CN114317476B - Biocatalysis production process of glucosyl glycerine and sucrose phosphorylase thereof - Google Patents

Abstract

The invention relates to the technical field of enzyme catalysis, in particular to a biocatalysis production process of glucosyl glycerine and sucrose phosphorylase thereof, wherein the amino acid sequence of the sucrose phosphorylase is shown as SEQ ID NO: 1. When the biocatalysis is used for producing the glucosyl glycerine, the sucrose mother liquor is taken, the buffer solution, the glycerine and the pure water are added, the crude enzyme solution of the sucrose phosphorylase is added for reaction at 37 ℃. Compared with wild sucrose phosphorylase, the sucrose phosphorylase in the biocatalysis production process has the advantages of higher reaction speed and higher activity, and can effectively shorten the reaction time, improve the production efficiency, reduce the energy consumption and comprehensively reduce the cost when the substrate is in high concentration.

Description

Biocatalysis production process of glucosyl glycerine and sucrose phosphorylase thereof

Technical Field

The invention relates to the technical field of enzyme catalysis, in particular to a biocatalysis production process of glucosyl glycerine and sucrose phosphorylase thereof.

Background

2-O- α -D-glucopyranosyl glycerol is mainly found in cyanobacteria for combating the osmotic pressure of saline-alkaline environments; it was also found to be present in plants Myrothamnus flabellifolia and to have the effect of reviving the plants. Such desert plants can survive many years of complete desiccation without losing tissue integrity. Glucosyl glycerols also have an important function of protecting cells and tissues (e.g. skin), which offers the potential for use in cosmetics. Since glucosyl glycoside has a high water binding capacity, it gives a strong moisturizing effect when applied to skin. In addition to cosmetic applications, glucosyl glycerols have great potential as low calorie sweeteners, which may have prebiotic effects.

It has been reported to be useful as a substitute sweetener in foods because of its low cariogenicity and caloric value compared to sucrose. In addition, glucosyl glycerols and derivatives thereof have been studied as therapeutic agents for diseases caused by protein misfolding and cancer treatment. In cosmetics, glucosyl glycerols are used as anti-aging agents and as moisture regulating compounds.

In order to support the development of industrial applications, glucosyl glycerols must be efficiently provided on a large scale. Microbial production of glucosyl glycerols may be limited by the output parameters achievable by biosynthesis. Specifically, in the engineering corynebacterium glutamicum, the concentration of the product is less than or equal to 2g/L, the yield and the productivity on the used substrate are low, and in the engineering cyanobacteria polycystic strain, the yield is even lower.

The existing preparation methods mainly comprise chemical synthesis methods, enzymatic in-vitro catalysis and biological synthesis methods. The chemical process may involve various starting compounds such as maltitol, isomaltose, trehalose, etc. The use of sodium periodate and sodium borohydride has been reported to catalyze maltitol to glucosyl glycerol, but at 18% yield. The yield of the method for catalyzing isomaltose by using acetic acid, lead tetraacetate and sodium borohydride is 12%. The yield is only 5% by taking trehalose as a raw material. And the byproducts have larger influence on the subsequent purification. Biosynthesis can be achieved by treatment of microorganisms such as stenotrophomonas (Stenotrophomonas rhizophila DSM 14405) or cyanobacteria by salt stress, which has the disadvantage of large-scale production difficulties.

Enzymatic catalysis mainly involves alpha-glucosidase, cyclodextrin glucanotransferase, glycosyl-glycerol-phosphate synthase and sucrose phosphorylase.

Among them, sucrose phosphorylase (EC2.4.1.7) is a specific transglycosidase. Mainly catalyzes two reactions: in the first category, the glucosyl group of glucose-1-phosphate may be transferred to a receptor. For example, glucose-1-phosphate and D-fructose are capable of producing sucrose under the catalysis of sucrose phosphorylase. Second, the glucosyl group in sucrose is transferred to the receptor. For example, sucrose and phosphoric acid are capable of producing glucose-1-phosphate and D-fructose under the catalysis of sucrose phosphorylase. In the absence of external influences, sucrose phosphorylase catalyzes the reversible conversion of sucrose and phosphate to glucose 1-phosphate and D-fructose. In the absence of phosphate, glycerol may intercept the glycosylase intermediate that reacts with sucrose to produce glycosylglycerol, with minimal hydrolysis side reactions occurring.

An efficient route for the preparation of glucosyl glycerols is sucrose phosphorylase. Sucrose is an excellent donor substrate for glycosylation of glycerol, has high yield (. Gtoreq.90%), and has good cost economy. Glucosyl glycerols are manufactured industrially by Bitop AG (dotmond, germany) using the biocatalytic process of lmSucp and formulated as commercial products for cosmetic applications, sold as Glycoin (50% solution of GG).

Leuconostoc mesenteroides-derived sucrose phosphorylase has a broad acceptor specificity when glucose-1-phosphate and sucrose are used as donors.

Bifidobacterium adolescentis sucrose phosphorylase uses only arabinose, arabitol and xylitol as acceptors when glucose-1-phosphate is used as glycosyl donor, and uses sucrose as glycosyl donor, the acceptors are wider and have higher activity.

Published patent WO2008034158 to the university of Gratz's technology is the first use of sucrose phosphorylase (SPase) to catalyze the conversion of sucrose and glycerol to glycerol glucoside at a concentration of about 0.29M for the glucosyl glycerol product at about 70g/L yield under reaction conditions of 0.3M sucrose and 2.0M glycerol. However, the method has the disadvantages of large enzyme dosage, low product concentration and the like.

The patent CN109576239A utilizes Thermoanaerobacterium thermosaccharolyticum source heat-resistant sucrose phosphorylase to carry out catalytic reaction under high temperature condition, avoids the decomposition of the substrate and the product by the enzyme and the growth of the bacteria, has the highest concentration of 189.8g/L and the highest conversion rate of 94 percent, and has better industrial production prospect. However, high conversions (60 g/L to 200g/L sucrose) are only obtained at low substrate concentrations, and the enzyme dosage is relatively high, in the case of a maximum concentration of 300g/L sucrose, the enzyme dosage is 150g/L, the mass ratio of substrate to enzyme is 2:1, the enzyme cost is relatively high, and the conversion rate of 28 hours of reaction under the condition is only 85.2 percent.

Disclosure of Invention

The invention aims to provide a biocatalysis production process of glucosyl glycerine and sucrose phosphorylase thereof.

In order to achieve the above purpose, the present invention provides the following technical solutions:

a sucrose phosphorylase, the amino acid sequence of which is shown in SEQ ID NO: 1.

The reaction speed of the sucrose phosphorylase is higher than that of the wild type sucrose phosphorylase. The conversion rate is more than 90 percent and is far higher than that of the wild type.

A biocatalysis production process of glucosyl glycerine specifically comprises the following steps: taking sucrose mother liquor, adding buffer solution, glycerol and pure water, adding the crude enzyme solution of the sucrose phosphorylase, and reacting at 37 ℃.

Experiments prove that the highest substrate concentration of the sucrose phosphorylase can reach 400g/L; and the conversion rate is greater than 90% in 3 hours.

The preparation method of the crude enzyme liquid comprises the following steps:

(1) By total gene synthesis, SEQ ID NO:1, and cloning the corresponding coding polynucleotide sequence of the protein shown in the formula 1 into a prokaryotic expression vector for expression so as to realize high expression in escherichia coli;

(2) Shaking flask fermentation

E.coli single colony containing the expression vector is selected and inoculated in 10mL of culture medium A after autoclaving, and is cultured at 30 ℃ and 250rpm overnight;

taking 1L triangular flask the next day, and mixing the materials according to the following weight ratio of 1:100 inoculation ratio example 100 is inoculated into 100mL of medium B after autoclaving, cultured at 30 ℃ until the cell OD 5-6 is reached, immediately placing the triangular flask in a 25 ℃ shaker, and culturing at 250rpm for 1 hour; IPTG was added to a final concentration of 0.1mM and incubation was continued at 25℃and 250rpm for 16 hours;

after the culture, the culture solution was centrifuged at 12000g for 20 minutes at 4℃to collect wet cells; then washing the bacterial precipitate twice with distilled water, collecting bacterial precipitate, and preserving at-70 ℃; simultaneously taking 2g of thalli, adding 6mL of pure water, performing SDS-PAGE detection after ultrasonic crushing, and preserving crude enzyme liquid at the temperature of minus 20 ℃;

(3) Fed-batch fermentation

Fed-batch fermentation was performed in a computer controlled bioreactor, with 200mL cultures prepared by primary inoculation of the strain, and inoculated at OD 2.0; the temperature was maintained at 37 ℃ throughout the fermentation, the dissolved oxygen concentration during the fermentation was automatically controlled at 30% by the stirring rate and aeration supply cascade, while the pH of the medium was maintained at 7.0 by 50% orthophosphoric acid and 30% aqueous ammonia;

during the fermentation, when the dissolved oxygen is greatly raised, feeding is started, and the feeding solution contains 9% w/v peptone, 9% w/v yeast extract and 14% w/v glycerol; when the OD600 was 35.0, induction was performed with 0.2mM IPTG for 16 hours; 2g of the cells were sonicated in 6mL of pure water, and then subjected to SDS-PAGE, and the crude enzyme solution was stored at-20 ℃.

Further, the medium a in the step (2) is: 10g/L of tryptone, 5g/L of yeast extract, 3.55g/L of disodium hydrogen phosphate, 3.4g/L of monopotassium phosphate, 2.68g/L of ammonium chloride, 0.71g/L of sodium sulfate, 0.493g/L of magnesium sulfate heptahydrate, 0.027g/L of ferric chloride hexahydrate, 5g/L of glycerol and 0.8g/L of glucose, and kanamycin is added to 50mg/L.

The culture medium B is as follows: 10g/L of tryptone, 5g/L of yeast extract, 3.55g/L of disodium hydrogen phosphate, 3.4g/L of monopotassium phosphate, 2.68g/L of ammonium chloride, 0.71g/L of sodium sulfate, 0.493g/L of magnesium sulfate heptahydrate, 0.027g/L of ferric chloride hexahydrate, 5g/L of glycerol and 0.3g/L of glucose, and kanamycin is added to 50mg/L.

Further, the culture medium used in the step (3) is: 24g/L of yeast extract, 12g/L of peptone, 0.4% of glucose, 2.31g/L of phosphatase and 12.54g/L of dipotassium hydrogen phosphate, and the pH value is 7.0.

Compared with the prior art, the invention has the beneficial effects that:

compared with wild sucrose phosphorylase, the sucrose phosphorylase has higher reaction speed and higher activity, and can effectively shorten the reaction time, improve the production efficiency, reduce the energy consumption and comprehensively reduce the cost in a high-concentration substrate.

The biocatalytic production process of the glucosyl glycerine has high conversion rate of more than 90 percent, is especially suitable for industrial mass production of the glucosyl glucoside, and can obtain better social benefit and economic value.

Drawings

FIG. 1 is a map of a substrate standard; sucrose for 10 minutes and glycerin for 16.8 minutes.

FIG. 2 shows the results of the test in example 2, wherein sucrose was used for 10 minutes, the target product was used for 11.7 minutes, fructose was used for 12.8 minutes, and glycerol was used for 16.8 minutes.

FIG. 3 shows the results of the comparative example, sucrose for 10 minutes, target product for 11.7 minutes, fructose for 12.8 minutes, and glycerin for 16.8 minutes.

FIG. 4 shows the results of the test in example 3, sucrose for 10 minutes, target product for 11.7 minutes, fructose for 12.8 minutes, and glycerol for 16.8 minutes

FIG. 5 shows the results of the test in example 4, sucrose for 10 minutes, the target product for 11.7 minutes, fructose for 12.8 minutes, and glycerin for 16.8 minutes.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The detection conditions referred to in the following examples are as follows.

Liquid phase detection conditions:

mobile phase: 5mM sulfuric acid

A detector: differential detector

Flow rate: 0.6mL/min

Column temperature: 50 DEG C

Differential detector cell temperature: 50 DEG C

250mm 4.6um femomei organic acid column was used.

EXAMPLE 1 preparation of crude enzyme solution

(1) By total gene synthesis, SEQ ID NO:1, and the corresponding coding polynucleotide sequence of the protein shown in SEQ ID NO:3 and cloning to prokaryotic expression vector for high expression in colibacillus, named ZY4.

Likewise, the control wild-type sucrose phosphorylase protein SEQ ID NO:2, the corresponding coding polynucleotide sequence SEQ ID NO:4, and cloning to prokaryotic expression vector for expression, named ZY3.

(2) Shaking flask fermentation

E.coli single colonies containing the expression vector were picked and inoculated into 10mL of autoclaved medium: 10g/L of tryptone, 5g/L of yeast extract, 3.55g/L of disodium hydrogen phosphate, 3.4g/L of monopotassium phosphate, 2.68g/L of ammonium chloride, 0.71g/L of sodium sulfate, 0.493g/L of magnesium sulfate heptahydrate, 0.027g/L of ferric chloride hexahydrate, 5g/L of glycerol and 0.8g/L of glucose, and kanamycin is added to 50mg/L. Culturing at 30℃and 250rpm overnight. Taking 1L triangular flask the next day, and mixing the materials according to the following weight ratio of 1: inoculation ratio of 100 example 100mL of autoclaved medium was inoculated: 10g/L of tryptone, 5g/L of yeast extract, 3.55g/L of disodium hydrogen phosphate, 3.4g/L of monopotassium phosphate, 2.68g/L of ammonium chloride, 0.71g/L of sodium sulfate, 0.493g/L of magnesium sulfate heptahydrate, 0.027g/L of ferric chloride hexahydrate, 5g/L of glycerol and 0.3g/L of glucose, and kanamycin is added to 50mg/L. The cells were cultured at 30℃until the cell OD 5-6 was reached, and the flask was immediately placed in a 25℃shaker at 250rpm for 1 hour. IPTG was added to a final concentration of 0.1mM and the incubation was continued at 25℃and 250rpm for 16 hours. After the completion of the culture, the culture was centrifuged at 12000g for 20 minutes at 4℃to collect wet cells. Then the bacterial cell precipitate is washed twice with distilled water, and the bacterial cells are collected and stored at-70 ℃. Meanwhile, 2g of thalli are added with 6mL of pure water for ultrasonic crushing, SDS-PAGE detection is carried out, and crude enzyme liquid is preserved at the temperature of minus 20 ℃.

(3) Fed-batch fermentation:

fed-batch fermentation was carried out in a computer-controlled bioreactor (Shanghai state of China) with a capacity of 15L and a working volume of 8L, using 24g/L yeast extract, 12g/L peptone, 0.4% glucose, 2.31g/L dihydrogenphosphate and 12.54g/L dipotassium hydrogen phosphate, pH 7.0. 200mL cultures were prepared from the primary seed strain and inoculated at OD 2.0. The temperature was maintained at 37℃throughout the fermentation, the dissolved oxygen concentration was automatically controlled at 30% by a stirring rate (rpm) and aeration supply cascade, and the pH of the medium was maintained at 7.0 by 50% (v/v) orthophosphoric acid and 30% (v/v) aqueous ammonia. During the fermentation process, when the dissolved oxygen is greatly raised, the feeding is started. The feed solution contained 9% w/v peptone, 9% w/v yeast extract, 14% w/v glycerol. When the OD600 was about 35.0 (wet weight about 60 g/L), induction was performed with 0.2mM IPTG for 16 hours. 2g of the cells were sonicated in 6mL of pure water, and then subjected to SDS-PAGE, and the crude enzyme solution was stored at-20 ℃.

Example 2 catalytic reaction application example

Firstly, preparing sucrose mother liquor (800 g/L), taking 800g of sucrose, adding water to a constant volume to 1L, and heating to aid dissolution for later use.

2ml of the total reaction system, 50mM MES pH7.0,0.9M sucrose with final concentration and 1.8M glycerol are prepared in a 5ml centrifuge tube, water is added to 1.8ml, pH is adjusted to 7, and then 0.2ml of new enzyme preparation ZY4 is added for shaking table reaction at 37 ℃. Sampling and detecting for 3 hours. The HPLC results are shown in FIG. 2. Sucrose 3 hours conversion was greater than 97% at glycerol excess.

Comparative example control of catalytic reactions

2ml of the total reaction system, 50mM MES pH7.0,0.9M sucrose with final concentration, 1.8M glycerol and 1.8ml of water are prepared in a 5ml centrifuge tube, pH is adjusted to 7, and then 0.2ml of new enzyme preparation ZY3 is added for shaking table reaction at 37 ℃. Sampling and detecting for 3 hours. The HPLC results are shown in FIG. 3. Sucrose 3 hours conversion was only 72% at glycerol excess.

Example 3 high concentration catalytic reaction example

Firstly, preparing sucrose mother liquor (800 g/L), taking 800g of sucrose, adding water to a constant volume to 1L, and heating to aid dissolution for later use.

2ml of the total reaction system, 50mM MES pH7.0,1.2M sucrose (reduced to 408g/L substrate concentration), 1.8M glycerol, water addition to 1.8ml, pH adjustment to 7, and addition of 0.2ml of fresh enzyme solution ZY4, and shaking reaction at 37 ℃. Sampling and detecting for 3 hours. Because of the high substrate concentration, the sample is diluted 20 times with pure water before injection. The HPLC results are shown in FIG. 4. Sucrose 3 hours conversion was greater than 90% at glycerol excess.

Example 4 high concentration catalytic reaction example

Firstly, preparing sucrose mother liquor (800 g/L), taking 800g of sucrose, adding water to a constant volume to 1L, and heating to aid dissolution for later use.

2ml of the total reaction system, 50mM MES pH7.0,0.9M sucrose (reduced to 306g/L substrate concentration), 1.8M glycerol, water addition to 1.8ml, pH adjustment to 7, and addition of 0.2ml of fresh enzyme solution ZY4, and shaking reaction at 37 ℃. Sampling and detecting for 3 hours. Because of the high substrate concentration, the sample is diluted 20 times with pure water before injection. The HPLC results are shown in FIG. 5. Sucrose 3 hours conversion was greater than 96%.

As can be seen from the comparison of the experimental results, the sucrose phosphorylase has a faster reaction speed than the wild sucrose phosphorylase, and can still effectively shorten the reaction time at high concentration of substrate, improve the production efficiency, reduce the energy consumption and comprehensively reduce the cost; the reaction system has high conversion rate and high reaction speed, can still keep the conversion rate of sucrose to be more than 90% in 3 hours reaction time when high-concentration sucrose reacts, and has no obvious byproducts except fructose. Is especially suitable for the industrial mass production of the glyceroglycosides, and can obtain better social benefit and economic value.

Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Sequence listing

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His Asp Gly Ile Gly Val Ile Asp Ile Gly Ser Asp Gln Leu Asp Arg

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Ser Leu Lys Gly Leu Val Pro Asp Glu Asp Val Asp Asn Leu Val Asn

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Ala Leu Gly Cys Asn Asp Gln His Tyr Ile Ala Ala Arg Ala Val Gln

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Phe Phe Leu Pro Gly Val Pro Gln Val Tyr Tyr Val Gly Ala Leu Ala

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Gly Lys Asn Asp Met Glu Leu Leu Arg Lys Thr Asn Asn Gly Arg Asp

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His Trp Thr Asp Ile Arg Pro Asn Asn Ala Val Thr Val Leu Asp Thr

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His Asp Gly Ile Gly Val Ile Asp Ile Gly Ser Asp Gln Leu Asp Arg

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Ser Leu Lys Gly Leu Val Pro Asp Glu Asp Val Asp Asn Leu Val Asn

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Thr Ile His Ala Asn Thr His Gly Glu Ser Gln Ala Ala Thr Gly Ala

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Ala Ala Ser Asn Leu Asp Leu Tyr Gln Val Asn Ser Thr Tyr Tyr Ser

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Ala Leu Gly Cys Asn Asp Gln His Tyr Ile Ala Ala Arg Ala Val Gln

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Phe Phe Leu Pro Gly Val Pro Gln Val Tyr Tyr Val Gly Ala Leu Ala

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Gly Lys Asn Asp Met Glu Leu Leu Arg Lys Thr Asn Asn Gly Arg Asp

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Arg Pro Val Val Lys Ala Leu Asn Ala Leu Ala Lys Phe Arg Asn Glu

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Ser Ile Ser Phe Thr Trp Arg Gly Glu Thr Ser Gln Ala Thr Leu Thr

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Met Leu Glu Trp Glu Asp Ser Ala Gly Asp His Arg Ser Asp Asp Leu

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gctggtacct cttgcttcat gaccccgaaa accttcaaac tgatctctcg tctgcgtgaa 660

gaaggtgtta aacgtggtct ggaaatcctg atcgaagttc actcttacta caaaaaacag 720

gttgaaatcg cttctaaagt tgaccgtgtt tacgacttcg ctctgccgcc gctgctgctg 780

cacgctctgt ctaccggtca cgttgaaccg gttgctcact ggaccgacat ccgtccgaac 840

aacgctgtta ccgttctgga cacccacgac ggtatcggtg ttatcgacat cggttctgac 900

cagctggacc gttctctgaa aggtctggtt ccggacgaag acgttgacaa cctggttaac 960

accatccacg ctaacaccca cggtgaatct caggctgcta ccggtgctgc tgcttctaac 1020

ctggacctgt accaggttaa ctctacctac tactctgctc tgggttgcaa cgaccagcac 1080

tacatcgctg ctcgtgctgt tcagttcttc ctgccgggtg ttccgcaggt ttactacgtt 1140

ggtgctctgg ctggtaaaaa cgacatggaa ctgctgcgta aaaccaacaa cggtcgtgac 1200

atcaaccgtc actactactc taccgctgaa atcgacgaaa acctgaaacg tccggttgtt 1260

aaagctctga acgctctggc taaattccgt aacgaactgg acgctttcga cggtaccttc 1320

tcttacacca ccgacgacga cacctctatc tctttcacct ggcgtggtga aacctctcag 1380

gctaccctga ccttcgaacc gaaacgtggt ctgggtgttg acaacaccac cccggttgct 1440

atgctggaat gggaagactc tgctggtgac caccgttctg acgacctgat cgctaacccg 1500

ccggttgttg cttaa 1515

Claims (4)

1. A sucrose phosphorylase, characterized in that: the amino acid sequence is shown in SEQ ID NO: 1.

2. A biocatalytic production process of glucosyl glycerine is characterized in that: taking sucrose mother liquor, adding buffer solution, glycerol and pure water, adding crude enzyme solution containing sucrose phosphorylase as defined in claim 1, and reacting at 37 ℃; the amino acid sequence of the sucrose phosphorylase is shown as SEQ ID NO: 1.

3. The biocatalytic production process of glucosyl glycerol according to claim 2, wherein the preparation method of the crude enzyme solution comprises the following steps:

(1) By total gene synthesis, SEQ ID NO:1, and cloning the corresponding coding polynucleotide sequence of the protein shown in the formula 1 into a prokaryotic expression vector for expression so as to realize high expression in escherichia coli;

(2) Shaking and fermenting:

e.coli single colony containing the expression vector is selected and inoculated in 10mL of culture medium A after autoclaving, and is cultured at 30 ℃ and 250rpm overnight; the culture medium A is as follows: 10g/L of tryptone, 5g/L of yeast extract, 3.55g/L of disodium hydrogen phosphate, 3.4g/L of monopotassium phosphate, 2.68g/L of ammonium chloride, 0.71g/L of sodium sulfate, 0.493g/L of magnesium sulfate heptahydrate, 0.027g/L of ferric chloride hexahydrate, 5g/L of glycerol and 0.8g/L of glucose, and adding kanamycin to 50mg/L;

taking 1L triangular flask the next day, and mixing the materials according to the following weight ratio of 1: inoculating 100 to 100mL of autoclaved culture medium B, culturing at 30deg.C until the cell OD 5-6 is reached, immediately placing the triangular flask in 25 deg.C shaking table, and culturing at 250rpm for 1 hr; IPTG was added to a final concentration of 0.1mM and incubation was continued at 25℃and 250rpm for 16 hours; the culture medium B is as follows: 10g/L of tryptone, 5g/L of yeast extract, 3.55g/L of disodium hydrogen phosphate, 3.4g/L of monopotassium phosphate, 2.68g/L of ammonium chloride, 0.71g/L of sodium sulfate, 0.493g/L of magnesium sulfate heptahydrate, 0.027g/L of ferric chloride hexahydrate, 5g/L of glycerol and 0.3g/L of glucose, and adding kanamycin to 50mg/L;

after the culture, the culture solution was centrifuged at 12000g for 20 minutes at 4℃to collect wet cells; then washing the bacterial precipitate twice with distilled water, collecting bacterial precipitate, and preserving at-70 ℃; simultaneously taking 2g of thalli, adding 6mL of pure water, performing SDS-PAGE detection after ultrasonic crushing, and preserving crude enzyme liquid at the temperature of minus 20 ℃;

alternatively, fed-batch fermentation:

fed-batch fermentation is carried out in a computer-controlled bioreactor, 200mL of culture is prepared by primary inoculation strain, and is connected to the bioreactor when the culture is OD2.0, wherein the strain is escherichia coli containing the expression vector and prepared in the step (1); the temperature was maintained at 37℃throughout the fermentation, the dissolved oxygen concentration during the fermentation was automatically controlled at 30% by the stirring rate and aeration supply cascade, and the pH of the medium was maintained at 7.0 by 50% v/v orthophosphoric acid and 30% v/v aqueous ammonia;

during the fermentation, when the dissolved oxygen is greatly raised, feeding is started, and the feeding solution contains 9% w/v peptone, 9% w/v yeast extract and 14% w/v glycerol; when the OD600 was 35.0, induction was performed with 0.2mM IPTG for 16 hours; 2g of the cells were sonicated in 6mL of pure water, and then subjected to SDS-PAGE, and the crude enzyme solution was stored at-20 ℃.

4. A biocatalytic production process of glucosyl glycerol according to claim 3, characterized in that: the culture medium used for fed-batch fermentation in the step (2) of the crude enzyme preparation method is as follows: 24g/L of yeast extract, 12g/L of peptone, 0.4% w/v glucose, 2.31g/L of phosphatase and 12.54g/L of dipotassium hydrogen phosphate, pH 7.0.

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