutinase was obtained. The protein concentration was measured by Bradford method and was 3.2 mg/mL.

The substrate piperazine (11.6 μmol, 2 g/L) and vinyl acetate (34.8 μmol) were added to N,N-dimethylformamide, and 78.8 μL of wild-type Aspergillus oryzae cutinase solution was added, and the final enzyme concentration was 0.5 mg/mL; the final volume of the catalytic system was 500 μL; the catalytic reaction was carried out at 30°C for 12 hours, and the yields of 1-acetylpiperazine and 1,4-diacetylpiperazine were determined by Agilent 8890GC gas chromatograph to be 48.94% and 1.93%, respectively.

A blank group was also set up in this comparative example, in which substrate piperazine (11.6 μmol, 2 g/L) and vinyl acetate (34.8 μmol) were added to N,N-dimethylformamide; 30 μL PBS buffer was added to the blank group; the final volume of the system was 500 μL; the catalytic reaction was carried out at 30° C. for 12 h, and the yields of 1-acetylpiperazine and 1,4-diacetylpiperazine were determined by Agilent 8890GC gas chromatograph to be 42.91% and 3.83%, respectively.

From the data obtained from the control group and Comparative Example 1, it can be seen that compared with the blank group, the yield of 1,4-diacetylpiperazine in the control group is lower. Since piperazine and vinyl acetate spontaneously undergo acetylation reaction to generate 1-acetylpiperazine and 1,4-diacetylpiperazine, the above results indicate that the wild-type cutinase has no obvious catalytic acetylation effect on 1,4-diacetylpiperazine.

From the data obtained in Example 3, Example 4 and Comparative Example 1, it can be seen that, compared with the blank group and the non-mutated group, the recombinant mutant cutinase has a significant effect on improving the yield of the double transacetyl product 1,4-diacetylpiperazine.

Example 5: Synthesis of 1,4-diacetylpiperazine using the cutinase mutant cutinaseM1

Step 1: Introduce the cutinaseM1-modified plasmid pET-28a-cutinaseM1 into Escherichia coli BL21, culture in LB-kana liquid medium at 37°C until _OD600 is between 0.6 and 0.8, add IPTG with a final concentration of 0.5mM, and induce at 16°C for 16h; collect the cells by centrifugation, the centrifugation conditions are: 4°C, 8000rpm for 5min; wash the cells by centrifugation with PBS, resuspend them, and obtain a recombinant Escherichia coli suspension.

Step 2: Use a high-pressure cell disruptor to disrupt the bacteria and centrifuge at 4°C and 13,500 rpm for 30 min. Discard the precipitate to obtain a crude enzyme solution.

Step 3: Use 5 column volumes of Binding Buffer to equilibrate the nickel column, repeatedly load the crude enzyme solution three times, use 5 column volumes of Washing Buffer to wash away the impurities, and use 3 column volumes of Elution Buffer to collect the target protein. Finally, the purified enzyme cutinase M1 was obtained. The protein concentration was measured by Bradford method and was 12.5 mg/mL.

Step 4: Add the substrate piperazine (8.7 μmol, 0.75 g/L) and vinyl acetate (26.1 μmol) to N,N-dimethylformamide, add 80 μL of cuitinaseM1 recombinant cutinase solution, and the final enzyme concentration is 1.0 mg/mL; the final volume of the catalytic system is 1000 μL; the catalytic reaction is carried out at 30° C. for 12 h, and the yield of 1-acetylpiperazine is 46.72% and the yield of 1,4-diacetylpiperazine is 9.11% as determined by Agilent 8890GC gas chromatograph.

Example 6: Synthesis of 1,4-diacetylpiperazine using the cutinase mutant cutinaseM2

Step 1: Introduce the cutinaseM2-modified plasmid pET-28a-cutinaseM2 into Escherichia coli BL21, culture in LB-kana liquid medium at 37°C until _OD600 is between 0.6 and 0.8, add IPTG with a final concentration of 0.5mM, and induce at 16°C for 16h; collect the cells by centrifugation, the centrifugation conditions are: 4°C, 8000rpm for 5min; wash the cells by centrifugation with PBS, resuspend them, and obtain a recombinant Escherichia coli suspension.

Step 2: Use a high-pressure cell disruptor to disrupt the bacteria and centrifuge at 4°C and 13,500 rpm for 30 min. Discard the precipitate to obtain a crude enzyme solution.

Step 3: Use 5 column volumes of Binding Buffer to equilibrate the nickel column, repeatedly load the crude enzyme solution three times, use 5 column volumes of Washing Buffer to wash away the impurities, and use 3 column volumes of Elution Buffer to collect the target protein. Finally, the purified enzyme cutinase M2 was obtained. The protein concentration was measured by Bradford method and was 15.9 mg/mL.

Step 4: Add the substrate piperazine (8.7 μmol, 0.75 g/L) and vinyl acetate (26.1 μmol) to N,N-dimethylformamide, add 62.8 μL of cuitinaseM2 recombinant cutinase solution, and the final enzyme concentration is 1.0 mg/mL; the final volume of the catalytic system is 1000 μL; the catalytic reaction is carried out at 30° C. for 12 h, and the yield of 1-acetylpiperazine is 53.55% and the yield of 1,4-diacetylpiperazine is 15.14% as determined by Agilent 8890GC gas chromatograph.

Example 7: Synthesis of 1,4-diacetylpiperazine using the cutinase mutant cutinaseM2

Step 1: Introduce the cutinaseM2-modified plasmid pET-28a-cutinaseM2 into Escherichia coli BL21, culture in LB-kana liquid medium at 37°C until _OD600 is between 0.6 and 0.8, add IPTG with a final concentration of 0.5mM, and induce at 16°C for 16h; obtain the cells by centrifugation, the centrifugation conditions are: 4°C, 8000rpm for 5min; wash the cells by centrifugation with PBS, resuspend them, and obtain a recombinant Escherichia coli suspension.

Step 2: Use a high-pressure cell disruptor to disrupt the bacteria and centrifuge at 4°C and 13,500 rpm for 30 min. Discard the precipitate to obtain a crude enzyme solution.

Step 3: Use 5 column volumes of Binding Buffer to equilibrate the nickel column, repeatedly load the crude enzyme solution three times, use 5 column volumes of Washing Buffer to wash away the impurities, and use 3 column volumes of Elution Buffer to collect the target protein. Finally, the purified enzyme cutinase M2 was obtained. The protein concentration was measured by Bradford method and was 15.9 mg/mL.

Step 4: Add substrate piperazine (8.7 μmol, 0.75 g/L) and vinyl acetate (26.1 μmol) to N,N-dimethylformamide, add 62.8 μL of cuitinaseM2 recombinant cutinase solution, and the final enzyme concentration is 1.0 mg/mL; the final volume of the catalytic system is 1000 μL; the catalytic reaction is carried out at 40° C. for 12 h, and the yield of 1-acetylpiperazine is 45.49% and the yield of 1,4-diacetylpiperazine is 18.55% as determined by Agilent 8890GC gas chromatograph.

Example 8: Synthesis of 1,4-diacetylpiperazine using the cutinase mutant cutinaseM2

Step 1: Introduce the cutinaseM2-modified plasmid pET-28a-cutinaseM2 into Escherichia coli BL21, culture in LB-kana liquid medium at 37°C until _OD600 is between 0.6 and 0.8, add IPTG with a final concentration of 0.5mM, and induce at 16°C for 16h; collect the cells by centrifugation, the centrifugation conditions are: 4°C, 8000rpm for 5min; wash the cells by centrifugation with PBS, resuspend them, and obtain a recombinant Escherichia coli suspension.

Step 2: Use a high-pressure cell disruptor to disrupt the bacteria and centrifuge at 4°C and 13,500 rpm for 30 min. Discard the precipitate to obtain a crude enzyme solution.

Step 3: Use 5 column volumes of Binding Buffer to equilibrate the nickel column, repeatedly load the crude enzyme solution three times, use 5 column volumes of Washing Buffer to wash away the impurities, and use 3 column volumes of Elution Buffer to collect the target protein. Finally, the purified enzyme cutinase M2 was obtained. The protein concentration was measured by Bradford method and was 15.9 mg/mL.

Step 4: Add substrate piperazine (8.7 μmol, 0.75 g/L) and vinyl acetate (26.1 μmol) to ethyl acetate, add 62.8 μL of cuitinase M2 recombinant cutinase solution, and the final enzyme concentration is 1.0 mg/mL; the final volume of the catalytic system is 1000 μL; the catalytic reaction is carried out at 40° C. for 12 h, and the yield of 1-acetylpiperazine is 60.25% and the yield of 1,4-diacetylpiperazine is 13.61% as determined by Agilent 8890GC gas chromatograph.

Although the present invention has been disclosed as above in the form of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims (9)

1. A cutinase mutant for improving the yield of 1,4-diacetylpiperazine, characterized in that the cutinase mutant is any one of the following mutants: Mutant cuitinase M1, the amino acid sequence of which is shown in SEQ ID NO.1; The mutant cuitinase M2, the amino acid sequence is shown in SEQ ID NO.2. 2. A gene encoding the cutinase mutant according to claim 1, characterized in that The coding gene of the mutant cuitinase M1 is shown in SEQ ID NO.3; The coding gene of the mutant cuitinase M2 is shown in SEQ ID NO.4. 3. A recombinant vector containing the coding gene according to claim 2. 4. A recombinant bacterium containing the recombinant vector according to claim 3. 5. Use of the cutinase mutant according to claim 1, the encoding gene according to claim 2, the recombinant vector according to claim 3 or the recombinant bacteria according to claim 4 in the synthesis of 1,4-diacetylpiperazine, characterized in that piperazine and an acetyl donor are dissolved in a solvent, and the cutinase mutant according to claim 1 is added to catalyze the production of 1,4-diacetylpiperazine. 6. The use according to claim 5, characterized in that the piperazine concentration is 0.1-5 g/L; the acetyl donor is vinyl acetate; the molar ratio of vinyl acetate to piperazine is 2.2-3:1; and the cutinase concentration is 0.1-10 mg/L. 7. The use according to claim 5, characterized in that the solvent is any one of N,N-dimethylformamide, acetonitrile, dimethyl sulfoxide, tetrahydrofuran, n-hexane, ethyl acetate and water, or a mixture of two or more thereof. 8. The use according to claim 5, characterized in that the catalytic reaction temperature is 30-40°C and the reaction time is 12-24 h. 9. A method for preparing the cutinase mutant according to claim 1, characterized in that it comprises the following steps: (1) constructing a plasmid vector containing a gene encoding the cutinase mutant according to claim 1; (2) Introducing the mutant plasmid into Escherichia coli BL21; (3) Selecting positive clones for fermentation culture, collecting cells by centrifugation, and the supernatant from cell wall disruption is the crude enzyme solution of the cutinase mutant; (4) Use a nickel column to purify the crude enzyme solution to obtain the target protein, and then use an ultrafiltration tube to concentrate it to obtain the purified enzyme.