CN115806956A - l-menthol-O-beta-galactosyl enzyme, coding gene, engineering bacterium and application thereof - Google Patents

Abstract

The invention discloses l-menthol-O-beta-galactosylation enzyme, a coding gene, a genetic engineering bacterium containing the l-menthol-O-beta-galactosylation enzyme and application of the genetic engineering bacterium in preparing l-menthol-O-beta-galactoside under the catalysis of microorganisms. The amino acid sequence of the l-menthol-O-beta-galactosylation enzyme is shown as SEQ ID NO. 2. The recombinant Escherichia coli for producing the l-menthol-O-beta-galactoside, namely the recombinant Escherichia coli (Escherichia coli) E.coli 28a-sth1275, can efficiently synthesize the l-menthol-beta-galactosyl glycosylase intracellularly. The recombinant strain is fermented to induce intracellular expression of l-menthol-O-beta-galactosyl transferase, and the fermented liquid is centrifuged to obtain cells which are then resuspended for direct use in preparing l-menthol-O-beta-galactoside. Catalyzing and synthesizing l-menthol-O-beta-galactoside by using l-menthol and lactose as substrates.

Description

l-menthol-O-beta-galactosyl enzyme, coding gene, engineering bacterium and application thereof

(I) technical field

The invention relates to l-menthol-O-beta-galactosylation enzyme, a coding gene thereof, a genetic engineering bacterium containing the l-menthol-O-beta-galactosylation enzyme, and application thereof in preparing l-menthol-O-beta-galactoside under the catalysis of microorganisms.

(II) technical background

l-menthol-O-beta-galactoside (hereinafter referred to as beta-MenG) is a substance formed by bonding l-menthol molecules and galactose molecules by glycosidic bonds, and the structural formula is shown in figure 1, and the glycosidic bonds couple one hydroxyl group of D-galactopyranose and l-menthol to form glycoside compounds. Galactose molecules are combined with hydroxyl groups of l-menthol to form glycoside compounds of two anomers, i.e., l-menthol-O-alpha-galactoside and l-menthol-O-beta-galactoside. Research shows that the solubility of the L-menthol-O-beta-glucoside in water is 27 times higher than that of the L-menthol-O-alpha-glucoside, and the solubility in water can reach 18.1g/L. The added menthol glycoside can be slowly degraded by microorganisms or glycosidase on the skin surface or the oral cavity, so that the menthol is released, the mint fragrance is prolonged, the cooling feeling and the drug effect are realized, and the menthol glycoside is suitable to be used as a novel and stable latent fragrance raw material.

The l-menthol-O-beta-galactoside has wide application prospect in the industries of food, medicine, cosmetics, tobacco industry and the like. But is limited to the production technology of beta-MenG, and the price of the high-purity product is high. The current production methods include chemical synthesis, enzymatic synthesis and direct extraction from plants or algae. Wherein the chemical method lacks the stereospecificity and regiospecificity, the products are various, and the yield is low. The catalytic synthesis of UDP-dependent glycosyltransferase for beta-glycosylation needs uridine diphosphate (UDP-G) as glycosyl donor, and the raw materials are expensive and difficult to be widely applied. The direct extraction method has too low yield, high cost and difficult industrialization. Currently, the beta-MenG production technology focuses on screening high-activity glycosidases from inexpensive glycosyl donors for biocatalytic conversion synthesis.

Disclosure of the invention

The invention aims to provide l-menthol-O-beta-galactosylation enzyme, a coding gene, a gene engineering bacterium containing the l-menthol-O-beta-galactosylation enzyme and application thereof in preparing l-menthol-O-beta-galactoside under the catalysis of microorganisms, and realizes the one-step glycosylation production of beta-MenG by an enzyme method.

The technical scheme adopted by the invention is as follows:

the amino acid sequence of the l-menthol-O-beta-galactosylating enzyme is shown in SEQ ID NO. 2. The l-menthol-O-beta-galactosyl transferase is derived from Streptococcus thermophilus.

SEQ ID NO.2：

MNMTEKIQTYLNDPKIVSVNTVDAHSDHKYFESLEEFSEGEMKLRQSLNGKWKIHYAQNT

NQVLKDFYKTEFDETDLNFINVPGHLELQGFGSPQYVNTQYPWDGKEFLRPPQVPQESNA

VASYVKHFTLNDALKDKKVFISFQGVATSIFVWVNGNFVGYSEDSFTPSEFEISDYLVEG

DNKLAVAVYRYSTASWLEDQDFWRLYGIFRDVYLYAIPKVHVQDLFVKGDYDYQTKAGQL

DIDLKTVGDYEDKKIKYVLSDYEGIVTEGDASVNGDGELSVSLENLKIKPWSAESPKLYD

LILHVLDDDQVVEVVPVKVGFRRFEIKDKLMLLNGKRIVFKGVNRHEFNARTGRCITEED

MLWDIKVMKQHNINAVRTSHYPNQTRWYELCDEYGLYVIDEANLETHGTWQKLGLCEPSW

NIPASEPEWLPACLDRANNMFQRDKNHASVIIWSCGNESYAGKDIADMADYFRSVDNTRP

VHYEGVAWCREFDYITDIESRMYAKPADIEEYLTTGKLVDLSSVSDKHFASGNLTNKPQK

PYISCEYMHTMGNSGGGLQLYTDLEKYPEYQGGFIWDFIDQAIYKTLPNGSEFLSYGGDW

HDRPSDYEFCGNGIVFADRTLTPKLQTVKHLYSNIKIAVDEKSVTIKNDNLFEDLSAYTF

LARVYEDGRKVSESEYHFDVKPGEEATFPVNFVVEASNSEQIYEVACVLREATEWAPKGH

EIVRGQYVVEKISTETPVKAPLNVVEGDFNIGIQGQNFSILLSRAQNTLVSAKYNGVEFI

EKGPKLSFTRAYTDNDRGAGYPFEMAGWKVAGNYSKVTDTQIQIEDDSVKVTYVHELPGL

SDVEVKVTYQVDYKGRIFVTANYDGKAGLPNFPEFGLEFAIGSQFTNLSYYGYGAEESYR

DKLPGAYLGRYETSVEKTFAPYLMPQESGNHYGTREFTVSDDNHNGLKFTALNKAFEFSA

LRNSTEQIENARHQYELQESDATWIKVLAAQMGVGGDDTWGAPVHDEFLLSSADSYQLSFMIEPLN。

The invention also relates to a gene encoding said l-menthol-O-beta-galactosylating enzyme.

Specifically, the nucleotide sequence of the coding gene is shown as SEQ ID NO. 1.

SEQ ID NO.1：

atgaacatgactgaaaaaattcaaacttatttaaacgatccaaagattgttagcgttaat

actgttgatgctcactcagatcataagtattttgaatctcttgaagaattttctgaaggg

gagatgaagttaagacaatctcttaatggaaaatggaaaattcactatgctcagaataca

aatcaggttttaaaagacttttataaaacagaatttgatgaaactgatttgaatttcatc

aatgtaccaggtcatttagagcttcaaggttttggttctccacaatatgtgaatacccaa

tatccttgggatggtaaagaattccttcgtccacctcaagttcctcaagaatcaaatgct

gttgcatcatacgttaaacattttaccttgaatgatgcattaaaagataaaaaagtattt

atctcattccaaggggttgctacttccatctttgtatgggtcaatggtaacttcgtagga

tacagtgaagattcatttacacctagtgaatttgaaattagtgattaccttgttgaaggt

gataacaagttggcggtagctgtttatcgttattctacagcaagctggttggaagaccaa

gacttctggagactttacggtatttttagagatgtttacttgtatgctattccaaaagtt

cacgttcaagatctctttgttaagggagattatgattaccaaacaaaagcaggtcaattg

gatattgatttgaagactgttggtgattatgaagacaagaagattaaatatgttctttca

gattatgaaggcatcgttacagaaggtgatgcatctgttaatggtgacggtgaactatct

gtaagtcttgaaaatcttaaaatcaaaccttggagtgctgaaagtcctaaactttacgat

ttgatccttcatgttttggatgatgaccaagttgttgaagtcgttccagttaaagttgga

tttagacgctttgaaattaaagataaacttatgcttttgaatggtaagagaattgtcttt

aaaggggttaacagacacgaatttaacgctagaacaggacgttgtatcactgaagaagat

atgctttgggatatcaaagtgatgaaacaacataacatcaatgctgttcgtacttcacac

tatcctaaccaaacacgttggtatgaattgtgtgatgaatatggactttatgttatcgat

gaagccaaccttgaaacacacggtacatggcaaaaacttggtctatgcgaaccttcatgg

aatatcccagctagtgaaccagaatggttgcctgcttgtttggatcgtgccaataacatg

ttccaacgcgataagaaccacgctagtgttatcatttggtcttgtggtaatgaatcatat

gctggtaaagatattgctgacatggctgattacttccgtagtgttgacaatactcgtcca

gttcactatgaaggtgttgcatggtgtcgtgagtttgattacattacagacatcgaaagt

cgtatgtatgcgaaaccagctgatatcgaagaatacctcacaactggtaaactagttgat

ctttcaagcgttagtgataaacactttgcttcaggtaacctaactaacaaacctcaaaaa

ccttatatttcatgtgaatacatgcacacaatgggtaactctggtggtggattgcaactc

tacactgacttagagaaatatccagaataccaaggtggatttatttgggacttcattgac

caagctatttacaaaacacttccaaatggtagcgaattcctatcatatggtggtgactgg

catgatagaccttctgactacgaattttgtggaaatggtatcgtctttgcagatcgtacc

ctaactccaaaacttcaaacagttaaacatctttactctaatattaagattgctgttgat

gaaaaatcagtaactatcaagaatgataatctcttcgaagatctttctgcttatactttc

ctagctagagtttacgaagatggtagaaaagttagtgaaagtgaatatcactttgatgtt

aaaccaggcgaagaagcaacattcccagttaactttgtagtcgaggcttcaaattctgaa

caaatttacgaagttgcttgtgttctgagggaagcaactgaatgggctcctaaaggtcat

gaaattgttcgtggtcaatatgttgttgaaaagattagcactgaaacaccagttaaagca

cctttgaatgttgttgaaggcgacttcaacatcggtattcaaggacaaaacttctcaatc

ttgctttcacgtgcacaaaatactttagtatctgctaagtataatggtgttgaattcatt

gagaaaggtcctaaacttagcttcactcgtgcttacactgacaacgatcgtggtgctgga

tatccattcgaaatggcaggctggaaggttgctggaaactatagtaaagttacagatact

caaattcaaatcgaagacgactctgttaaagtgacttatgttcatgaattgccaggcttg

tctgatgtcgaagttaaggtaacttatcaagttgattacaagggtcgaatctttgttact

gcaaactatgatggtaaagcaggtttgccaaacttccctgaatttggtctagaatttgct

atcggttcacaatttacaaaccttagctattatggatacggtgcagaagaaagctaccgt

gataaacttcctggtgcctatcttggtcgatatgaaacatctgttgaaaagacatttgct

ccatatctaatgccacaagaatctggtaatcactatggtactcgtgaattcacagtatct

gatgataaccataatggtcttaaattcaccgcacttaataaagcattcgaattcagtgct

ttgcgtaacagtactgaacaaattgaaaatgctcgtcaccaatatgagttgcaagaatct

gatgctacatggattaaagttcttgctgctcaaatgggtgtaggtggtgacgacacatgg

ggtgctccagttcatgacgaattcttgcttagctcagcagatagctatcaattaagcttcatgattgaaccactaaattag。

The invention also relates to a recombinant vector containing the coding gene and a gene engineering bacterium.

The construction method of the genetic engineering bacteria comprises the following steps: cloning l-menthol-O-beta-galactosylation enzyme gene (sth 1275) shown in SEQ ID NO.1 onto a pET28a plasmid, constructing a pET28a-sth1275 recombinant expression plasmid, and transforming into Escherichia coli E.coli BL21 (DE 3) to obtain recombinant Escherichia coli E.coli BL21 (DE 3) (pET 28a-sth 1275), namely the genetically engineered bacterium.

The invention also relates to application of the genetic engineering bacteria in preparation of l-menthol-beta-galactoside by microbial catalysis.

Specifically, the application is as follows: taking fermentation liquor obtained by fermentation culture of the genetic engineering bacteria or wet bacteria obtained by fermentation centrifugation as a catalyst, taking l-menthol as a substrate and alpha-lactose as an auxiliary substrate, reacting at 30-45 ℃ to obtain reaction liquid containing l-menthol-beta-galactoside, and separating and purifying the reaction liquid to obtain the l-menthol-beta-galactoside.

The catalyst can be recombinant escherichia coli E.coli BL21 (DE 3) (pET 28a-sth 1275) cell fermentation liquor or a solid microbial inoculum or crude enzyme liquid obtained after cell homogenization, wherein the solid microbial inoculum is prepared by adding an adsorbent into the fermentation microbial inoculum, and the adsorbent is light calcium carbonate or turf; the crude enzyme liquid after cell homogenization is obtained by carrying out high-pressure homogenization on fermentation liquor. The wet thallus content in the fermentation liquor or the bacterial suspension is 5-100 g/L.

The preparation method of the wet thallus shake flask comprises the following steps: (1) Inoculating recombinant escherichia coli containing a beta-galactosidase gene into a seed culture medium containing 50mg/L kanamycin, and culturing at 30-37 ℃ and 180-250 rpm to a logarithmic growth metaphase to obtain a seed solution; the final concentration composition of the seed culture medium is as follows: 5g/L yeast powder, 10g/L, naHPO peptone ₄ ·12H ₂ O 8.9g/L、KH ₂ PO ₄ 3.4g/L、NH ₄ Cl ₂ .67g/L、Na ₂ SO ₄ 0.71g/L、MgSO ₄ ·7H ₂ O0.49 g/L, deionized water as solvent, pH 6.8-7.0;

(2) Fermentation culture: inoculating the seed liquid into a fermentation culture medium containing 50mg/L kanamycin in an inoculation amount of 5% of volume concentration, and culturing for 4-6 h at 30-37 ℃; adding IPTG with the final concentration of 0.3mM, continuing to ferment for 12-18 h at the temperature of 22-25 ℃, taking the fermentation liquor for centrifugation, and collecting wet bacterial cells; the fermentation medium comprises the following components in final mass concentration: yeast powder 12g/L, peptone 15g/L, glycerin 10g/L, na ₂ HPO ₄ ·12H ₂ O 8.9g/L、KH ₂ PO ₄ 3.4g/L、NH ₄ Cl 2.67g/L、Na ₂ SO ₄ 0.71g/L、MgSO ₄ ·7H ₂ O0.3 g/L, deionized water as solvent, and pH6.8-7.0.

In the reaction system, the final concentration of the L-menthol is 10-15 g/L, and the final concentration of the alpha-lactose is 300-400 g/L.

Specifically, the reaction pH is 6.0-8.0, and the reaction time is 8-24 h.

Compared with the prior reported technology, the method comprises a plant extraction method, a chemical synthesis method, a microorganism direct fermentation method and a beta-glycosylase biological catalysis method, and the beneficial effects of the method are mainly reflected in that: (1) the beta-MenG product is synthesized by adopting a one-step biological enzyme method, and has the characteristics of high regio-specific and stereospecific glycosylation; (2) firstly, the beta-MenG is synthesized by glycosidase catalysis; (3) the enzyme has high catalytic activity, and the yield of the product beta-MenG is 0.52g/L.

Description of the drawings

FIG. 1 is a molecular structural formula of l-menthol-O-beta-galactoside;

FIG. 2 is a schematic structural diagram of the pET28a-sth1275 vector;

FIG. 3 is a TLC analysis of a silica gel column purified menthol glycoside sample;

FIG. 4 is a HPLC analysis of a silica gel column purified menthol glycoside sample;

FIG. 5 is a mass spectrometry profile of a purified l-menthol glycoside sample;

FIG. 6 shows a sample of purified l-menthol glycoside ¹ H NMR analysis spectrum;

FIG. 7 shows a sample of purified l-menthol glycoside ¹³ C NMR analysis spectrum;

FIG. 8 shows a sample of purified l-menthol glycoside ¹ H, ¹³ C-COSY NMR spectrum;

FIG. 9 is a HSQC NMR analysis spectrum of a purified l-menthol glycoside sample;

FIG. 10 is a HMBC NMR spectrum of a purified l-menthol glycoside sample.

(V) detailed description of the preferred embodiment

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

in the examples of the present invention, unless otherwise specified, all methods used are conventional ones, and all reagents used are commercially available.

LB medium: 5.0g/L yeast powder, 10.0g/L, naCl 10.0.0 g/L peptone, deionized water as solvent, and 6.5-7.0 pH value.

Seed culture medium: 5g/L yeast powder, 10g/L, naHPO peptone ₄ ·12H ₂ O 8.9g/L、KH ₂ PO ₄ 3.4g/L、NH ₄ Cl 2.67g/L、Na ₂ SO ₄ 0.71g/L、MgSO ₄ ·7H ₂ O0.49 g/L, deionized water as solvent, and pH6.8-7.0.

The final concentration of the fermentation medium is as follows: yeast powder 12g/L, peptone 15g/L, glycerin 10g/L, na ₂ HPO ₄ ·12H ₂ O 8.9g/L、KH ₂ PO ₄ 3.4g/L、NH ₄ Cl 2.67g/L、Na ₂ SO ₄ 0.71g/L、MgSO ₄ ·7H ₂ O0.3 g/L, deionized water as solvent, and pH6.8-7.0.

Example 1: construction of recombinant escherichia coli containing l-menthol-O-beta-galactosyl transferase

1. Construction of Escherichia coli 28a-st1275 containing l-menthol-O-beta-galactosyl transferase

Extracting the genome DNA of common Streptococcus thermophilus (such as Streptococcus thermophilus separated from common fermented yoghourt) by using a bacterial genome DNA extraction kit, and performing PCR amplification by using the extracted genome DNA as a template and using the following primers:

ASCC1275-lacZ-F：

GCCTGGTGCCGCGCGGCAGCCATATGAACATGACTGAAAAAATTC

ASCC1275-lacZ-R：

CTTGTCGACGGAGCTCGAATTCGGATCCCTAATTTAGTGGTTCAATC

the PCR amplification is carried out by adopting high-efficiency Fidelity enzyme Phanta Max Super-Fidelity DNA Polymerase of Nanjing Nuozhen biological technology limited, and the PCR amplification program is as follows: 3min at 95 ℃; 15s at 95 ℃,15 s at 58 ℃ and 1.5min at 72 ℃ for 30 cycles; 5min at 72 ℃.

The obtained PCR product was purified using a PCR product recovery Kit, and cloned between Nde I and BamH I of pET28a + plasmid using One-Step Cloning Kit One of Biotech, inc. of Nanjing Novowed. The pET28a + plasmid was digested with Nde I and BamH I from TaKaRa, and left to stand at 37 ℃ for 4 hours, after which the digested linearized pET28a + plasmid was purified using a DNA gel recovery kit. The purified PCR product and the restriction enzyme purified linear pET28a + plasmid were ligated, transformed into E.coli BL21 (DE 3) high-performance competent cells, and screened on LB plate containing 50mg/L kanamycin at the final concentration. The colony PCR verification positive clone is inoculated to a fresh LB liquid culture medium, and the plasmid is extracted for sequencing analysis, so that the successful connection of the l-menthol-O-beta-galactosylating enzyme gene is verified, the nucleotide sequence of the gene is shown as SEQ ID NO.1, and the amino acid sequence of the gene is shown as SEQ ID NO. 2. The recombinant expression vector contained in this positive clone was designated pET28a-sth1275 (FIG. 2).

Coli BL21 (DE 3) (pET 28a-sth 1275) was designated as E.coli (Escherichia coli) 28a-sth1275, a positive clone E.coli containing recombinant plasmid pET28 a-sth1275.

2. Preparation of fermentation broth microbial inoculum for producing beta-MenG

Recombinant E.coli 28a-sth1275 was cultured to the middle of logarithmic growth at 37 ℃ and 220rpm in a seed medium containing 50. Mu.g/ml kanamycin to obtain a seed solution.

Inoculating the freshly cultured seed liquid into a fermentation medium containing 50mg/L kanamycin in an inoculation amount of 5% by volume, and culturing at 37 ℃ for 4 h; adding alpha-lactose with the final concentration of 20g/L, controlling the fermentation temperature to be 25 ℃, and continuing to ferment for 12h to obtain fermentation liquor containing 30g/L of wet thalli to be used as a catalytic microbial inoculum for producing beta-MenG.

Diluting fresh fermentation liquor by 3 times with production water to make the wet thallus content be 10g/L, and breaking cells by adopting a high-pressure cell homogenizer to obtain a crude enzyme liquid which is required to be used for catalytic reaction as soon as possible and is prevented from being stored for a long time.

Example 2: application of microbial inoculum in synthesis of beta-MenG

1. Activity detection of catalyst

1.0g of the recombinant E.coli 28a-sth1275 wet cells prepared in example 1 was resuspended in 20mL of 2.5mM phosphate buffer pH 7.0; l-menthol with a final concentration of 20g/L and lactose with a final concentration of 300g/L are added, shaking table catalysis is carried out in a water bath at 220rpm and 45 ℃ for 8h, the reaction solution is used for HPLC analysis, and the concentration of the formed product beta-MenG is 0.52g/L.

High performance liquid chromatography detection conditions. Sample pretreatment: boiling 1mL of reaction solution; centrifuging at 10000 Xg for 5min, filtering with 0.22 μm filter membrane, and adding the filtrate into liquid sample bottle; taking methanol: water = 7: 3 as a mobile phase, a detector is a refractive index detector RID-20A, a chromatographic column is a silica gel column ZORBAX SIL 4.6 × 250mmol/L, the concentration is 5 μm, the column temperature is 40 ℃, and the flow rate is 1 mL/min.

2. Purification and identification of l-menthol-O-beta-galactose purification

50g of wet E.coli 28a-sth1275 cells prepared in example 1 were resuspended in 500mL of 2.5mM phosphate buffer pH7.0, and disrupted with a high-pressure cell homogenizer to obtain a crude enzyme solution. Then 10g l-menthol and 150g lactose are added, the reaction temperature is 45 ℃, and the reaction time is 4h. The progress of the reaction was monitored by thin layer chromatography TLC. After the reaction is finished, the reaction liquid is placed in boiling water bath for 5min, and then is centrifuged at 8000 Xg for 10min, and then supernatant is collected. Adding ethyl acetate with volume of 3 times, extracting for 2 times, combining the two extraction solutions, evaporating and concentrating, and separating and purifying the concentrated sample by silica gel column chromatography to obtain 0.2g of white powder.

Dissolving the separated menthol glycoside sample in ethyl acetate, developing by TLC, and using l-menthol-alpha-glucoside standard as a control, wherein the sample 1# in the figure is the standard of l-menthol-alpha-glucoside and l-menthol; sample No.2 was spotted as a purified menthol glycoside sample. TLC analysis showed R of purified menthol glycoside product _f The value is the same as l-menthol-alpha-glucoside, and the band is single, indicating that the product is an l-menthol glucoside product.

The purity of the separated and purified l-menthol glycoside sample was analyzed by HPLC, and the results are shown in FIG. 4. The results indicate that the HPLC purity of the isolated and purified sample was >95% for further mass spectrometry and nuclear magnetic detection for structural identification.

The structure of purified l-menthol-glycoside product was analyzed by MS and NMR, and the mass spectrum result is shown in FIG. 5, in which the molecular ion peak of the product is [ M + Na ] ⁺ ]m/z 341.37, indicating that the molecular weight of the glycoside product is 318, it can be concluded that a galactose is attached to the l-menthol molecule.

Dissolving l-menthol glycoside sample in deuterated DMSO, transferring the solution into a nuclear magnetic tube for nuclear magnetic resonance spectroscopy, and combining ¹ H、 ¹³ C. COSY, HMBC and HSQC spectra, the analysis results are shown in figures 6-10, and the nuclear magnetic analysis result shows that the compound is l-menthol-O-beta-galactoside, wherein the chemical shift attribution is as follows: ¹ HNMR(600MHz,DMSO-d6)δ4.70(d,J＝4.7Hz,1H),4.64(d,J＝5.5Hz,1H),4.44(dd,J＝5.8,5.1Hz,1H),4.31–4.28(m,1H),4.13(d,J＝7.6Hz,1H),3.65(td,J＝3.8,3.4,1.1Hz,1H),3.55(ddd,J＝10.5,7.1,5.8Hz,1H),3.42–3.32(m,9H),3.32–3.24(m,2H),3.24–3.16(m,1H),2.25(pd,J＝7.0,2.5Hz,1H),2.06–1.98(m,1H),1.60(ddq,J＝28.3,12.8,3.1Hz,2H),1.21–1.09(m,1H),0.95(td,J＝12.9,3.3Hz,1H),0.92–0.80(m,7H),0.80–0.74(m,1H),0.72(d,J＝6.8Hz,3H)； ¹³ C NMR(151MHz,DMSO-d6)δ101.36,76.69,75.26,74.04,70.92,68.34,60.54,47.92,40.97,34.56,31.39,24.93,23.10,22.76,21.42,16.01。

thin Layer Chromatography (TLC) monitoring method. And (3) carrying out TLC thin plate spotting on 10 mu L, preparing a color developing agent by using n-butyl alcohol, isopropanol and water (volume ratio) = 10: 5: 4 as a developing agent and anisaldehyde, ethanol and concentrated sulfuric acid (volume ratio) = 1: 19: 1, soaking a sample plate with the color developing agent after development, and baking at 160 ℃ until color development is achieved.

Analytical method of high performance liquid chromatography HPLC. Taking 1mL of sample solution, filtering with a 0.22 mu m filter membrane, and adding the filtrate into a liquid phase sample bottle; taking methanol: water = 7: 3 as a mobile phase, a detector is a refractive index detector RID-20A, a chromatographic column is a silica gel column ZORBAX SIL 4.6 × 250mmol/L, the concentration is 5 μm, the column temperature is 40 ℃, and the flow rate is 1 mL/min.

Claims (9)

1.l-menthol-O-beta-galactosylating enzyme, the amino acid sequence of which is shown in SEQ ID NO. 2.

2. A gene encoding the l-menthol-O- β -galactosylating enzyme of claim 1.

3. The encoding gene of claim 2, wherein the nucleotide sequence of the encoding gene is represented by SEQ ID No. 1.

4. A recombinant vector comprising the coding gene of claim 2.

5. A genetically engineered bacterium containing the coding gene of claim 2.

6. The use of the genetically engineered bacterium of claim 5 in the microbial catalytic production of l-menthol- β -galactoside.

7. The use according to claim 6, characterized in that the use is: taking fermentation liquor obtained by fermentation culture of the genetically engineered bacteria or wet thalli obtained by fermentation centrifugation as a catalyst, taking l-menthol as a substrate and alpha-lactose as an auxiliary substrate, reacting at the temperature of 30-45 ℃ to obtain reaction liquid containing l-menthol-beta-galactoside, and separating and purifying the reaction liquid to obtain the l-menthol-beta-galactoside.

8. The use according to claim 7, wherein the final concentration of L-menthol is 10-15 g/L and the final concentration of α -lactose is 300-400 g/L in the reaction system.

9. The use according to claim 7, wherein the reaction pH is from 6.0 to 8.0 and the reaction time is from 8 to 24 hours.

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