CN112226428B - Oleate hydratase mutant and its application in the preparation of 10-hydroxystearic acid - Google Patents

Abstract

The invention relates to an oleic acid hydratase mutant and application thereof in preparation of 10-hydroxystearic acid, and discloses an oleic acid hydratase mutant with remarkably improved activity and stability, a coding gene and an amino acid sequence thereof, a recombinant expression vector and a recombinant expression transformant containing the gene sequence, a recombinant oleic acid hydratase mutant catalyst, and practical application of the oleic acid hydratase mutant or the recombinant oleic acid hydratase mutant catalyst in preparation of 10-hydroxystearic acid and 10-carbonyl stearic acid by catalytic conversion of oleic acid. Compared with the prior art, the oleic acid hydratase mutant has remarkably high activity and stability, so that the dosage of a catalyst is greatly reduced, and in the enzymatic oxidation preparation process of 10-carbonyl stearic acid, cheap hydrogen peroxide is used as an oxidant, so that the cost is low, the method is green and environment-friendly, and the method has a good application prospect in the production of 10-carbonyl stearic acid.

Description

Oleic acid hydratase mutant and its application in the preparation of 10-hydroxystearic acid

technical field

The invention belongs to the technical field of bioengineering, in particular to a mutant of oleic acid hydratase derived from Paracoccus aminophilus, its coding gene, a recombinant expression vector containing the gene sequence and a recombinant expression transformant; recombinant oleic acid The hydratase mutant catalyst also relates to the oleic acid hydratase mutant or recombinant oleic acid hydratase mutant catalyst in the preparation of 10-hydroxystearic acid, and the cascade preparation of 10-carbonyl stearic acid with 10-hydroxystearic acid dehydrogenase Applications in fatty acids.

Background technique

As an important natural product, oleic acid is a typical renewable resource with low price and abundant sources. With the successful research and development of high-purity oleic acid production technology and the promotion of high-oleic acid plants, the production of oleic acid in my country is increasing year by year. In 2015, the output of my country's high-purity oleic acid industry was about 16,500 tons (Research Report on the Development of China's Oleic Acid Industry), a year-on-year increase of 22.2%. With the continuous expansion of the oleic acid market, its production scale is still expanding.

Oleic acid is a long-chain fatty acid containing carbon-carbon double bonds. By modifying its functional groups, products such as 10-hydroxystearic acid and 10-carbonyl stearic acid can be obtained. Among them, 10-hydroxystearic acid and 10-carbonylstearic acid can be used as chemical intermediates in the synthesis of fine chemicals and medicines, and have high industrial application value. It has been reported that 10-hydroxystearic acid has beneficial cosmetic effects and can be used to treat or prevent any symptoms caused by the negative development of healthy skin's physiological homeostasis, as well as to promote hair growth and hair loss protection, improve skin age spots and obvious pores.

In 2011, Oh et al. used whole cells of wild-type Stenotrophomonas nitritireducens to catalyze the hydration reaction of oleic acid under anaerobic conditions. The substrate concentration was 30 g L ^-1 , and the wet cell loading capacity was 20 g L ^-1 , the reaction can be completely converted in 4 hours, but only 63% can be converted in 4 hours under aerobic conditions (Biotechnol. Lett., 2011, 33:993-997). In 2019, Xu Jianhe and others excavated oleic acid hydratase (PaOH for short) from Paracoccus aminophilus. When the concentration of oleic acid was 90g/L and the loading capacity of lyophilized enzyme powder was 5kU/L, the conversion rate was higher than 95% after 4 hours of reaction; On this basis, use hydroxystearate dehydrogenase to catalyze the dehydrogenation reaction of the hydration product 10-hydroxystearate to generate 10-carbonylstearate, and couple lactate dehydrogenase to catalyze the oxidation of pyruvate to realize the cycle of coenzyme NAD ⁺ Regeneration, continue to react for 2h, the final yield of 10-carbonylstearic acid is 95.8% (Chinese patent CN110004133A).

At present, the activity of oleic acid hydratase is relatively low. Due to the lack of effective high-throughput screening methods, there are few studies on the modification of activity and stability; In the acid process, expensive pyruvate is used as the cosubstrate of coenzyme cycle regeneration, and the cost of coenzyme cycle is high, which is not conducive to its industrial utilization.

Contents of the invention

In view of the reported defects of oleic acid hydratase, such as low activity, poor stability, and low space-time yield of catalytic reaction, the present invention establishes an efficient and simple high-throughput screening method for oleic acid hydratase, which is derived from Paracoccus aminophilus Starting from the oleic acid hydratase PaOH, its activity and stability were improved by means of molecular engineering, and oleic acid hydratase mutants with higher catalytic performance were obtained. On this basis, the oleic acid hydratase mutant was used in the preparation of 10-hydroxystearic acid, which improved the coenzyme regeneration process of the enzymatic conversion of 10-hydroxystearic acid to prepare 10-carbonylstearic acid, and effectively reduced the 10 - Production cost of carbonylstearic acid.

The overall goal of the invention is to establish an efficient high-throughput screening method, improve the activity and thermal stability of oleic acid hydratase by means of molecular transformation, and establish a relatively cheap and suitable coenzyme circulation system to reduce production costs.

The purpose of the present invention can be achieved through the following technical solutions:

One of the technical solutions of the present invention is to obtain the oleic acid hydratase mutant with significantly improved catalytic performance.

The present invention uses an oleic acid hydratase (named PaOH) derived from Paracoccus aminophilus JCM 7686 as the parent, and finds the key amino acids near the substrate binding site through the comparison of amino acid sequences and spatial structures Residues, using the strategy of site-directed saturation mutation, successfully obtained mutants with improved stability and catalytic activity, and on this basis, carried out directed evolution transformation through error-prone PCR and other transformation strategies, and combined A batch of oleic acid hydratase mutants with significantly improved stability and catalytic activity were identified through preliminary quantitative screening and secondary screening by gas chromatography.

Wherein, the nucleic acid sequence of the oleic acid hydratase PaOH is shown in SEQ ID No.1, and its amino acid sequence is shown in SEQ ID No.2. The strain Paracoccus aminophilus (Paracoccus aminophilus) JCM 7686 was purchased from the Japan Culture Collection of Microorganisms.

The currently known oleic acid hydratases are all flavoproteins, and no colored compounds are involved in the reaction that catalyzes the conversion of oleic acid to 10-hydroxystearic acid. Therefore, it is difficult to screen mutants directly by high-throughput spectrophotometry. In the present invention, the reaction catalyzed by oleic acid hydratase is coupled with the enzymatic oxidation reaction of the product 10-hydroxystearic acid, and a rapid high-throughput primary screening method with a microplate reader is established. The high-throughput primary screening method of the microplate reader is carried out in the microplate, adding the oleic acid hydratase mutant to the buffer solution containing the oleic acid substrate, reacting for a certain period of time, and controlling the reaction conversion rate within 10%. Then take an appropriate amount of the hydration reaction solution and add it to the mixture containing 10-hydroxystearate dehydrogenase, NAD ⁺ , phenazineethosulfate (PES) and 3-(4,5-dimethyl-2-thiazolyl)- 2,5-diphenyltetrazolium bromide (3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide, MTT, thiazolium blue) color reaction solution. 10-hydroxystearate dehydrogenase can catalyze the oxidation of 10-hydroxystearate and convert NAD ⁺ into NADH at the same time; then NADH can reduce PES from oxidized state to reduced state, and regenerate NAD ⁺ at the same time; and reduced PES MTT can also be converted from an oxidized state to a reduced state, and the oxidized PES can be regenerated at the same time. Repeatedly, 10-hydroxystearic acid can be completely converted into 10-carbonyl stearic acid, and the same amount of reduced MTT can be generated at the same time. The reduced MTT is blue-purple and has a maximum absorption peak at 580nm. It can be quickly detected by a microplate reader, converted to obtain the yield of 10-hydroxystearic acid in the oleic acid hydration reaction, and then calculate the activity of the oleic acid hydratase mutant .

The oleic acid hydratase mutant described in the present invention is the 15th threonine, the 122nd phenylalanine, the 233rd phenylalanine, the 332nd phenylalanine of the amino acid sequence shown in SEQ ID No.2. The protein corresponding to the new amino acid sequence formed by replacing one or more amino acid residues in histidine, 444th tyrosine, 451st aspartic acid, and 622th glutamic acid with other amino acid residues .

Specifically, the oleic acid hydratase mutant is a protein composed of any of the following amino acid sequences:

(1) replacing the 233rd phenylalanine in the amino acid sequence shown in SEQ ID No.2 with leucine;

(2) replacing the 233rd phenylalanine in the amino acid sequence shown in SEQ ID No.2 with leucine, and replacing the 122nd phenylalanine with leucine;

(3) replacing the 233rd phenylalanine in the amino acid sequence shown in SEQ ID No.2 with leucine, and replacing the 622nd glutamic acid with glycine;

(4) Replace the 233rd phenylalanine with leucine in the amino acid sequence shown in SEQ ID No.2, replace the 122nd phenylalanine with leucine, and replace the 15th threonine with alanine amino acid;

(5) Replace the 233rd phenylalanine with leucine in the amino acid sequence shown in SEQ ID No.2, replace the 122nd phenylalanine with leucine, and replace the 15th threonine with serine ;

(6) Replace the 233rd phenylalanine with leucine in the amino acid sequence shown in SEQ ID No.2, replace the 122nd phenylalanine with leucine, and replace the 15th threonine with threonine Paragine;

(7) Replace the 233rd phenylalanine with leucine in the amino acid sequence shown in SEQ ID No.2, replace the 122nd phenylalanine with leucine, and replace the 15th threonine with glycine ;

(8) Replace the 233rd phenylalanine with leucine in the amino acid sequence shown in SEQ ID No.2, replace the 122nd phenylalanine with leucine, and replace the 15th threonine with half Cystine;

(9) Replace the 233rd phenylalanine with leucine in the amino acid sequence shown in SEQ ID No.2, replace the 122nd phenylalanine with leucine, and replace the 15th threonine with iso Leucine;

(10) replacing the 332nd histidine in the amino acid sequence shown in SEQ ID No.2 with tyrosine;

(11) replacing the 444th tyrosine in the amino acid sequence shown in SEQ ID No.2 with phenylalanine, and replacing the 451st aspartic acid with glutamic acid;

(12) The 332nd histidine in the amino acid sequence shown in SEQ ID No.2 is replaced with tyrosine, and the 451st aspartic acid is replaced with threonine.

The method for obtaining the oleic acid hydratase mutant of the present invention can be obtained by conventional methods in the field, preferably by isolating from a transformant recombinantly expressing the above-mentioned oleic acid hydratase mutant; or obtaining it artificially.

The second technical solution of the present invention provides the coding gene of the oleic acid hydratase mutant and a recombinant expression vector containing the coding gene. The coding gene encodes and expresses the modified oleic acid hydratase mutant as described in Technical Solution 1, and its source includes: cloning the gene sequence of the series of oleic acid hydratase mutants described in Technical Solution 1 through genetic engineering technology or obtain the nucleic acid molecule encoding the oleic acid hydratase mutant as described in Technical Scheme 1 by the method of artificial complete sequence synthesis.

The recombinant expression vector can be constructed by linking the nucleotide sequence of the oleic acid hydratase mutant gene of the present invention to various commercially available empty vectors by conventional methods in the art. The commercially available empty vector can be various conventional plasmid vectors in the art, as long as the recombinant expression vector can normally replicate in the corresponding expression host and express the corresponding oleic acid hydratase mutant. For different expression hosts, the preferred plasmid vectors are different. Those of ordinary skill in the art will know how to select appropriate vectors, promoters, enhancers and host cells. For Escherichia coli host, the plasmid vector is preferably plasmid pET-28a(+). The Escherichia coli recombinant expression vector of the present invention can be obtained by the following method: the oleic acid hydratase mutant gene sequence DNA fragment obtained by PCR amplification is double-digested with restriction endonucleases EcoR I and Xho I, At the same time, the empty plasmid pET28a was also double-digested with restriction endonucleases EcoR I and Xho I, and the DNA fragment of the oleic acid hydratase mutant and the empty plasmid after the above digestion were recovered, and ligated with T4 DNA ligase to construct A recombinant expression vector containing the nucleic acid molecule encoding the oleic acid hydratase mutant for expression in Escherichia coli is obtained.

The third technical solution of the present invention provides a recombinant expression transformant comprising the oleic acid hydratase mutant gene of the present invention or its recombinant expression vector. The recombinant expression transformant can be prepared by transforming the above-mentioned recombinant expression vector into corresponding host cells by conventional techniques in the art. The host cell is a conventional host cell in the art, as long as the recombinant expression vector can stably replicate itself and the oleic acid hydratase mutant gene encoded by it can be effectively expressed. In the present invention, Escherichia coli is preferred as the host cell, more preferably Escherichia coli E. coli BL21 (DE3) is used for high-efficiency expression of the oleic acid hydratase mutant described in the present invention.

The fourth technical solution of the present invention provides a recombinant oleic acid hydratase mutant catalyst, and the recombinant oleic acid hydratase mutant catalyst is any one of the following forms:

(1) cultivating the recombinant expression transformant of the present invention, and isolating the transformant cells containing the oleic acid hydratase mutant;

(2) cultivating the recombinant expression transformant of the present invention, and isolating the crude enzyme solution containing the oleic acid hydratase mutant;

(3) crude enzyme powder obtained by drying the crude enzyme liquid of the oleic acid hydratase mutant.

The culture method and conditions of the recombinant expression transformant are conventional methods and conditions in the art, which include the following steps: cultivating the recombinant expression transformant of the invention to obtain a recombinant oleic acid hydratase mutant. For recombinant Escherichia coli, the preferred medium is LB medium: peptone 10g/L, yeast extract 5g/L, NaCl 10g/L, pH 6.5-7.0. A preferred culture method is: inoculate the recombinant Escherichia coli constructed as above into LB medium containing kanamycin, and cultivate overnight at 37° C. with shaking at 180 rpm. Insert 1-2% (v/v) inoculum into a 500mL Erlenmeyer flask equipped with 100mL LB medium (containing kanamycin), place 37°C, 180rpm shaker for shaking culture, when the OD of the culture solution When the ₆₀₀ reaches 0.6-0.8, add isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.1-0.5mmol/L as an inducer, and after induction at 16-25°C for 16-24h, culture The solution was centrifuged, the precipitate was collected, and then washed twice with physiological saline to obtain recombinant expression transformant cells. The harvested recombinant cells are freeze-dried to obtain freeze-dried cells containing the oleic acid hydratase mutant. Suspend the harvested recombinant cells in 5-10 times the volume (v/w) of buffer solution, ultrasonically break, and centrifuge to collect the supernatant to obtain the crude enzyme solution of the recombinant oleic acid hydratase mutant. The collected crude enzyme solution is frozen at -80°C, and then dried at low temperature using a vacuum freeze dryer to obtain freeze-dried enzyme powder. Store the obtained lyophilized enzyme powder in a refrigerator at 4°C for convenient use.

The activity determination method of oleic acid hydratase mutant described in the present invention: react in 2mL round-bottomed EP tube, add the enzyme liquid of suitably dilution in the potassium phosphate buffer solution (100mM, pH 6.5) containing substrate oleic acid After the reaction was carried out at 30° C. in a shaking reactor at 1,000 rpm for 5 min, 20 μL of sulfuric acid (20%, w/v) was added to terminate the reaction. Then add an equal volume of ethyl acetate containing 2mM internal standard palmitic acid, shake and extract, and centrifuge at a centrifugal force of 16,200×g for 2 minutes. Use a pipette gun to suck out 350 μL of the upper organic phase into a new EP tube, add an appropriate amount of anhydrous sodium sulfate to dry overnight. Take 40 μL of the extraction supernatant and add it to the inner tube of the gas chromatography analysis bottle, then add 40 μL of methanol/ether (1:1) mixture and 20 μL of trimethylsilyl diazomethane, and keep warm at 20°C-40°C After 15 min, gas chromatographic analysis was carried out to detect the contents of substrates and products.

The activity unit (U) of oleic acid hydratase is defined as: the amount of enzyme required to catalyze the conversion of 1.0 μmol of oleic acid to 10-hydroxystearic acid per minute under the above reaction conditions.

The fifth technical solution of the present invention provides the application of the recombinant oleic acid hydratase mutant or the catalyst of the oleic acid hydratase mutant in the synthesis of 10-hydroxystearic acid, that is, the use of the recombinant oleic acid hydratase mutant enzyme Method conversion method for preparing 10-hydroxystearic acid.

The specific reaction conditions for the oleic acid hydratase mutant to catalyze oleic acid to generate 10-hydroxystearic acid, such as substrate concentration, buffer composition, pH, enzyme dosage, etc., can be selected according to the conventional conditions of this type of reaction in the art. Preferably, the reaction buffer is KPB buffer, pH 6.0-7.5, the buffer concentration is 0.05-0.2mol/L, more preferably KPB buffer with a concentration of 0.1mol/L, pH 6.5; the reaction temperature is 15-35°C , more preferably at 30° C.; the concentration of the substrate oleic acid in the reaction solution is 10-100 g/L, and the activity of the mutant oleic acid hydratase is 0.1-5 kU/L.

After the reaction is finished, the product can be extracted using conventional methods in the art. Add acid to the reaction solution, adjust the pH of the reaction solution to strong acidity, preferably, add concentrated hydrochloric acid, adjust the pH of the reaction solution to about 2, perform multiple extractions with ethyl acetate, combine the extracts, and remove the solvent by rotary evaporation , and then add an appropriate amount of methanol for recrystallization, filter with suction, and dry the filter cake to obtain the target product.

The sixth technical solution of the present invention provides the application of the recombinant oleic acid hydratase mutant or the oleic acid hydratase mutant catalyst in the synthesis of 10-carbonyl stearic acid, that is, to provide the recombinant oleic acid hydratase mutant enzyme Method conversion to prepare 10-hydroxystearic acid, and then add 10-hydroxystearate dehydrogenase to prepare the method for 10-carbonylstearic acid. Among them, the present invention adopts a green and cheap coenzyme recycling method, which reduces the cost of coenzyme application.

The preparation route of described 10-carbonylstearic acid can refer to following way:

Use the recombinant oleic acid hydratase mutant of the present invention as a catalyst to catalyze the hydration of oleic acid to generate 10-hydroxystearic acid; when the conversion rate of the reaction reaches the expected level, adjust the pH of the reaction system, and then add 10-hydroxystearic acid Dehydrogenase MlADH, NAD(P)H oxidase SmNOX _Var , catalase, hydrogen peroxide and coenzyme NAD ⁺ catalyze in situ the conversion of 10-hydroxystearic acid generated in the first step into 10-carbonyl hard Fatty acid.

The 10-hydroxystearate dehydrogenase can be conventionally selected in the field. The present invention provides a source of 10-hydroxystearate dehydrogenase: it is derived from Micrococcus luteus, named It is MlADH, its amino acid sequence is shown in SEQ ID No.3. The specific reaction conditions of described 10-hydroxystearate dehydrogenase catalyzing 10-hydroxystearic acid to generate 10-carbonylstearic acid such as substrate concentration, buffer solution composition, pH, enzyme dosage etc. can be according to this type of reaction in this area Choose from normal conditions. Preferably, the pH of the reaction buffer is 7.5-9.0, more preferably 8.0; the reaction temperature is 15-35°C, more preferably 30°C; the activity of the 10-hydroxystearate dehydrogenase is 0.1-10kU /L.

10-hydroxystearate dehydrogenase catalyzes the reaction of 10-hydroxystearic acid to 10-carbonylstearic acid, which requires the participation of coenzyme NAD ⁺ , the concentration of coenzyme NAD ⁺ is 0.1-0.5mM, and the coenzyme NAD ⁺ is reduced during the reaction for NADH. The in situ regeneration of NAD ⁺ during the reaction can be realized by coupling with the reaction catalyzed by NAD(P)H oxidase. The NAD(P)H oxidase uses oxygen as a substrate to catalyze the oxidation of NADH to generate NAD ⁺ , and at the same time the oxygen is reduced to generate water. Since oxygen is limited in the system, hydrogen peroxide is decomposed by catalase to provide oxygen.

The NAD(P)H oxidase is derived from a mutant of Streptococcus mutans, named SmNOX _Var , and its amino acid sequence is shown in SEQ ID No.4. The SmNOX _Var catalyzes the oxygen oxidation of NADH to generate NAD ⁺ , and the specific reaction conditions such as buffer composition, pH, enzyme dosage, etc. can be selected according to the conventional conditions of this type of reaction in the art. Preferably, the upper limit of the activity of the SmNOX _Var is 0.1-10kU/L.

The catalase was purchased from Sangon Bioengineering (Shanghai) Co., Ltd., with an activity of 2000-5000 U/mg. The catalase can catalyze the decomposition of hydrogen peroxide to generate oxygen under the same conditions as the catalytic reaction of SmNOX _Var .

After the reaction is finished, the product can be extracted using conventional methods in the art. Add acid to the reaction solution to adjust the pH of the reaction solution to strong acidity. Preferably, add concentrated hydrochloric acid to adjust the pH of the reaction solution to about 2, then perform multiple extractions with ethyl acetate, combine the extracts, and remove by rotary evaporation. solvent, and then add an appropriate amount of methanol for recrystallization, filter with suction, and dry the filter cake to obtain the target product.

Compared with the prior art, the present invention has the advantages that: the present invention provides an oleic acid hydratase mutant with significantly improved activity and thermal stability, which can efficiently catalyze oleic acid to generate 10-hydroxystearic acid, and the 10- In the synthesis of hydroxystearic acid and 10-carbonyl stearic acid, the amount of catalyst is less, the concentration of substrate oleic acid is as high as 100g/L, and the conversion rate of more than 99% is achieved; and a greener and cheaper coenzyme is provided The recycling method uses cheap hydrogen peroxide as an oxidant, and the enzymatic cascade realizes the cyclic regeneration of the expensive coenzyme NAD+, which effectively reduces the cost of enzymatic cascade production of 10-carbonyl stearic acid, and has a good industrial application prospect.

Description of drawings

Figure 1: Schematic diagram of the cascade catalyzed conversion of oleic acid to 10-carbonyl stearic acid by oleic acid hydratase mutants and 10-hydroxystearate dehydrogenase.

Detailed ways

The various reaction or detection conditions described in the summary of the present invention can be combined or changed according to common knowledge in the field, and can be verified through experiments. The technical solutions and technical effects of the present invention will be clearly and completely described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited to these embodiments, and any changes or equivalent substitutions that do not depart from the concept of the present invention are included in within the protection scope of the present invention.

The sources of material in the following examples are:

The parent recombinant plasmid pET28a-PaOH contains the nucleic acid sequence shown in the sequence table SEQ ID No. 1, which is also disclosed in the patent CN110004133A.

Plasmid vector pET28a was purchased from Novagen.

E.coli DH5α, E.coli BL21(DE3), 2×Taq PCR MasterMix, and Agarose Gel DNA Recovery Kit were purchased from Beijing Tiangen Biochemical Technology Co., Ltd.

Both restriction endonucleases EcoR I and Xho I are commercially available from New England Biolabs (NEB).

Unless otherwise stated, the specific experiments in the following examples were carried out according to conventional methods and conditions in the art, or according to the commercial instructions of the kits.

Example 1 High-throughput screening method for oleic acid hydratase mutants

Transform the oleic acid hydratase mutant gene DNA transformed into Escherichia coli E.coli BL21 (DE3) competent cells, and pick the single colony grown on the transformed plate with a sterilized toothpick until each well contains 200 μL LB medium Incubate in a 96-well deep-well plate at 37°C and 220 rpm, which is the primary plate. After the primary plate is cultured for 12 hours, the growth of the bacterial solution in each well tends to be consistent after exponential growth, and then it can be transferred to the secondary deep-well plate. Aspirate 20 μL of the first-level plate bacterial liquid with a row gun, transfer it to a 96-well deep-well plate containing 600 μL LB medium in each well, and cultivate for 2.5 hours. After the OD ₆₀₀ reaches 1-2, add 50 μL of LB medium containing IPTG, The final concentration of IPTG in the secondary plate was 0.1 mM. After induction at 16° C. for 20 h, the cells were collected by centrifugation at 2395×g for 10 min, and the supernatant was discarded. The primary plate was stored at 4°C after transfer to the secondary plate.

Add 200 μL of lysate (containing 750 mg/L Lysozyme and 10 mg/L DNase I, dissolved in 100 mM, pH 7.5 KPB) to each well of the secondary plate, close the lid, shake the suspended cells vigorously on the shaker, and then place the secondary plate Place on a shaker at 30°C to incubate, shake at 180rpm to fully lyse the cells, and react for 1 hour.

The screening of oleic acid hydratase mutants was carried out in two steps: in the first step, 300 μL of potassium phosphate buffer (100 mM, pH 7.5) containing oleic acid substrate was added to the above cell lysis mixture, and the final concentration of oleic acid was 8 mM, Place on a shaker at 30°C to incubate and shake at 180rpm for 1h.

In the second step, on the basis of the reaction in the first step, take 20 μL of the reaction solution from each well into the corresponding hole of the microtiter plate, and then add 180 μL of the colorimetric reaction solution. The colorimetric reaction solution contains KPB (100mM, pH 7.5), 50U/mL MlADH, 0.8mMNAD ⁺ , 1mM PES and 2mM MTT, placed in a microplate reader, shaken for 5min, and then detected, read at 580nm absorbance value. The first column in each deep-well plate is the female parent, and based on the female parent, the oleic acid hydratase mutant whose absorbance value is significantly higher than that of the female parent is selected, which is the preferred high-activity or high-stability mutant.

Example 2 Semi-rational design and construction of oleic acid hydratase mutants

Homology modeling was carried out on the parent PaOH of oleic acid hydratase, and molecular docking was carried out between it and the substrate molecule, and then site-directed saturation mutation was carried out on the amino acids near the substrate pocket to improve the enzyme activity. Through Uniprot, NCBI BLAST and spatial structure modeling, in the three-dimensional structure of the oleic acid hydratase PaOH of the amino acid sequence shown in the sequence table SEQ ID No.1, the amino acid residues around the binding site of the substrate oleic acid include : 108 glycine, 110 glutamic acid, 112 asparagine, 214 methionine, 233 phenylalanine, 245 cysteine, 377 tryptophan, 442 tryptophan and 537 phenylalanine. The amino acid residues at these positions were subjected to site-directed saturation mutation using site-directed saturation mutagenesis technology.

The primers used are shown in Table 1:

Table 1 Primer list

Wherein N represents any one of the four bases A/T/C/G, K represents any one of the two bases G/T, and M represents any one of the two bases C/A. In single-point saturation mutation, NNK needs more than 90 clones to achieve 95% coverage.

Using pET28a-PaOH as a template, PCR amplification was performed with PrimeStar HS premix to construct a site-directed saturation mutation library. PCR system (20 μL): 10 μL of 2×PrimeStar HS premix, 1 μL of upstream and downstream primers (10 μM), 0.5 ng of pET28a-PaOH plasmid, 1 μL of dimethyl sulfoxide, add sterilized distilled water to make up to 20 μL. PCR reaction program: (1) Pre-denaturation at 95°C for 5 minutes; (2) Denaturation at 94°C for 30 seconds; (3) Annealing at 55°C for 30 seconds; (4) Extension at 72°C for 8 minutes; steps (2) to (4) were performed for 15 cycles in total ;Finally extend at 72°C for 10min, and store the product at 4°C. After the reaction, add 1 μL of restriction endonuclease Dpn I to 20 μL of the PCR product, and incubate at 37°C for 2 hours to fully digest and degrade the template, and transform the digested product into Escherichia coli E.coli BL21(DE3) Competent cells were evenly spread on LB agar plates containing 50 μg/mL kanamycin, and placed in a 37°C incubator for static culture for about 12 hours. The obtained monoclonal colony was picked into a 96-well deep-well plate for culture, and the expressed protein was screened for high-throughput activity according to the method described in Example 1, and the mutants with higher activity were purified and characterized. The corresponding genes were sequenced.

The activity determination method of described oleic acid hydratase mutant: react in 2mL round-bottom EP tube, add the enzyme liquid of suitably dilution in the potassium phosphate buffer solution (100mM, pH 6.5) containing substrate oleic acid, at 30 After shaking at 1,000 rpm for 5 min at °C, 20 μL of sulfuric acid (20%, w/v) was added to terminate the reaction. Then add an equal volume of ethyl acetate containing 2mM internal standard palmitic acid, shake and extract, and centrifuge at a centrifugal force of 16,200×g for 2 minutes. Use a pipette gun to suck out 350 μL of the upper organic phase into a new EP tube, add an appropriate amount of anhydrous sodium sulfate to dry overnight. Take 40 μL of the extraction supernatant and add it to the inner tube of the gas chromatography analysis bottle, then add 40 μL of methanol/ether (1:1) mixture and 20 μL of trimethylsilyl diazomethane, and keep warm at 20°C-40°C 15min, then carry out gas chromatography analysis, detect the content of substrate oleic acid and product 10-hydroxystearic acid.

Through screening, it was found that the stability of the mutant PaOH _F233L obtained by replacing the 233rd phenylalanine of oleic acid hydratase PaOH with leucine and the activity of catalyzing the hydration reaction of oleic acid were improved.

Example 3 Random mutation screening of oleic acid hydratase mutants with improved stability

On the basis of the mutant PaOH _F233L described in Example 2, error-prone PCR was carried out on it to further improve the stability and activity of the enzyme.

The primers used were:

Upstream primer sequence: G GAATTC ATGAGCCCCAAGACCTCCAAACCC

Downstream primer sequence: CCG CTCGAG TCACTTGCGGGTCCTCTCTTTGA

Wherein, the sequence indicated by the underline of the upstream primer is the restriction site of EcoR I, and the sequence indicated by the underline of the downstream primer is the restriction site of Xho I.

Using pET28a-PaOH as a template, error-prone PCR was performed with rTaq DNA polymerase to construct a random mutation library. PCR system (50 μL): rTaq DNA polymerase 0.5 μL, 10×PCR buffer (Mg ²⁺ Plus) 5.0 μL, dNTP Mixture (2.0 mM each) 4.0 μL, final concentration of 75 μM MnCl ₂ , pET28a-PaOH plasmid 0.5ng , 2 μL each of upstream and downstream primers (10 μM), add sterilized distilled water to make up to 50 μL. PCR reaction program: (1) Pre-denaturation at 95°C for 5 minutes; (2) Denaturation at 94°C for 30 seconds; (3) Annealing at 50°C for 30 seconds; (4) Extension at 72°C for 2 minutes; steps (2) to (4) were performed for 20 cycles in total ;Finally extend at 72°C for 10min, and store the product at 4°C. The PCR products were analyzed and verified by agarose gel electrophoresis, and then purified and recovered by gel cutting. The recovered DNA fragment of the target gene and the empty plasmid pET28a were double-digested with restriction endonucleases EcoR I and Xho I at 37°C for 12 hours, respectively. The double-digestion product was verified by agarose gel electrophoresis analysis, and then purified and recovered by gel cutting. The obtained linearized pET28a plasmid and the purified target gene DNA fragment were ligated at 16°C overnight with T4 DNA ligase. Transform the ligation product into Escherichia coli E.coli BL21(DE3) competent cells, spread evenly on the LB agar plate containing 50 μg/mL kanamycin, and place it in a 37°C incubator for static culture for about 12h . The obtained monoclonal colony was picked into a 96-well deep-well plate for culture, and the expressed protein was screened for high-throughput activity according to the method described in Example 1, and mutants with higher activity and stability were purified Characterization and sequencing of the corresponding genes.

Through screening, mutants with significantly improved activity on oleic acid were obtained, and then the thermostability of these mutants was characterized, and a series of mutants with improved thermostability were selected, the sequences of these mutants and the catalysis of these mutants for oleic acid The activity and stability of the hydration reactions are listed in Table 1. In Table 1, sequence numbers correspond to the series of sequences following Table 1, respectively. In the activity column, one plus sign "+" indicates that the activity of the mutant protein to oleic acid is increased by 1-2 times compared with the parent PaOH; two plus signs "++" indicate the activity of the mutant protein on oleic acid The activity is increased by 2-3 times; three plus signs "+++" indicate that the activity of the mutant protein to oleic acid is increased by 3-4 times. In the thermal stability column, one plus sign "+" corresponds to 10.0-20.0% of the residual activity of the mutant protein after incubation at 30°C for 1 hour; two plus signs "++" correspond to the mutation The residual activity of the bulk protein remains 20.1-30.0%; the three plus signs "+++" correspond to 30.1-40.0% residual activity of the mutant protein after incubation at 30°C for 1 hour.

Table 1: List of oleic acid hydratase mutant sequences and corresponding activity improvements

The amino acid sequences of the oleic acid hydratase mutants corresponding to the sequence numbers are as follows:

(1) replacing the 233rd phenylalanine in the amino acid sequence shown in SEQ ID No.2 with leucine;

(2) replacing the 233rd phenylalanine in the amino acid sequence shown in SEQ ID No.2 with leucine, and replacing the 122nd phenylalanine with leucine;

(3) replacing the 233rd phenylalanine in the amino acid sequence shown in SEQ ID No.2 with leucine, and replacing the 622nd glutamic acid with glycine;

(4) Replace the 233rd phenylalanine with leucine in the amino acid sequence shown in SEQ ID No.2, replace the 122nd phenylalanine with leucine, and replace the 15th threonine with alanine amino acid;

(5) Replace the 233rd phenylalanine with leucine in the amino acid sequence shown in SEQ ID No.2, replace the 122nd phenylalanine with leucine, and replace the 15th threonine with serine ;

(6) Replace the 233rd phenylalanine with leucine in the amino acid sequence shown in SEQ ID No.2, replace the 122nd phenylalanine with leucine, and replace the 15th threonine with threonine Paragine;

(7) Replace the 233rd phenylalanine with leucine in the amino acid sequence shown in SEQ ID No.2, replace the 122nd phenylalanine with leucine, and replace the 15th threonine with glycine ;

(8) Replace the 233rd phenylalanine with leucine in the amino acid sequence shown in SEQ ID No.2, replace the 122nd phenylalanine with leucine, and replace the 15th threonine with half Cystine;

(9) Replace the 233rd phenylalanine with leucine in the amino acid sequence shown in SEQ ID No.2, replace the 122nd phenylalanine with leucine, and replace the 15th threonine with iso Leucine;

(10) replacing the 332nd histidine in the amino acid sequence shown in SEQ ID No.2 with tyrosine;

(11) replacing the 444th tyrosine in the amino acid sequence shown in SEQ ID No.2 with phenylalanine, and replacing the 451st aspartic acid with glutamic acid;

(12) The 332nd histidine of the amino acid sequence shown in SEQ ID No.2 is replaced with tyrosine, and the 451st aspartic acid is replaced with threonine;

Example 4 Expression and activity determination of recombinant oleic acid hydratase mutant PaOH _M6

The recombinant Escherichia coli E. coli BL21(DE3)/pET28a-PaOH _M6 expressing the mutant M6 obtained in Example 3 was inoculated into LB medium containing 50 μg/mL kanamycin, and cultured on a shaking table at 37°C for 12 After that, the inoculation amount of 1% (v/v) was inserted into a 500mL Erlenmeyer flask equipped with 100mL LB medium (containing 50μg/mL kanamycin), placed at 37°C and shaken at 180rpm for shaking culture. When the OD ₆₀₀ of the culture medium reached 0.6, IPTG with a final concentration of 0.2 mM was added as an inducer, and induced at 16°C for 24 hours. The culture solution was centrifuged at 8000×g for 10 min, the cells were collected, and washed twice with saline to obtain resting cells. Suspend the cells obtained in 100mL of culture medium in 10mL of potassium phosphate buffer (100mM, pH 8.0), and perform the following ultrasonic disruption in an ice-water bath: power 400W, working for 4s, intermittent for 6s, for 99 cycles, 12000 at 4°C Centrifuge at ×g for 40 minutes to collect the supernatant crude enzyme solution with an activity of 9.0 U/mL; freeze-dry the crude enzyme solution to obtain crude enzyme powder with an activity of 1.9 U/mg. In addition, the harvested cells were freeze-dried to obtain a freeze-dried cell viability of 0.5 U/mg.

Embodiment 5 PaOH and PaOH _M6 catalytic performance comparison

Add 0.1 mg/mL PaOH pure enzyme or PaOH _M6 pure enzyme to 0.5 mL KPB (100 mmol/L, pH 6.5), and add oleic acid at a final concentration of 10 g/L. The reaction was shaken at 1000 rpm for 12 hours at 30°C. After the reaction, adjust the pH to below 2 with sulfuric acid (20%, w/v), add 0.5mL ethyl acetate (containing 2mM internal standard palmitic acid) for extraction, repeat the extraction 3 times, combine the extracts, add an appropriate amount of anhydrous Sodium sulfate was dried overnight, and the results of gas chromatography analysis showed that the conversion rate of the parent enzyme PaOH catalyzed reaction was 40%, while the conversion rate of the mutant PaOH _M6 catalyzed reaction was 99%.

The specific analysis conditions of the product are as follows: use a gas chromatograph for analysis, the chromatographic column is Rxi-5Sil MS, nitrogen is used as the carrier gas, the temperature of the injection port and the hydrogen flame detector are both 280 ° C, and the initial column temperature is 180°C, rising to 250°C at 4°C/min. The sample injection volume was 1 μL, the split ratio was 25:1, and the constant pressure mode was controlled. The retention time of oleic acid was 12.24min, and that of 10-hydroxystearic acid was 16.34min.

Example 6 PaOH _M6 Catalyzed Conversion of Oleic Acid to 10-Hydroxystearic Acid

The reaction was carried out in a 250mL three-necked flask, and the reaction system was 100ml, containing KPB buffer (100mM, pH6.5), 10g oleic acid, 0.2g Tween-80, and oleic acid and Tween-80 were emulsified with a high-pressure homogenizer in advance , and then add 0.5 kU of the crushed supernatant crude enzyme liquid of oleic acid hydratase PaOH _M6 as described in Example 4, the reaction is carried out at 25° C. and 200 rpm with stirring, and the reaction is carried out for 4 hours, and the conversion rate is 99.5%.

Example 7 PaOH _M6 Catalyzed Conversion of Oleic Acid to 10-Hydroxystearic Acid

The reaction was carried out in a 250mL three-necked flask, and the reaction system was 100ml, containing KPB buffer (100mM, pH6.5), 5g oleic acid, 0.2g Tween-80, oleic acid and Tween-80 were emulsified with a high-pressure homogenizer in advance , and then add 0.5 kU of the crushed supernatant crude enzyme solution of oleic acid hydratase PaOH _M6 as described in Example 4, and the reaction is carried out at 40° C. and 200 rpm with stirring, and the reaction is carried out for 2 hours, and the conversion rate is 96.8%.

Example 8 PaOH _M6 Catalyzed Conversion of Oleic Acid to 10-Hydroxystearic Acid

The reaction was carried out in a 1L mechanically stirred reactor, the reaction system was 600ml, containing KPB buffer (100mM, pH6.5), 30g oleic acid, 1.2g Tween-80, oleic acid and Tween-80 were homogenized by high pressure in advance agent emulsification, and then add 1.5 kU of the crushed supernatant crude enzyme solution of oleic acid hydratase PaOH _M6 as described in Example 4, and the reaction is carried out at 30° C. and 200 rpm with stirring for 2 hours. Use concentrated hydrochloric acid to adjust the pH of the reaction solution to below 2, use an equal volume of ethyl acetate to extract, repeat 3 times, combine the extracts, add anhydrous sodium sulfate to dry, remove the solvent by rotary evaporation, recrystallize with methanol, and purify to obtain 27.8g 10-Hydroxystearic Acid with a purity of 99.0%.

Example 9 MlADH catalyzes the conversion of 10-hydroxystearic acid into 10-carbonylstearic acid

The reaction was carried out in a 250mL mechanically stirred reactor, and the reaction system was 100ml, containing KPB buffer (100mM, pH 8.0), 5g of 10-hydroxystearic acid as described in Example 8, 0.2g Tween-80, 10- Hydroxystearic acid and Tween-80 were pre-emulsified using a high-pressure homogenizer, and then 0.5mM NAD ⁺ , 0.4kU MlADH, 2kU SmNOX _Var and 2kU lyophilized enzyme powder of catalase were added, and mixed at a rate of 25μL/min 15% H ₂ O ₂ was added to start the second step of reaction, and the reaction was continued for 4 hours at 30° C. with stirring at 200 rpm, and the conversion rate was 98.7%.

Example 10 PaOH _M6 and MlADH cascade catalyzed conversion of oleic acid into 10-carbonyl stearic acid

As shown in Figure 1, the oleic acid hydratase mutant (PaOH _M6 ) and 10-hydroxystearate dehydrogenase (M1ADH) of the present invention are used to catalyze the conversion of oleic acid into 10-carbonyl stearic acid.

The reaction was carried out in a 1L mechanically stirred reactor, the reaction system was 600ml, containing KPB buffer (100mM, pH6.5), 30g oleic acid, 1.2g Tween-80, oleic acid and Tween-80 were homogenized by high pressure in advance agent emulsification, and then add 1.5kU of the broken supernatant crude enzyme liquid of oleic acid hydratase PaOH _M6 as described in Example 4, the reaction is carried out at 30°C and 200rpm stirring, and the reaction is 2 hours, and the oleic acid is hydrated to generate 10-hydroxystearin acid. Then add NaOH solution to adjust the pH of the reaction system to be 8.0, then add 0.1mM NAD ⁺ , 2.4kU MlADH, 9kU SmNOX _Var and 12kU lyophilized enzyme powder of catalase, and add 15% of H ₂ O ₂ , start the second step of reaction, and continue to react for 4h. Use concentrated hydrochloric acid to adjust the pH of the reaction solution to below 2, use an equal volume of ethyl acetate to extract, repeat 3 times, combine the extracts, add anhydrous sodium sulfate to dry, remove the solvent by rotary evaporation, recrystallize with methanol, and purify to obtain 25.3g 10-Carbonyl Stearic Acid with a purity of 99.0%.

The above descriptions of the embodiments are for those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments, and apply the general principles described here to other embodiments without creative efforts. Therefore, the present invention is not limited to the above-mentioned embodiments. Improvements and modifications made by those skilled in the art according to the disclosure of the present invention without departing from the scope of the present invention should fall within the protection scope of the present invention.

sequence listing

<110> East China University of Science and Technology

Suzhou Baifuan Enzyme Technology Co., Ltd.

<120> Mutant oleic acid hydratase and its application in the preparation of 10-hydroxystearic acid

<160> 4

<170> SIP Sequence Listing 1.0

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<211> 1965

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gccgggctgg cagcggcgtt ctacctgatc cgtgatggcg ggatgaaagg gcaagacatc 240

accattctgg atgcgctgga tgtcacgggc ggctcgctcg acggggctgg caatcccgag 300

gatggctata tcatccgcgg cggccgcgag atgaacttta attacgacaa cctttgggac 360

atgttccagg acgtgcaggc gctggagctg cccgagggct acagcgtgct cgacgagtat 420

cgccaactga acgatgccga tcccaactgg tcaaagtctc ggctgatgca caatcagggc 480

gagattcgcg atttctcaac cttcggcctg accaagccgc agcaatggga gctgatccgc 540

ctgctcttga agcgcaaaga ggatctcgat gatctgacca tcgaggatta tttcagcccc 600

ggcttcctgc agtcgaactt ttggttcctg tggcgctcga tgttcgcctt tgagaactgg 660

cagagcttgc tggagatgaa gctttatacc caccgctttc tcgattccat cgacgggttt 720

gcggatatgt cctgcctcgt tttcccgaag tataatcagc atgatacctt cgttaagccg 780

ctggtcgacc acctaaagaa gctcggcgtt caggtccagt tcgcgacccg tgtctctgat 840

ttggaaatga ccgaagacgc aggcaagcgc agtgtgacgg gcattctggc cagcgtgaac 900

ggtcaggaac accgcatccc agtcgatgaa aaggatgtgg tctttgcgct gaccggctcg 960

atgaccgagg gcaccgccta tggcgatatg gatcatgccc ccgtgatgga gcgcgggcgc 1020

tcggatccgg gaccagacag tgattgggct ttgtggcaaa acctcgccgc gaaatcgccg 1080

atctttggca atcccaagaa gttctatggc gatatcgaca agtcgatgtg ggaatccggc 1140

acgctgacgt gcaagccctc gcccctgact gaccggctga cagagctgtc ggtcaacgac 1200

ccctattccg gcaagaccgt gaccggcggc atcattacct tcaccgactc gaattgggtg 1260

atgagcgtga cctgtaaccg ccagccgcat ttcctcggcc agcccaagga tgttctggtg 1320

ctctgggtct atgcgctgct gatggacaag gatggcaaca aggtcaaaaa gcccatgccc 1380

gcctgcaccg ggcgcgagat tttggccgag ctgtgccatc atttgggcat tcccgacgat 1440

caattcgagg ccgtcgccgc gaagaccaag gtacggttgg cgttgatgcc ctatattacc 1500

tcgatgttca tgccgcgtgc caaaggtgac cgtccccatg tcgtgcccga gggctgcacg 1560

aacctcgcgc tgatgggcca gttcgtcgag acggcgaatg atatcgtctt caccatggat 1620

agctcgatcc gcacggcgcg cattggcgtc tatacgctgt tggggctgcg caagcaggtg 1680

cccgatatca gcccggtgca atatgatatc cgcaccttga tcaaggccgc ccgcacggtg 1740

aacaacaacc agcccttccc gggtgaacgc ctgctgcatc gtctcttggg aaagacctac 1800

tatgcccata tcctgccgcc gctgcccgat cgcacccaaa ccaccccgcga cgctgccgag 1860

accgaactga aggcgtttct cggtactggc ggcactgctc tggcggccgt gggcggttgg 1920

ctgcaaaggg ttcgcgagga cctcaaagag aggacccgca agtga 1965

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Gly Asn Pro Glu Asp Gly Tyr Ile Ile Arg Gly Gly Arg Glu Met Asn

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Phe Asn Tyr Asp Asn Leu Trp Asp Met Phe Gln Asp Val Gln Ala Leu

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Asp Ala Asp Pro Asn Trp Ser Lys Ser Arg Leu Met His Asn Gln Gly

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Glu Ile Arg Asp Phe Ser Thr Phe Gly Leu Thr Lys Pro Gln Gln Trp

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Thr Ile Glu Asp Tyr Phe Ser Pro Gly Phe Leu Gln Ser Asn Phe Trp

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Gly Leu Thr Gln Glu Lys Ala Lys Arg Phe Gly Tyr Asn Pro Glu Val

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Thr Ala Phe Thr Asp Phe Gln Lys Ala Ser Phe Ile Glu His Asp Asn

370 375 380

Tyr Pro Val Thr Leu Lys Ile Val Tyr Asp Lys Asp Ser Arg Leu Val

385 390 395 400

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Met Phe Ser Leu Ala Ile Gln Glu Lys Val Thr Ile Glu Arg Leu Ala

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Claims (12)

1. An oleic acid hydratase mutant which is a protein consisting of any one of the following amino acid sequences:

(1) Substituting phenylalanine 233 of the amino acid sequence shown in SEQ ID No.2 with leucine;

(2) The phenylalanine at position 233 of the amino acid sequence shown in SEQ ID No.2 is replaced by leucine, and the phenylalanine at position 122 is replaced by leucine;

(3) The 233 rd phenylalanine and 622 nd glutamic acid of the amino acid sequence shown in SEQ ID No.2 are replaced by leucine and glycine respectively;

(4) Substituting phenylalanine at position 233, phenylalanine at position 122, and threonine at position 15 of the amino acid sequence shown in SEQ ID No.2 with leucine;

(5) Substituting phenylalanine at position 233, phenylalanine at position 122, and threonine at position 15 of the amino acid sequence shown in SEQ ID No.2 with leucine;

(6) Substituting phenylalanine at position 233 of the amino acid sequence shown in SEQ ID No.2 with leucine, phenylalanine at position 122 with leucine, and threonine at position 15 with asparagine;

(7) The phenylalanine at position 233, the phenylalanine at position 122 and the threonine at position 15 of the amino acid sequence shown in SEQ ID No.2 are replaced by leucine, leucine and glycine respectively;

(8) The phenylalanine at position 233, the phenylalanine at position 122 and the threonine at position 15 of the amino acid sequence shown in SEQ ID No.2 are replaced by leucine, leucine and cysteine respectively;

(9) The phenylalanine at position 233, the phenylalanine at position 122, and the threonine at position 15 of the amino acid sequence shown in SEQ ID No.2 were replaced with leucine, and isoleucine, respectively.

2. An isolated nucleic acid encoding the oleic acid hydratase mutant of claim 1.

3. A recombinant expression vector comprising the nucleic acid of claim 2.

4. A recombinant expression transformant comprising the recombinant expression vector of claim 3.

5. A recombinant oleic hydratase mutant catalyst which is in any one of the following forms:

(1) Culturing the recombinant expression transformant according to claim 4, and isolating a transformant cell containing the oleic acid hydratase mutant according to claim 1;

(2) Culturing the recombinant expression transformant according to claim 4, and isolating a crude enzyme solution containing the oleic acid hydratase mutant according to claim 1;

(3) Culturing the recombinant expression transformant according to claim 4, isolating a crude enzyme solution containing the oleic acid hydratase mutant according to claim 1, and drying the crude enzyme solution to obtain a crude enzyme powder.

6. Use of the oleic acid hydratase mutant of claim 1 or the recombinant oleic acid hydratase mutant catalyst of claim 5 in the preparation of 10-hydroxystearic acid comprising the steps of:

catalyzing the hydration reaction of the substrate oleic acid to produce 10-hydroxystearic acid using the oleic acid hydratase mutant of claim 1 or the recombinant oleic acid hydratase mutant catalyst of claim 5.

7. Use of an oleic acid hydratase mutant according to claim 1 or a recombinant oleic acid hydratase mutant catalyst according to claim 5 in the preparation of 10-carbonyl stearic acid comprising the steps of:

(1) Catalyzing the hydration reaction of the substrate oleic acid to produce 10-hydroxystearic acid using the oleic acid hydratase mutant of claim 1 or the recombinant oleic acid hydratase mutant catalyst of claim 5;

(2) Catalyzing the oxidation of the reaction product of step (1) with 10-hydroxystearic acid dehydrogenase having an amino acid sequence as set forth in SEQ ID No.3 to produce 10-carbonyl stearic acid.

8. Use according to claim 7, characterized in that: the 10-hydroxystearic acid dehydrogenase catalyzes the reaction of oxidizing 10-hydroxystearic acid to generate 10-carbonyl stearic acid by using coenzyme NAD ⁺ In the course of the reaction, NAD ⁺ Converting to NADH, coupling the reaction with oxygen reduction reaction catalyzed by NADH oxidase to oxidize coenzyme NADH to regenerate NAD ⁺ 。

9. Use according to claim 8, characterized in that: coenzyme NAD ⁺ The concentration of (B) is 0.1-0.5 mM.

10. Use according to claim 8, characterized in that: oxygen is obtained by catalase-catalyzed decomposition of hydrogen peroxide.

11. The use of claim 7, wherein in step (2) the oxidation of 10-hydroxystearic acid to 10-carbonyl stearic acid is catalyzed by a 10-hydroxystearic acid dehydrogenase having an amino acid sequence as shown in SEQ ID No.3, wherein NAD (P) H oxidase is addedSmNOX _Var Catalase, hydrogen peroxide and coenzyme NAD ⁺ ；

The NAD (P) H oxidase is derived from Streptococcus mutans (S.) (Streptococcus mutans) Is namedSmNOX _Var The amino acid sequence is shown in SEQ ID No. 4.

12. Use according to claim 7, characterized in that:

the steps (1) and (2) are carried out step by step, after the step (1) is finished, the product 10-hydroxystearic acid is extracted by a conventional method, and then the reaction of the step (2) is carried out, so that the 10-hydroxystearic acid is oxidized to generate 10-carbonyl stearic acid; or the like, or, alternatively,

the steps (1) and (2) are carried out in series, after the step (1) is finished, the pH value of the reaction liquid is adjusted to a required range, and then the reaction of the step (2) is carried out, so that 10-hydroxystearic acid is oxidized to generate 10-carbonyl stearic acid.

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