CN114317476A - Biological catalysis production process of glucosyl glycerol and sucrose phosphorylase thereof - Google Patents

Abstract

The invention relates to the technical field of enzyme catalysis, in particular to a glucose-based glycerol biocatalytic production process and sucrose phosphorylase thereof, wherein the amino acid sequence of the sucrose phosphorylase is shown as SEQ ID NO: 1 is shown. When the glucose-based glycerol is produced by biological catalysis, the sucrose mother liquor is taken, the buffer solution, the glycerol and the pure water are added, the crude enzyme solution of the sucrose phosphorylase is added, and the reaction is carried out at 37 ℃. Compared with wild sucrose phosphorylase, the sucrose phosphorylase in the biocatalytic production process 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 when the substrate is at high concentration.

Description

Biological catalysis production process of glucosyl glycerol and sucrose phosphorylase thereof

Technical Field

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

Background

2-O-alpha-D-glucopyranosyl glycerol is mainly found in blue algae and is used for resisting osmotic pressure of saline-alkali environment; it was also found to be present in the plant Myrothamnus flabellifolia and has a rejuvenating effect on the plant. Such desert plants can survive years after being completely dry without losing tissue integrity. Glucosyl glycerol also has an important function of protecting cells and tissues (e.g., skin), which provides the possibility of potential applications in cosmetics. Since glycerol glucoside has a high water binding ability, it produces a strong moisturizing effect when applied to the skin. In addition to cosmetic applications, glucosylglycerols have great potential as low-calorie sweeteners, which may have prebiotic effects.

It has been reported to be useful as a replacement sweetener in food products because of its low cariogenicity and caloric value compared to sucrose. In addition, glucosylglycerols and derivatives thereof have been studied as therapeutic agents for diseases caused by protein misfolding and cancer treatment. In cosmetics, glucosyl glycerol is used as an anti-aging agent and a moisture regulating compound.

To support the development of industrial applications, glucosyl glycerol must be efficiently provided on a large scale. Microbial production of glucosylglycerols may be limited by the output parameters attainable for biosynthesis. In particular, the product concentration in the engineered Corynebacterium glutamicum is less than or equal to 2g/L, the yield and productivity on the substrate used are low, and even lower in the engineered cyanobacteria polycystic algae strains.

The existing preparation methods mainly comprise a chemical synthesis method, an enzymatic in vitro catalysis method and a biological synthesis method. Chemical methods may involve various starting compounds such as maltitol, isomalt, trehalose, and the like. It has been reported that sodium periodate and sodium borohydride can catalyze maltitol to glucosyl glycerol, but the yield is 18%. The yield of the method for catalyzing isomaltose by using acetic acid, lead tetraacetate and sodium borohydride is 12%. The yield was only 5% with trehalose as the starting material. And the byproducts have large influence on the subsequent purification. The biosynthesis method can be synthesized by treating microorganisms such as Stenotrophomonas rhizophila DSM14405 or blue algae by salt stress, and has the disadvantage of difficult scale-up.

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

Among them, sucrose phosphorylase (EC2.4.1.7) is a specific transglycosidase. Two reactions are mainly catalyzed: in the first category, the glucose group of glucose-1-phosphate can be transferred to an acceptor. For example, glucose-1-phosphate and D-fructose are capable of producing sucrose under the catalysis of sucrose phosphorylase. In the second category, the glucose group in sucrose is transferred to an acceptor. For example, sucrose and phosphate are capable of producing glucose-1-phosphate and D-fructose under the catalysis of sucrose phosphorylase. Sucrose phosphorylase catalyzes the reversible conversion of sucrose and phosphate to glucose 1-phosphate and D-fructose in the absence of external influences. In the absence of phosphate, glycerol can intercept the glucosylase intermediate that reacts with sucrose to produce glucosylglycerol, with minor hydrolysis side reactions occurring.

One effective route to glucosylglycerol production is sucrose phosphorylase. Sucrose is an excellent donor substrate for glycerol glycosylation, has high yield (more than or equal to 90 percent) and has better cost economy. Glucosyl glycerol was manufactured industrially by Bitop AG (dottmond, germany) using a biocatalytic process of LmSucP and formulated as a commercial product for cosmetic applications, sold as Glycoin (50% solution of GG).

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

When glucose-1-phosphate is used as a glycosyl donor, only arabinose, arabitol and xylitol can be used as acceptors, and when sucrose is used as the glycosyl donor, the acceptors have wider range and higher activity.

WO2008034158 published by the university of Gredz technology is the first time sucrose phosphorylase (SPase) is used for catalyzing the conversion of sucrose and glycerol into glycerol glucoside, and under the reaction conditions of 0.3M sucrose and 2.0M glycerol, the concentration of the glucose-based glycerol product is about 0.29M, namely, the yield is about 70 g/L. However, the method has the disadvantages of large enzyme dosage, low product concentration and the like.

The patent CN109576239A utilizes Thermoanaerobacterium thermosaccharolyticum to carry out catalytic reaction on heat-resistant sucrose phosphorylase under high temperature, avoids the decomposition of substrates and products and the growth of miscellaneous bacteria by miscellaneous enzymes, has the highest concentration of 189.8g/L and the highest conversion rate of 94 percent, and has better industrial production prospect. However, high conversion (60g/L-200g/L sucrose) can be obtained only at low substrate concentration, and the enzyme dosage is higher, in the example, the highest concentration is 300g/L sucrose, the enzyme dosage is 150g/L, and the mass ratio of the substrate to the enzyme is 2: 1, the enzyme cost is comparatively 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 biological catalysis production process of glucosyl glycerol and sucrose phosphorylase thereof.

In order to achieve the purpose, the invention provides the following technical scheme:

a sucrose phosphorylase having an amino acid sequence as set forth in SEQ ID NO: 1 is shown.

The reaction rate of the sucrose phosphorylase of the present invention 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 biocatalytic production process of glucosyl glycerol specifically comprises the following steps: taking a sucrose mother liquor, adding a 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 400 g/L; and the conversion rate is more than 90% in 3 hours.

Wherein, the preparation method of the crude enzyme solution comprises the following steps:

(1) by a method of whole gene synthesis, the nucleotide sequence shown in SEQ ID NO: 1, and cloning to a prokaryotic expression vector for expression so as to realize high expression in escherichia coli;

(2) shake flask fermentation

Selecting a single escherichia coli colony containing an expression vector, inoculating the single escherichia coli colony into 10mL of culture medium A subjected to autoclaving, and carrying out overnight culture at 30 ℃ and 250 rpm;

taking a 1L triangular flask the next day, and carrying out the following steps: 100 of the inoculation ratio example is inoculated into 100mL of culture medium B after autoclaving, the culture is carried out at 30 ℃ until the thallus OD 5-6, and the triangular flask is immediately placed in a shaker at 25 ℃ and cultured for 1 hour at 250 rpm; IPTG was added to a final concentration of 0.1mM and incubation was continued at 25 ℃ for 16 h at 250 rpm;

after the culture is finished, centrifuging the culture solution at 4 ℃ and 12000g for 20 minutes to collect wet thalli; then washing the thallus precipitate twice with distilled water, collecting thallus and preserving at-70 ℃; meanwhile, 2g of thalli is taken and added with 6mL of pure water for ultrasonic crushing, SDS-PAGE detection is carried out, and the crude enzyme liquid is preserved at the temperature of minus 20 ℃;

(3) fed-batch fermentation

Fed-batch fermentation is carried out in a bioreactor controlled by a computer, 200mL of culture is prepared by primary inoculation of strains, and the strains are inoculated when OD2.0 is reached; during the whole fermentation process, the temperature is kept at 37 ℃, the dissolved oxygen concentration during the fermentation process is automatically controlled at 30 percent by controlling the stirring speed and the aeration supply cascade, and the pH value of the culture medium is maintained at 7.0 by 50 percent of orthophosphoric acid and 30 percent of ammonia water;

during the fermentation, when a large dissolved oxygen rise occurs, feeding is started, and the feeding solution contains 9% w/v peptone, 9% w/v yeast extract and 14% w/v glycerol; when OD600 was 35.0, induction with 0.2mM IPTG for 16 hours; taking 2g of thallus, adding 6mL of pure water, carrying out ultrasonic disruption, carrying out SDS-PAGE detection, and storing the crude enzyme liquid at-20 ℃.

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

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

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

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

compared with wild sucrose phosphorylase, the sucrose phosphorylase of the invention 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 biological catalysis production process of the glucosyl glycerol has high conversion rate which is more than 90 percent, is particularly suitable for the industrial large-scale production of the glycerol glucoside, and can obtain better social benefit and economic value.

Drawings

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

FIG. 2 shows the results of the measurement in example 2, in which sucrose was used for 10 minutes, the objective 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 measurement of the comparative example, sucrose at 10 minutes, the objective product at 11.7 minutes, fructose at 12.8 minutes, and glycerin at 16.8 minutes.

FIG. 4 shows the results of the measurement in example 3, in which sucrose was used for 10 minutes, the objective product was used for 11.7 minutes, fructose was used for 12.8 minutes, and glycerin was used for 16.8 minutes

FIG. 5 shows the results of the measurement in example 4, in which sucrose was used for 10 minutes, the objective product was used for 11.7 minutes, fructose was used for 12.8 minutes, and glycerol was used for 16.8 minutes.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present 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

A 250mm x 4.6um femomelatic acid column was used.

EXAMPLE 1 preparation of crude enzyme solution

(1) By a method of whole gene synthesis, the nucleotide sequence shown in SEQ ID NO: 1, or a corresponding coding polynucleotide sequence of the protein shown in SEQ ID NO: 3, assembling, cloning into a prokaryotic expression vector for expression so as to realize high expression in the Escherichia coli, and naming the ZY 4.

Likewise, a control wild-type sucrose phosphatase protein of SEQ ID NO: 2, the corresponding coding polynucleotide sequence of SEQ ID NO: 4, assembling, cloning to a prokaryotic expression vector for expression, and naming as ZY 3.

(2) Shake flask fermentation

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

(3) Fed-batch fermentation:

the fed-batch fermentation was carried out in a computer-controlled bioreactor (Shanghai Seisaku) with a reactor capacity of 15L and a working volume of 8L, using 24g/L yeast extract, 12g/L peptone, 0.4% glucose, 2.31g/L catalase phosphate and 12.54g/L dipotassium hydrogen phosphate as the medium, pH 7.0. 200mL of the culture was prepared for the primary inoculum and inoculated at OD 2.0. Throughout the fermentation, the temperature was maintained at 37 ℃, the dissolved oxygen concentration during fermentation was automatically controlled at 30% by the agitation rate (rpm) and aeration supply cascade, while 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 a large amount of dissolved oxygen rises, feeding is started. The feed solution contained 9% w/v peptone, 9% w/v yeast extract, 14% w/v glycerol. When OD600 was about 35.0 (wet weight about 60g/L), induction was carried out with 0.2mM IPTG for 16 hours. Taking 2g of thallus, adding 6mL of pure water, carrying out ultrasonic disruption, carrying out SDS-PAGE detection, and storing the crude enzyme liquid at-20 ℃.

Example 2 application example of catalytic reaction

Firstly, preparing a cane sugar mother solution (800g/L), taking 800g of cane sugar, adding water to a constant volume of 1L, heating and dissolving assisting for later use.

2ml of total reaction system with the final concentration of 50mM MES pH7.0, 0.9M sucrose and 1.8M glycerol is prepared in a 5ml centrifuge tube, water is supplemented to 1.8ml, the pH is adjusted to 7, then new enzyme preparation ZY40.2ml is added, and shaking table reaction at 37 ℃ is carried out. Sampling and detecting for 3 hours. The HPLC results are shown in FIG. 2. At glycerol excess, the 3 hour conversion of sucrose was greater than 97%.

Comparative example control for catalytic reaction

2ml of total reaction system with the final concentration of 50mM MES pH7.0, 0.9M sucrose and 1.8M glycerol is prepared in a 5ml centrifuge tube, water is supplemented to 1.8ml, the pH is adjusted to 7, then new enzyme preparation ZY30.2ml is added, and shaking table reaction at 37 ℃ is carried out. Sampling and detecting for 3 hours. The HPLC results are shown in FIG. 3. At glycerol excess, sucrose conversion was only 72% at 3 hours.

Example 3 high concentration catalytic reaction example

Firstly, preparing a cane sugar mother solution (800g/L), taking 800g of cane sugar, adding water to a constant volume of 1L, heating and dissolving assisting for later use.

2ml of total reaction system is prepared in a 5ml centrifuge tube, the final concentration is 50mM MES pH7.0, 1.2M sucrose (equivalent to 408g/L substrate concentration) and 1.8M glycerol are supplemented with water to 1.8ml, after the pH is adjusted to 7, new enzyme preparation ZY40.2ml is added, and the shaking table reaction is carried out at 37 ℃. Sampling and detecting for 3 hours. Due to the high concentration of the substrate, the sample was diluted 20 times with pure water before injection. The HPLC results are shown in FIG. 4. At glycerol excess, sucrose conversion was greater than 90% at 3 hours.

EXAMPLE 4 high concentration catalytic reaction example

Firstly, preparing a cane sugar mother solution (800g/L), taking 800g of cane sugar, adding water to a constant volume of 1L, heating and dissolving assisting for later use.

2ml of total reaction system is prepared in a 5ml centrifuge tube, the final concentration is 50mM MES pH7.0, 0.9M sucrose (equivalent to 306g/L substrate concentration) and 1.8M glycerol are supplemented with water to 1.8ml, after the pH is adjusted to 7, new enzyme preparation ZY40.2ml is added, and shaking table reaction is carried out at 37 ℃. Sampling and detecting for 3 hours. Due to the high concentration of the substrate, the sample was diluted 20 times with pure water before injection. The HPLC results are shown in FIG. 5. The conversion rate of sucrose is more than 96 percent in 3 hours.

The comparison of the experimental results shows that the sucrose phosphorylase has higher reaction speed than the wild sucrose phosphorylase, can effectively shorten the reaction time at high-concentration substrates, improves the production efficiency, reduces the energy consumption and comprehensively reduces the cost; the reaction system has high conversion rate and high reaction speed, can still keep the conversion rate of the sucrose to be more than 90 percent within 3 hours when the high-concentration sucrose is reacted, and simultaneously has no other obvious byproducts except the fructose. Is particularly suitable for the industrial large-scale production of the glycerol glucoside, and can obtain better social benefit and economic value.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments 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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acccactaca aattcgctgg taaaacccgt ctggtttggg tttctttcac cccgcagcag 480

gttgacatcg acaccgactc tgacaaaggt tgggaatacc tgatgtctat cttcgaccag 540

atggctgctt ctcacgtttc ttacatccgt ctggacgctg ttggttacgg tgctaaagaa 600

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 (8)

1. A sucrose phosphorylase enzyme characterized by: the amino acid sequence is shown as SEQ ID NO: 1 is shown.

2. The sucrose phosphorylase according to claim 1, wherein: the substrate concentration and the reaction speed are higher than those of the wild sucrose phosphorylase.

3. The sucrose phosphorylase according to claim 1, wherein: the conversion rate is more than 90%.

4. A biocatalytic production process of glucosyl glycerol is characterized in that: taking a sucrose mother liquor, adding a buffer solution, glycerol and pure water, adding a crude enzyme solution of sucrose phosphorylase described in claims 1 to 3, and reacting at 37 ℃.

5. The process for the biocatalytic production of glucosylglycerols according to claim 4, characterized in that: the highest substrate concentration of the sucrose phosphorylase is less than or equal to 400g/L, and the conversion rate is more than 90% within 3 hours.

6. The process of claim 4, wherein the crude enzyme solution is prepared by the steps of:

(1) by a method of whole gene synthesis, the nucleotide sequence shown in SEQ ID NO: 1, and cloning to a prokaryotic expression vector for expression so as to realize high expression in escherichia coli;

(2) shake flask fermentation

Selecting a single escherichia coli colony containing an expression vector, inoculating the single escherichia coli colony into 10mL of culture medium A subjected to autoclaving, and carrying out overnight culture at 30 ℃ and 250 rpm;

taking a 1L triangular flask the next day, and carrying out the following steps: 100 of the inoculation ratio example is inoculated into 100mL of culture medium B after autoclaving, the culture is carried out at 30 ℃ until the thallus OD 5-6, and the triangular flask is immediately placed in a shaker at 25 ℃ and cultured for 1 hour at 250 rpm; IPTG was added to a final concentration of 0.1mM and incubation was continued at 25 ℃ for 16 h at 250 rpm;

after the culture is finished, centrifuging the culture solution at 4 ℃ and 12000g for 20 minutes to collect wet thalli; then washing the thallus precipitate twice with distilled water, collecting thallus and preserving at-70 ℃; meanwhile, 2g of thalli is taken and added with 6mL of pure water for ultrasonic crushing, SDS-PAGE detection is carried out, and the crude enzyme liquid is preserved at the temperature of minus 20 ℃;

(3) fed-batch fermentation

Fed-batch fermentation is carried out in a bioreactor controlled by a computer, 200mL of culture is prepared by primary inoculation of strains, and the strains are inoculated when OD2.0 is reached; during the whole fermentation process, the temperature is kept at 37 ℃, the dissolved oxygen concentration during the fermentation process is automatically controlled at 30 percent by controlling the stirring speed and the aeration supply cascade, and the pH value of the culture medium is maintained at 7.0 by 50 percent of orthophosphoric acid and 30 percent of ammonia water;

during the fermentation, when a large dissolved oxygen rise occurs, feeding is started, and the feeding solution contains 9% w/v peptone, 9% w/v yeast extract and 14% w/v glycerol; when OD600 was 35.0, induction with 0.2mM IPTG for 16 hours; taking 2g of thallus, adding 6mL of pure water, carrying out ultrasonic disruption, carrying out SDS-PAGE detection, and storing the crude enzyme liquid at-20 ℃.

7. The process for the biocatalytic production of glucosylglycerols according to claim 6, characterized in that: in the step (2) of the crude enzyme solution preparation method, the culture medium A is: 10g/L tryptone, 5g/L yeast extract, 3.55g/L disodium hydrogen phosphate, 3.4g/L potassium dihydrogen phosphate, 2.68g/L ammonium chloride, 0.71g/L sodium sulfate, 0.493g/L magnesium sulfate heptahydrate, 0.027g/L ferric chloride hexahydrate, 5g/L glycerol, 0.8g/L glucose, and kanamycin to 50 mg/L;

the culture medium B comprises: 10g/L tryptone, 5g/L yeast extract, 3.55g/L disodium hydrogen phosphate, 3.4g/L potassium dihydrogen phosphate, 2.68g/L ammonium chloride, 0.71g/L sodium sulfate, 0.493g/L magnesium sulfate heptahydrate, 0.027g/L ferric chloride hexahydrate, 5g/L glycerol, 0.3g/L glucose, and kanamycin to 50 mg/L.

8. The process for the biocatalytic production of glucosylglycerols according to claim 6, characterized in that: the culture medium used in step (3) of the crude enzyme solution preparation method is as follows: 24g/L yeast extract, 12g/L peptone, 0.4% glucose, 2.31g/L catalase phosphate and 12.54g/L dipotassium phosphate, pH 7.0.

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