CN104762334A - Method for producing peroxyacetic acid by enzyme catalytic reaction - Google Patents

Abstract

The invention discloses a method for producing peroxyacetic acid by enzymatic method. The method uses short-chain acetate and hydrogen peroxide as substrates, and acetyl xylan esterase perhydrolysis catalyzes the reaction to generate peracetic acid.

Description

Method for producing peracetic acid by enzyme-catalyzed reaction

technical field

The present invention belongs to the technical field of genetic engineering, and specifically relates to the production of peroxyacetic acid from an acetate substrate by acetylxylan esterase AXE (Genbank: KC292495) with perhydrolysis activity.

Background technique

Peroxyacetic acid (PAA), also known as peracetic acid, peroxyacetic acid, and acetylhydroperoxide, is a highly active and highly oxidizing organic acid with simple structure, easy synthesis, and relatively stable relative stability among peroxyorganic acids. The oxidation ability of peracetic acid is higher than that of hydrogen peroxide, and it can be used as a bactericidal disinfectant, oxidizing agent, and epoxy oxidizing agent. It has unique functions and is a broad-spectrum, efficient, quick-acting, and cheap sterilizing agent. It is widely used in medical devices Disinfection, sterilization, environment, surface, air and other epidemic site disinfection and preventive disinfection. (Han Shuangzhan, Synthesis and application of peracetic acid. Hebei Chemical Industry 2008 (6): 41-43.) Due to the existence of active oxygen in its structure, in addition to being used as a bactericidal disinfectant, peracetic acid is used in papermaking, textiles, chemical synthesis, Environmental engineering, energy engineering, polymer industry and biomimetic chemistry have extensive application research, especially as epoxy oxidants and high-efficiency fungicides in organic synthesis, with outstanding performance and broad prospects for development and utilization. (Zhang Tengyun, et al. Research progress on the synthesis and industrial application of peracetic acid. Chemical Industry Progress 2007(26):194-197)

There are many ways to synthesize peracetic acid, but the methods currently used in industry are mainly hydrogen peroxide method and acetaldehyde oxidation method. The hydrogen peroxide method is to use acetic acid and hydrogen peroxide to react, under the catalysis of strong acid, to prepare peracetic acid. Sulfuric acid is often used as a catalyst, the reaction process is violent, and high concentration of acetic acid is often required, and the obtained peracetic acid is strongly acidic. The acetaldehyde oxidation method is based on the oxidation of acetaldehyde to generate acetic acid, by changing the conditions of acetaldehyde oxidation and lowering the reaction temperature to obtain peracetic acid. But this reaction is more complicated, and to obtain higher conversion rate and peracetic acid yield, it is necessary to add an appropriate catalyst. And because peracetic acid has strong oxidizing and corrosive properties, it will cause combustion and explosion when exposed to open flames and high heat; contact with reducing agents, metal powder and wood may cause combustion and explosion hazards, and there are strict requirements for packaging, transportation and storage of peracetic acid. (Liu Jiqi, Properties, Preparation and Application of Peracetic Acid. Henan Journal of Preventive Medicine 2004(03):171-173)

Therefore, people began to actively explore the biological method to produce peracetic acid. The advantages of enzymatic or microbial production of peracetic acid are rapid and mild reaction, no need for high concentration hydrogen peroxide or substrate, and problems such as transportation and storage can be solved. Esterases and lipases, which catalyze the hydrolysis of esters, are reported to often have overlapping catalytic activity with non-heme haloperoxides, which catalyze peroxidation reactions in the presence of hydrogen peroxide to produce peroxycarboxylic acids. The response is as follows:

                                                

For example, O. Kirk et al. (Biocatalysis, 11:65-77 (1994)) studied hydrolytic enzymes (lipases, esterases and proteases) catalyzing the perhydrolysis of acyl substrates with hydrogen peroxide to form peroxycarboxylic acids; recently Several protease and lipase combinations have been reported to generate in situ peracids (eg, peracetic acid) at concentrations suitable for use as disinfectants and/or commercial bleaches (US Patent Applications 11/413,246 and 11/588,523). However, most of the known enzyme biocatalyzed production of peracetic acid has a low concentration and does not have effective application value.

To realize the biological preparation of peracetic acid, the most important thing is to obtain an enzyme that is easy to obtain, highly active, and highly expressed, so as to meet the requirements of high production efficiency, rapid and sensitive response, and then realize large-scale production. However, this problem is still a key issue restricting the preparation of peracetic acid by biological methods.

Contents of the invention

The inventors found that acetylxylan esterase AXE with independent intellectual property rights (patent application number CN201210403666.2) has perhydrolysis activity, and based on this, proposed the present invention. Therefore, the inventor requests to incorporate the content of the patent application CN201210403666.2 into this application, including the amino acid sequence SEQ ID NO: 2 of the acetylxylan esterase in the application; active homotrimers and hexamers Protein structure; and the construction process of genetically engineered bacteria Escherichia coli ( Escherichia coli ) BL21-pET-Cah, as well as the obtained strains.

The technical problem to be solved by the present invention is to obtain an easy-to-obtain, high-activity, high-expression enzyme for the enzymatic production of peracetic acid.

The technical purpose of the present invention is to provide the application of the esterase in the perhydrolysis reaction. Specifically, the enzyme can be used to catalyze the perhydrolysis reaction, more specifically, in the presence of hydrogen peroxide, the enzyme can be used to catalyze short-chain acetate to generate peroxyacetic acid.

Specifically, the technical solutions of the present invention include:

There is the application of the acetylxylan esterase of the amino acid sequence represented by SEQ ID NO:1 in the perhydrolysis reaction.

Wherein, the amino acid sequence represented by SEQ ID NO:1 in this application is consistent with the amino acid sequence represented by SEQ ID NO:2 described in Chinese patent application CN201210403666.2. First of all, the inventors have discovered that the enzyme has perhydrolysis activity and can catalyze the generation of peroxyacids from acyl substrates in the presence of a peroxygen source; secondly, the perhydrolysis reaction should be understood as, in the presence of a peroxygen source, such as hydrogen peroxide , the enzyme catalyzes the formation of peroxyacids from acyl substrates.

In a preferred reaction, the enzyme catalyzes the formation of peracetic acid from an acetyl substrate in the presence of hydrogen peroxide.

A method for enzymatically producing peracetic acid, using acetyl substrates and hydrogen peroxide as substrates, contacting acetyl xylan esterase with the amino acid sequence represented by SEQ ID NO: 1 with the substrates to perform a perhydrolysis reaction to generate peracetic acid.

In the method of the present invention, the carrier containing the esterase protein can be used as an enzyme catalyst to contact with the substrate for perhydrolysis reaction to generate peroxyacetic acid.

For the carrier containing the protein as an enzyme catalyst, it can be understood that the above protein can be purified and immobilized after being expressed in an expression system; the above protein can also be expressed and the expressed strain can be crushed to obtain a crude enzyme solution, React the crude enzyme solution with the substrate to obtain the target product, or immobilize the crude enzyme solution or whole cells, contact the substrate with it and react to obtain the target product.

In the method of the present invention, the acetyl substrate is selected from one of ethyl acetate, glycerol monoacetate, glycerol diacetate, and glycerol triacetate.

The method of the present invention, wherein, in the perhydrolysis reaction system, in milligrams per milliliter, the quantitative ratio of the acetyl substrate: hydrogen peroxide: enzyme is 1:0.5-8.5:0.015-0.075.

For this ratio relationship, it should be understood that the present invention can be realized under the condition that the presence of the substrate species is satisfied, and the ratio provided by the present invention is a preferred ratio.

The method of the present invention, wherein the reaction conditions of the perhydrolysis reaction are: pH 6.5-9.5, preferably pH 8.0.

The method of the present invention, wherein, the reaction time of the perhydrolysis reaction is within 5 minutes to about 2 hours, preferably within 5 minutes.

The method of the present invention, wherein the temperature of the perhydrolysis reaction is 20-37°C, preferably 20°C.

For the understanding of the time, temperature, and pH value of perhydrolysis in the present invention, it should be understood that the present invention can be realized under the condition that there are substrate species, and the conditions provided by the present invention are preferred conditions.

The method of the present invention further includes the process of expressing the protein and obtaining the crude enzyme solution according to the coding or amino acid sequence of the protein.

The method of the present invention, wherein, comprises the following steps in sequence:

(1) Escherichia coli ( Escherichia coli ) BL21 / pET-CAH was used as the starting strain for seed culture; the Escherichia coli was preserved in the China Center for Type Culture Collection, and the preservation number was: CCTCC NO: M2012408; among them, the seed culture The base was LB medium; the culture conditions were: 250 mL Erlenmeyer flask, the liquid volume was 50 mL, the culture temperature was 37°C, the shaker speed was 200 r/min, and the culture was 12 h; in addition, the crude enzyme solution obtained by breaking the wall was also The catalytic reaction can be further carried out by immobilization.

The invention has the beneficial effects of providing a brand-new enzyme preparation for the catalytic synthesis of peracetic acid by a biological method.

In addition, the scheme of the present invention can obtain a higher final concentration of peracetic acid than that produced by existing biological methods.

Description of drawings

Figure 1 is the standard curve of p-nitrophenol (p-NP) concentration-OD405 absorbance value.

Figure 2 is the standard curve of bovine serum albumin (BSA) concentration-OD595 absorbance value.

Figure 3 is the standard curve of peracetic acid (PAA) concentration-OD405 absorbance value.

Figure 4 shows the experimental results of optimizing the dosage of the most suitable recombinant esterase AX.

Fig. 5 is the optimization experiment result of the optimum triacetin concentration.

Figure 6 shows the optimal hydrogen peroxide concentration optimization experiment results.

Figure 7 is the optimum pH optimization experiment results.

Detailed ways

The present invention will be further described below in conjunction with embodiment. The examples listed are for illustrative purposes only, and it is not intended that the spirit and scope of the invention be limited to the details and modifications thereof.

The strain source used in the present invention:

The genetically engineered bacterium ( Escherichia coli ) BL21/pET-CAH is owned by our laboratory and preserved in the China Center for Type Culture Collection with the preservation number: CCTCC NO: M2012408. For the specific construction process of the strain and the identification process of the enzyme, please refer to the patent CN 102952787 A.

Example 1 This example illustrates the induced expression method of esterase.

The specific scheme of utilizing genetically engineered bacteria to induce the expression of esterase is as follows:

(1) Starting strain: Escherichia coli BL21/pET-CAH;

(2) Seed culture:

Medium: LB medium: yeast powder 5g/L, peptone 10 g/L, sodium chloride 10 g/L;

Culture conditions: 250 mL Erlenmeyer flask, liquid volume 50 mL, culture temperature 37°C, shaker speed 200 r/min, culture 12 h;

(3) Fermentation culture:

Medium composition: LB medium: yeast powder 5g/L, peptone 10 g/L, sodium chloride 10 g/L;

Culture conditions: inoculum size 1.5% (v/v), fermentation temperature 37 ℃, when OD reaches 0.6~0.8, add isopropyl-β-D-thiogalactopyranoside (IPTG) to induce, the final IPTG Concentration 1.0 mmol/L, shaker speed 180~250 r/min, fermentation 2-10 h.

(4) Acquisition of crude enzyme solution:

Take out the fermentation broth, centrifuge at 8000rpm for 3min, add 1/8 volume of the original fermentation broth Tris-HCl (pH 7.0), concentrate 8 times; ultrasonic crushing 400W, 3s working time, 7s gap time, ultrasonic 4min (24 times); centrifuge 6500 rpm, 10 min. The supernatant is the crude enzyme solution.

(5) Determination of enzyme activity:

Using p-nitrophenol acetate as the substrate, 1 enzyme activity unit (U) was defined as: the amount of enzyme required to release 1 μL p -NP per minute under certain reaction conditions.

Preparation of standard curve: preparation of solution A: 50 mmol/L phosphate buffer (pH7.0), which contains 0.6% Triton X-100 and 0.1% gum arabic; preparation of solution B: accurately weigh 0.0139 g p NP standard Substances were dissolved in solution A, and the volumetric flask was adjusted to 250 mL to prepare solution B with a standard substance concentration of 1 μmol/mL. Use solution A to dilute solution B in a gradient manner to prepare p NP solutions with different concentrations. Add 10 μL of buffer solution to the microtiter plate, and then add 240 μL of pNP standard solutions of different concentrations in sequence, and add solution A to the control. The absorbance at a wavelength of 405 nm was measured with a microplate reader. Take the concentration of p NP as the abscissa and the absorbance value as the ordinate to make a standard curve (Figure 1).

The measurement steps are as follows: add 240 μL of substrate solution (A solution and B solution are mixed at 9:1), 10 μL of appropriately diluted enzyme solution in the reaction system, place in a water bath, the temperature is 40 °C, and the reaction time is 10 min ; Measure the absorbance at 405 nm using a microplate reader. Three parallel experiments were set up, the average value was taken, and 10 μL distilled water was used as the control group. The p -NP standard curve and equation are shown in Figure 1.

The measurement results showed that the 100-fold diluted enzyme solution had absorbance values at A405nm of 1.449, 1.482, and 1.509, respectively. Calculated by the standard curve (Figure 1), the corresponding activity was 224.96U/mL.

(6) Determination of protein amount

Protein amounts were determined using the Brandford method.

Preparation of protein standard song: Prepare Coomassie Brilliant Blue solution: 100 mg Coomassie Brilliant Blue G-250 was dissolved in 50 mL 95 % ethanol, then 100 mL 85 % (v/v) H ₃ PO ₄ was added, and finally diluted with distilled water to 1 l, can be used after filtering with filter paper; preparation of 0.1 g/l BSA: weigh 0.01 g of BSA, dissolve in 10 mL of distilled water, and prepare 0.01, 0.02, 0.03, 0.04, 0.06, 0.08 mg/mL with normal saline before use ; Take seven 2 mL centrifuge tubes and number them (0, 1, 2, 3, 4, 5, 6), respectively add 0.3 mL of the diluted BSA solution of each concentration above to the centrifuge tubes 1-6, and finally add Add 1.2 mL of Coomassie brilliant blue solution to each centrifuge tube. In a No. 0 centrifuge tube, add 1.2 mL of Coomassie brilliant blue solution and 0.3 mL of normal saline. After mixing the tubes, measure A ₅₉₅ with tube 0 as a control. The measured standard curve is shown in Figure 2.

The specific steps of protein detection are as follows: firstly, the standard curve equation is made with the concentration of bovine serum albumin (BSA) standard sample against the OD595 absorbance value, as shown in Figure 2. Then remove 300 μL of appropriately diluted enzyme solution, add 1.2 mL of Coomassie Brilliant Blue working solution, mix well, place at room temperature for 15 min, and measure the absorbance at 595 nm. Two parallel samples were made for each group, and distilled water was used as a blank.

The measurement results showed that the OD595 absorbance values of the 5-fold diluted enzyme solution after reaction were 0.252 and 0.277. After calculation according to the standard curve in Figure 2, the protein concentration is: 0.16 mg/mL. Therefore, the specific enzyme activity of the acetyl xylan esterase crude enzyme solution of the engineering bacteria fermentation broth was 1406 U/mg.

Example 2 This example illustrates the method of replacing the expression vector of the genetically engineered bacteria with pET-28a, constructing a new genetically engineered bacteria E. coli BL21pET28a-Cah, and inducing the expression of the esterase in a 5L tank scale.

(1) Cloning of gene Cah:

According to the Cah gene (Genbank: KC292495) published in the patent application number CN201210403666.2, the sequence design of the primers:

P1: 5'-CATG CCATGG GCATGCAATTATACGACT-3'.

P2: 5'-CC CTCGAG GCCTTTCAGATGCGCTT-3'.

Restriction sites Nco I and Xho I were introduced at both ends of the primers, and the genetically engineered bacteria ( Escherichia coli ) BL21/pET-CAH plasmid was extracted according to the instructions of the plasmid extraction kit (OMEGA company), and the genetically engineered bacteria ( Escherichia coli) ) BL21/pET-CAH plasmid DNA as a template to complete the PCR reaction;

The 8-tube reaction system for PCR is: Buffer 22.5 μL, ddH2O 141.3 μL, MgCl2 13.5 μL, dNTP-mix 18 μL, primer P1 4.5 μL, primer P2 4.5 μL, Taq enzyme 2.7 μL; Add 2 μL template DNA to each tube; PCR reaction conditions: pre-denaturation at 94 °C for 4 min; perform 30 cycle reactions: denaturation at 94 °C for 45 s, annealing at 54.1 °C for 30 s, extension at 72 °C for 60 s; Store at 4°C after completion; after purification by a PCR purification kit (TaKaRa Company), Nco I and Xho I digested the recombinant recovered product to recover a 957 bp fragment, Nco I and Xho I digested the pET28a vector, recovered a large fragment product, and The double digestion product was ligated under the action of T4 DNA ligase to obtain the recombinant plasmid pET28a-Cah.

(2) Acquisition of recombinant genetically engineered bacteria:

The recombinant plasmid pET28a-Cah was transformed into E. coli BL21 (DE3), spread on a plate containing 50 μg/mL kanamycin, picked positive transformants, and carried out colony PCR identification to obtain recombinant E. coli , named E. .coli BL21pET28a-Cah, the recombinant plasmid of E.coli BL21pET28a-Cah was extracted and verified by sequencing.

(3) Method for inducing expression of recombinant genetic engineering bacteria E. coli BL21pET28a-Cah 5L tank scale

Seed culture:

Medium: LB medium: yeast powder 5g/L, peptone 10 g/L, sodium chloride 10 g/L;

Culture conditions: 250 mL Erlenmeyer flask, liquid volume 50 mL, culture temperature 37 ℃, shaker speed 200 r/min, culture 12 h;

Fermentation culture:

Medium composition: self-induction medium: yeast powder 25g/L, tryptone 15g/L, sodium chloride 10g/L, glycerol (v/v) 0.1%, glucose (w/v) 0.2%, lactose ( w/v) 0.2%;

Culture conditions: 5L fermenter, liquid volume 3L, inoculum size 3.3% (v/v), fermentation temperature 30 ℃, pH 7.5, stirring speed 200 r/min, aeration 1.5 vvm for 16 h.

(4) Acquisition of crude enzyme solution: refer to the acquisition method of crude enzyme solution in Example 1

(5) Determination of enzyme activity: identify with reference to the detection method in Example 1. The results show that after 16 hours of self-induced expression in a 5L tank, an esterase activity of 2.28×10 ⁴ U/ml can be obtained. By replacing the expression vector with pET-28a, and performing self-induced expression of the esterase in a 5L tank (replacing the expensive IPTG inducer), indicating that the cost of producing esterase medium can be further reduced, thereby reducing the cost of enzymatically producing peracetic acid, which is more conducive to realizing industrial production.

Example 3 This example illustrates the production of peracetic acid and the detection of peracetic acid catalyzed by recombinant esterases using different short-chain acetates and hydrogen peroxide as substrates.

Steps:

(1) Obtain the enzyme liquid according to the method in Example 1;

(2) Reaction system and operation process for preparing peracetic acid catalyzed by esterase: short-chain acetate substrate (ethyl acetate, glycerol monoacetate, glycerol diacetate, glycerol triacetate) 0.1g , hydrogen peroxide 100 μL (1mol/l), esterase AX 30 μL (0.3g/ml), 0.1mol/l pH7.4 phosphate buffer, a total of 1 mL reaction system. In the blank control, the enzyme solution was replaced with an equal amount of buffer solution;

(3) Make 3 parallel reactions under each condition, and take samples at 5 minutes, 10 minutes, and 15 minutes;

(4) Product treatment: Centrifuge at 12,000 rpm after the reaction, and take the supernatant after 2 minutes, which is the reaction product;

(5) Perform peracetic acid detection. The concentration of peracetic acid was detected by oxidation of 2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS). Mix 100 μL of the diluted reaction product with 100 μL of 0.5 g/l ABTS solution and 50 μL of 1.5 mol/l acetic acid solution containing 0.03 mg/ml potassium iodide in a 96-well plate, react at room temperature for 10 minutes, and measure its absorbance at 405 nm .

Preparation of peracetic acid standard song: prepare 0.5g/l ABTS solution (protect from light) and 1.5mol/l acetic acid solution containing 0.03mg/ml potassium iodide: add 0μL, 5μL, 10 μL, 15 μL, 20 μL, 25 μL, 30 μL, 35 μL, 40 μL, 50 μL ABTS solution, 100 μL, 95 μL, 90 μL, 85 μL, 80 μL, 75 μL, 70 μL, 65 μL, 60 μL, 50 μL ddHO, add 50 μL _1.5 to number 1~10 A mol/l acetic acid solution containing 0.03 mg/ml potassium iodide and 100 μL standard peracetic acid solution were mixed evenly and then reacted at room temperature for 10 min. A ₄₀₅ was measured with No. 0 tube as a control. The measured standard curve is shown in Figure 3.

The experimental results are as follows: the concentration of peracetic acid obtained after reacting at 25°C for 5 minutes is shown in Table 1. Triacetin is used as the substrate to produce the highest concentration of peracetic acid, so triacetin is selected as the substrate.

Table 1. Concentration of peracetic acid produced by different acetate substrates

Example 4 This example illustrates the optimization of conditions for the production of peracetic acid catalyzed by recombinant esterase using triacetin and hydrogen peroxide as substrates

1. Optimization steps for the dosage of recombinant esterase AX:

(1) Obtain enzyme liquid according to the method in embodiment 1;

(2) Reaction system and conditions: optimize the amount of recombinant esterase AXE, 1ml reaction system contains 250mmol/l triacetin, 1mol/l hydrogen peroxide reacts at 25℃, pH:7.4 for 5min;

(3) Obtain and detect peracetic acid samples according to the method in Example 3.

The result shows: as shown in Figure 4, along with the increase of the amount of esterase, the amount of the peracetic acid of catalytic production also increases, and when esterase increases to 0.30mg/ml, the amount of the peracetic acid of generation increases slowly , so considering the practical application and cost, the optimum dosage of recombinant esterase in this reaction system is 0.30mg/ml, which can produce 8259ppm peracetic acid.

2. Optimization of triacetin concentration.

Steps:

(1) Obtain enzyme liquid according to the method in embodiment 1;

(2) Reaction system and conditions: optimize the concentration of triacetin, 1ml reaction system contains recombinant esterase AXE 0.3mg/ml, 1mol/l hydrogen peroxide reacts at 25℃, pH:7.4 for 5min;

(3) Obtain and detect peracetic acid samples according to the method in Example 3.

The result shows: as shown in Figure 5, along with the concentration of triacetin increases to 300mmol/l from 100mmol/l, the amount of the peracetic acid of production increases thereupon, but when triacetin concentration is higher than 300mmol When /l, the amount of peracetic acid does not increase significantly, so the concentration of triacetin in this reaction system is optimal 300mmol/l, can produce 9613ppm peracetic acid.

3. Optimization of hydrogen peroxide concentration

Steps:

(1) Obtain enzyme liquid according to the method in embodiment 1;

(2) Reaction system and conditions: optimize the concentration of hydrogen peroxide, 1ml reaction system contains 300mmol/l triacetin, and react 0.3mg/ml recombinant esterase AX at 25℃, pH:7.4 for 5min;

(3) Obtain and detect peracetic acid samples according to the method in Example 3.

The results show that: as shown in Figure 6, with the increase of hydrogen peroxide concentration, the amount of peracetic acid produced by catalysis first increases and then decreases, which may be caused by the rapid inactivation of esterase caused by high concentration of hydrogen peroxide . The optimal concentration of hydrogen peroxide in the reaction system is 1mol/l, which can produce 9613ppm peracetic acid.

4. Optimization of temperature

Steps:

(1) Obtain enzyme liquid according to the method in embodiment 1;

(2) Reaction system and conditions: at different temperatures, 1ml reaction system contains 300mmol/l triacetin, recombinant esterase AX 0.3mg/m, 1mol/l hydrogen peroxide reacts at pH:7.4 for 5min;

(3) Obtain and detect peracetic acid samples according to the method in Example 3.

The results show that, as shown in Table 2, at 20°C, the amount of peracetic acid produced reaches a maximum of 10481ppm, which has significant application value.

Table 2. Optimization of reaction temperature

temperature reflex peracetic acid ppm 20°C 10481.63 25°C 9818.63 30℃ 9730.00 37°C 8407.54

5. pH optimization operation steps:

(1) Obtain enzyme liquid according to the method in embodiment 1;

(2) Reaction system and conditions: under different pH conditions, 1ml reaction system contains 300mmol/l triacetin, recombinant esterase AX 0.3mg/ml, 1mol/l hydrogen peroxide at 20°C for 5min;

(3) Obtain and detect peracetic acid samples according to the method in Example 3.

The results showed that, as shown in Figure 7, when the pH was 8.0, the amount of peracetic acid produced reached a maximum of 11385ppm.

Example 5 This example illustrates the steps of producing peracetic acid by immobilizing AX esterase.

(1) Obtain enzyme liquid according to the method in embodiment 1;

(2) Immobilization of recombinant esterase AX: select polyacrylamide resin, use glutaraldehyde cross-linking method to immobilize recombinant esterase, obtain immobilized AX esterase, the activity of p-nitrophenyl acetate is : 17.5U/g

(3) Reaction system and conditions: 1ml reaction system contains 300mmol/l triacetin, 1mol/l hydrogen peroxide, 0.5g immobilized enzyme, react at pH 8.0, 20℃ for 5min;

(4) Obtain and detect peracetic acid samples according to the method in Example 3.

The results showed that 0.5g of the immobilized recombinant esterase AXE could produce 2678ppm of peracetic acid. Compared with free esterase AXE, it is more convenient and recyclable. The peracetic acid produced after repeating 10 times was about 72% of the initial reaction.

The present invention provides a process using acetylxylan esterase AXE (free or immobilized) having perhydrolytic activity in the presence of hydrogen peroxide (at a concentration of at least 200 mmol/l) at acidic to neutral Concentrated aqueous peracetic acid is generated in situ from suitable short-chain acetates, including glycerides, under reaction conditions.

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the

the

Tyr Pro Ala Asp Gly Val Lys Val Phe Arg Leu Thr Tyr Arg Ser Phe

50 55 60 60

the

the

Gly Lys Ala Glu Ile Glu Gly Trp Tyr Ala Val Pro Asp Arg Gln Gly

65 70 75 80

the

the

Pro His Pro Ala Ile Val Lys Tyr His Gly Tyr Asn Ala Ser Tyr Asp

85 90 95

the

the

Gly Asp Ile His Asp Ile Val Asn Trp Ala Leu His Gly Tyr Ala Ala

100 105 110

the

the

Phe Gly Met Leu Val Arg Gly Gln His Ser Ser Thr Asp Thr Ser Val

115 120 125

the

the

Ser Pro His Gly His Val Pro Gly Trp Met Thr Lys Gly Ile Leu Asp

130 135 140

the

the

Lys Asp Thr Tyr Tyr Tyr Arg Gly Val Tyr Leu Asp Ala Val Arg Ala

145 150 155 160

the

the

Leu Glu Val Ile Ser Gly Phe Asp Glu Val Asp Glu Thr Arg Ile Ala

165 170 175

the

the

Val Ile Gly Gly Ser Gln Gly Gly Gly Leu Ser Ile Ala Ala Ala Ala

180 185 190

the

the

Leu Ser Asp Ile Pro Arg Ala Val Ala Ala Asp Tyr Pro Tyr Leu Ser

195 200 205

the

the

Asn Phe Glu Arg Ala Ile Asp Val Ala Leu Asp Glu Pro Tyr Leu Glu

210 215 220

the

the

Ile Asn Ser Phe Phe Arg Lys Asn Gly Ser Pro Glu Thr Glu Lys Thr

225 230 235 240

the

the

Ala Met Asn Thr Leu Ala Tyr Phe Asp Ile Met Asn Leu Ala Asp Arg

245 250 255

the

the

Val Lys Val Pro Val Leu Met Ser Ile Gly Leu Ile Asp Arg Val Thr

260 265 270

the

the

Pro Pro Ser Thr Val Phe Ala Ala Tyr Asn His Leu Glu Thr Glu Lys

275 280 285

the

the

Gln Leu Lys Val Tyr Arg Tyr Phe Gly His Glu Tyr Ile Pro Ser Phe

290 295 300

the

the

His Thr Glu Lys Leu Ala Phe Leu Lys Ala His Leu Lys Gly

305 310 315

Claims (10)

1. there is the application of the acetylxylan esterase of the aminoacid sequence represented by SEQ ID NO:1 in the perhydrolysis reaction. 2. A method for enzyme-catalyzed production of peracetic acid, with acetyl substrate and hydrogen peroxide as substrate, characterized in that, with acetyl xylan esterase according to claim 1 as enzyme catalyst, contact with substrate The perhydrolysis reaction generates peracetic acid. 3. The method according to claim 2, characterized in that, the carrier containing the acetyl xylan esterase according to claim 1 is used as an enzyme catalyst, and is contacted with a substrate to carry out a perhydrolysis reaction to generate peracetic acid. 4. The method according to claim 2 or 3, wherein the acetyl substrate is selected from one of ethyl acetate, glycerol monoacetate, glycerol diacetate, and glycerol triacetate. 5. The method according to claim 4, characterized in that, in the perhydrolysis reaction system, in milligrams per milliliter, the ratio of the acetyl substrate: hydrogen peroxide: enzyme is 1:0.5- 8.5: 1.5-75. 6. according to the described method of claim 2 or 3, it is characterized in that, the reaction condition of described perhydrolysis reaction is: pH value 6.5-9.5, is preferably pH 8.0. 7. The method according to claim 2 or 3, characterized in that the temperature of the perhydrolysis reaction is 20-37°C, preferably 20°C. 8. The method according to claim 2 or 3, characterized in that the reaction time of the perhydrolysis reaction is within 5 minutes to about 1 hour, preferably within 5 minutes. 9. The method according to claim 2, characterized in that it further comprises the process of expressing the acetylxylan esterase and obtaining a crude enzyme solution according to the coding or amino acid sequence of the protein. 10. The method according to claim 9, characterized in that, comprising the steps in turn: (1) Escherichia coli ( Escherichia coli ) BL21 / pET-CAH was used as the starting strain for seed culture; the Escherichia coli was preserved in the China Center for Type Culture Collection, and the preservation number was: CCTCC NO: M2012408; among them, the seed culture The base is LB medium; the culture conditions are: 250 mL Erlenmeyer flask, liquid volume 50 mL, culture temperature 37°C, shaker speed 200 r/min, culture 12 h; (2) Fermentation culture, the composition of the fermentation medium is liquid LB medium; the culture conditions are: inoculate the seed culture medium according to the volume ratio of 1.5% inoculum, the fermentation temperature is 37 ℃, when the OD reaches 0.6~0.8, add isopropyl -Induction by β-D-thiogalactopyranoside (IPTG), the final concentration of IPTG is 1.0 mmol/L, the speed of the shaker is 180-250 r/min, and the fermentation is 2-10 h; (3) To obtain the crude enzyme liquid, take out the fermentation liquid, centrifuge at 8000rpm for 3 minutes, add 1/8 of the volume of the original fermentation liquid Tris-HCl (pH 7.0), concentrate 8 times; ultrasonic crushing 400W, 3s working time, 7s interval time, Sonicate for 4 minutes; centrifuge at 6500 rpm for 10 minutes, and the supernatant is the crude enzyme solution.