Glucosyltransferase mutants, genes encoding the same, recombinant vectors, recombinant strains and enzyme preparations, and use thereof

Abstract

The application relates to the field of biological medicine, and discloses a glucosyltransferase mutant, a coding gene of the glucosyltransferase mutant, a recombination carrier, a recombination strain, an enzyme preparation, and application of the glucosyltransferase mutant. The glucosyltransferase mutant is a point mutation at the R position of the amino acid sequence shown in SEQ ID NO:1, and 4-8 histidines are added at the N end. A method for preparing gastrodin comprises the following steps: contacting the glucosyltransferase mutant with p-hydroxybenzyl alcohol and uridine diphosphate glucose. The glucosyltransferase mutant provided by the application has higher specificity for the substrate p-hydroxybenzyl alcohol, thereby obviously improving the conversion rate of the conversion of p-hydroxybenzyl alcohol into gastrodin, and having a good industrial application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a glycosyltransferase mutant and its encoding gene, a recombinant vector, a recombinant strain, an enzyme preparation, a method for preparing gastrodin, and the application and method of preparing gastrodin. BACKGROUND

[0002] Glycosyltransferase can specifically transfer sugar groups from active intermediates (such as UDPG) to acceptor molecules, has good catalytic efficiency and regioselectivity, and is an ideal catalyst for the synthesis and preparation of glycoside compounds. Glycosyltransferase widely exists in nature, especially in plants, and plays an important role in the regulation of plant secondary metabolites. Many important natural product ingredients in plants exist in the form of glycosides. The catalytic mechanism, structure-activity relationship and selectivity mechanism of glycosyltransferase as an important catalyst for glycosylation of natural products have been studied to some extent. The good catalytic properties of glycosyltransferase have been successfully applied to the production and synthesis of some glycoside natural products, and it is an enzyme with great industrial application value.

[0003] Gastrodin, also known as 4-hydroxymethyl phenyl-β-D-glucopyranoside, is the main bioactive component of traditional Chinese medicine Gastrodia elata Blume, and its content is the preferred indicator for evaluating the quality of Gastrodia elata Blume. Today, gastrodin has been widely used in the fields of medical drugs and health foods. Gastrodin can be used as a single treatment drug or combined with other drugs, mainly for dilating blood vessels, enhancing blood vessel elasticity, improving cerebrovascular diseases and mental diseases caused by insufficient blood supply, and early senile dementia. Related preparations include gastrodin injection, gastrodin tablets, acetyl gastrodin tablets, etc. In addition, due to its effects of improving sleep, lowering blood pressure, and enhancing immunity, it is also used as a raw material for some health products. According to statistics, there are 117 kinds of health foods with gastrodin as the main active ingredient, and such health products are deeply welcomed by the public.

[0004] At present, the demand for Gastrodin as the main pharmaceutical ingredient of Gastrodia is growing, and with the further expansion of the application range, the market prospect is broad. However, as far as the industrialized production of Gastrodin is concerned, it mainly relies on plant extraction method and chemical synthesis method, which cannot meet the long-term, environmental protection and sustainable development of modern industrial production. Therefore, it is urgent to develop a new mode of green and sustainable production of Gastrodin, among which the microbial synthesis method is the most important way to realize the green, safe and sustainable industrial production of Gastrodin. Gastrodin is composed of 4-hydroxybenzyl alcohol and glucose in structure, and the last step of Gastrodin synthesis is glycosylation of 4-hydroxybenzyl alcohol, and glucosyltransferase plays an important role. However, the specificity of the existing glucosyltransferase needs to be improved, and the conversion rate of 4-hydroxybenzyl alcohol to Gastrodin is low. SUMMARY

[0005] The purpose of the present application is to overcome the problems of the prior art that the specificity of the glucosyltransferase needs to be improved and the conversion rate of 4-hydroxybenzyl alcohol to Gastrodin is low, and to provide a glucosyltransferase mutant and a coding gene thereof, a recombinant vector, a recombinant strain, an enzyme preparation, and their applications in preparing Gastrodin and a method for preparing Gastrodin. The glucosyltransferase mutant has higher specificity and can effectively improve the conversion rate of 4-hydroxybenzyl alcohol to Gastrodin.

[0006] In order to achieve the above-mentioned purpose, the first aspect of the present application provides a glucosyltransferase mutant, which is a point mutation at R position of the amino acid sequence shown in SEQ ID NO: 1, and 4-8 histidines are added at the N terminal thereof.

[0007] Preferably, the R position is the 15th position, and 6 histidines are added at the N terminal of the amino acid sequence shown in SEQ ID NO: 1.

[0008] Preferably, the glucosyltransferase mutant has an enzyme with the amino acid sequence shown in SEQ ID NO: 2.

[0009] The second aspect of the present application provides a gene encoding a glucosyltransferase mutant, which has a nucleotide sequence encoding the above-mentioned glucosyltransferase mutant.

[0010] Preferably, the gene has a nucleotide sequence encoding an enzyme with the amino acid sequence shown in SEQ ID NO: 1.

[0011] More preferably, the gene has the nucleotide sequence shown in SEQ ID NO: 2.

[0012] The third aspect of the present application provides a recombinant vector containing the above-mentioned gene.

[0013] Preferably, the expression vector of the recombinant vector is pET28a plasmid.

[0014] The fourth aspect of the present application provides a recombinant strain, which contains the gene or the recombinant vector described above.

[0015] Preferably, the recombinant strain is Escherichia coli and / or Bacillus subtilis.

[0016] More preferably, the recombinant strain is Escherichia coli.

[0017] The fifth aspect of the present application provides a method for preparing a glucosyltransferase mutant, which comprises inoculating the recombinant strain described above into a fermentation medium for fermentation.

[0018] The sixth aspect of the present application provides an enzyme preparation comprising the glucosyltransferase mutant prepared by the method described above.

[0019] The seventh aspect of the present application provides the use of at least one of the glucosyltransferase mutant, the gene, the recombinant vector, the recombinant strain, the glucosyltransferase mutant prepared by the method, and the enzyme preparation described above in the preparation of gastrodin.

[0020] The eighth aspect of the present application provides a method for preparing gastrodin, which comprises the following steps: contacting at least one of the glucosyltransferase mutant, the gene, the recombinant vector, the recombinant strain, the glucosyltransferase mutant prepared by the method, and the enzyme preparation described above with p-hydroxybenzyl alcohol and uridine diphosphate glucose.

[0021] Preferably, the weight ratio of the p-hydroxybenzyl alcohol to the uridine diphosphate glucose is 1:1.5-2.5; and the contacting condition comprises a temperature of 25-35℃ and a time of 4-8h.

[0022] Through the technical solutions described above, the present application has the following beneficial effects:

[0023] The glucosyltransferase mutant provided by the present application has higher specificity for the substrate p-hydroxybenzyl alcohol compared to the original glucosyltransferase, which significantly improves the conversion rate of p-hydroxybenzyl alcohol into gastrodin, and the yield of gastrodin can be increased by about 31%; the glucosyltransferase mutant is of great significance for the green, safe, and sustainable industrial production of gastrodin, and has good industrial application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 It is the high performance liquid chromatogram of the p-hydroxybenzyl alcohol standard sample in Example 3.

[0025] Figure 2 High performance liquid chromatogram of the Gastrodin standard sample in Example 3;

[0026] Figure 3 High performance liquid chromatogram of the reaction solution after the E. coli BL21(DE3) / pET28a-slyUGTmut catalyzed synthesis of Gastrodin in Example 3;

[0027] Figure 4 High performance liquid chromatogram of the reaction solution after the E. coli BL21(DE3) / pET28a-slyUGT catalyzed synthesis of Gastrodin in Comparative Example 1. DETAILED DESCRIPTION

[0028] The endpoints of the ranges and any values disclosed herein are not limited to the precise values stated. The endpoints of the ranges and the values are approximate values and should be understood to include values approximately near these ranges and values within these ranges. For ranges with endpoints, the endpoints are included in the ranges. For ranges without endpoints, the range extends to the endpoints of the nearest lower and upper ranges. The endpoints of the ranges and the values are not limited to the precise values stated.

[0029] The first aspect of the present application provides a glucose transferase mutant, which is a point mutation at position R of the amino acid sequence shown in SEQ ID NO: 1 and addition of 4-8 histidines at the N-terminal thereof.

[0030] The inventors of the present application found, in the process of research, that the glucose transferase based on the amino acid sequence shown in SEQ ID NO: 1 as a UGT original enzyme, after substitution of amino acid residues at a certain position R of the amino acid sequence of the original enzyme and addition of 4-8 histidines at the N-terminal, the obtained glucose transferase mutant (UGT mutant) can effectively improve the specificity for the substrate p-hydroxybenzyl alcohol, has high catalytic efficiency, and further, when applied to the production of Gastrodin, the conversion rate of p-hydroxybenzyl alcohol to Gastrodin is significantly improved, which has important significance for green, safe and sustainable industrial production of Gastrodin, and has good industrial application prospect.

[0031] In the present application, the amino acid sequence shown in SEQ ID NO: 1 can be referred to as UGT original enzyme or slyUGT, and the amino acid sequence of the UGT original enzyme is:

[0032] MAQIPHIAILPSPGMGHLIPLVEFAKRIFLHHHFSVSLILPTDGPISNAQKIFLNSLPSSMD

[0033] YHLLPPVNFDDLPEDVKIETRISLTVSRSLTSLRQVLESIIESKKTVALVVDLFGTDAFD

[0034] VAIDLKISPYIFFPSTAMGLSLFLHLPNLDETVSCEYRDLPDPIQIPGCTPIHGKDLLDPV

[0035] QDRNDESYKWLLHHAKRYGMAEGIIVNSFKELEGGAIGALQKDEPGKPTVYPVGPLIQ

[0036] MDSGSKVDGSECMTWLDEQPRGSVLYISYGSGGTLSHEQLIEVAAGLEMSEQRFLWV

[0037] VRCPNDKIANATFFNVQDSTNPLEFLPKGFLERTKGFGLVLPNWAPQARILSHESTGGF

[0038] LTHCGWNSTLESVVHGVPLIAWPLYAEQKMNAVMLSEDIKVALRPKVNEENGIVGRL

[0039] EIAKVVKGLMEGEEGKGVRSRMRDLKDAAAKVLSEDGSSTKALAELATKLRKKCQMIDVANH (SEQ ID NO: 1).

[0040] According to the application, preferably, the R position is the 15th position, and 6 histidines are added at the N terminal of the amino acid sequence shown in SEQ ID NO: 1. The inventors have found that, in this preferred embodiment, the catalytic specificity of the glucose transferase mutant is further improved, and the conversion rate of hydroxybenzyl alcohol into gastrodin is improved.

[0041] According to the application, preferably, the glucose transferase mutant has the enzyme of the amino acid sequence shown in SEQ ID NO: 2, i.e., the methionine at the 15th position of the amino acid sequence shown in SEQ ID NO: 1 is replaced by leucine.

[0042] In the application, the amino acid sequence shown in SEQ ID NO: 2 can be referred to as slyUGT mutant or slyUGTmut:

[0043] Amino acid sequence of slyUGT mutant:

[0044] MHHHHHHAQIPHIAILPSPGLGHLIPLVEFAKRIFLHHHFSVSLILPTDGPISNAQKIFLNS

[0045] LPSSMDYHLLPPVNFDDLPEDVKIETRISLTVSRSLTSLRQVLESIIESKKTVALVVDLFG

[0046] TDAFDVAIDLKISPYIFFPSTAMGLSLFLHLPNLDETVSCEYRDLPDPIQIPGCTPIHGKD

[0047] LLDPVQDRNDESYKWLLHHAKRYGMAEGIIVNSFKELEGGAIGALQKDEPGKPTVYP

[0048] VGPLIQMDSGSKVDGSECMTWLDEQPRGSVLYISYGSGGTLSHEQLIEVAAGLEMSEQ

[0049] RFLWVVRCPNDKIANATFFNVQDSTNPLEFLPKGFLERTKGFGLVLPNWAPQARILSH

[0050] ESTGGFLTHCGWNSTLESVVHGVPLIAWPLYAEQKMNAVMLSEDIKVALRPKVNEEN

[0051] GIVGRLEIAKVVKGLMEGEEGKGVRSRMRDLKDAAAKVLSEDGSSTKALAELATKLRKKCQMIDVANH(SEQ ID NO:2)。

[0052] The above-mentioned glucosyltransferase mutant can be obtained by artificial synthesis, or can be obtained by first synthesizing the coding gene thereof and then expressing it biologically. Illustratively, the glucosyltransferase mutant provided in the present application is obtained by mutating the coding gene of the amino acid sequence shown in SEQ ID NO: 1 using a site-directed saturation mutation technique, transforming a host Escherichia coli after connecting an expression vector, inducing expression, and then detecting positive mutations with improved catalytic activity by high-performance liquid detection method.

[0053] The second aspect of the present application provides a gene encoding a glucosyltransferase mutant, which has a nucleotide sequence encoding the above-mentioned glucosyltransferase mutant.

[0054] According to the present application, preferably, the gene has a nucleotide sequence encoding an enzyme having the amino acid sequence shown in SEQ ID NO: 2.

[0055] It is well known in the art that of the 20 different amino acids that make up proteins, 18 are encoded by 2-6 codons each, except Met (ATG) or Trp (TGG) which are encoded by single codons, respectively (Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, 2nd edition, 1989, see page 950, Appendix D). That is, due to the degeneracy of the genetic code, the codon that determines an amino acid is not unique, and substitution of the third nucleotide in the triplet codon often does not change the amino acid, so the nucleotide sequence of a gene encoding the same protein can be different. More preferably, the gene has the nucleotide sequence shown in SEQ ID NO: 3.

[0056] Nucleotide sequence of slyUGT mutant:

[0057] ATGCATCATCACCACCACCACGCTCAAATCCCTCACATTGCTATCCTGCCTTCTCCC

[0058] GGTCTCGGACACCTGATCCCTCTGGTGGAGTTTGCAAAACGAATCTTCCTGCACCA

[0059] TCATTTCTCGGTCAGCCTTATTCTTCCTACGGATGGTCCTATCTCTAACGCACAGAA

[0060] GATTTTCCTTAATTCTCTGCCCTCCTCCATGGACTACCACCTTCTTCCTCCTGTTAAT

[0061] TTTGACGACCTCCCCGAGGATGTTAAAATTGAGACGCGGATCTCGCTTACAGTGTC

[0062] GAGAAGCCTCACAAGCCTTAGACAAGTGCTCGAATCGATTATCGAGTCTAAAAAA

[0063] ACGGTTGCTCTCGTGGTgGACCTTTTCGGAACGGATGCCTTCGACGTCGCTATTGAC

[0064] CTTAAGATTTCGCCCTACATCTTCTTCCCCTCGACAGCAATGGGCCTTTCGCTGTTT

[0065] CTCCACCTGCCTAATCTCGATGAAACTGTGTCTTGTGAATACCGTGACCTCCCCGA

[0066] CCCTATCCAAATCCCCGGTTGCACTCCCATTCACGGTAAGGATCTCCTGGACCCCG

[0067] TCCAAGACCGGAATGACGAATCGTATAAGTGGCTTCTCCATCATGCTAAGCGGTAT

[0068] GGCATGGCTGAAGGTATTATTGTCAATTCCTTTAAGGAACTTGAAGGTGGCGCCAT

[0069] CGGTGCACTCCAAAAAGACGAACCCGGCAAACCTACCGTGTATCCTGTCGGTCCTC

[0070] TCATTCAAATGGACTCGGGTAGCAAGGTGGACGGATCCGAATGCATGACCTGGCT

[0071] CGACGAACAGCCCCGTGGTTCGGTCCTTTACATCTCTTATGGTTCGGGTGGTACAC

[0072] TTAGCCACGAGCAACTGATTGAAGTGGCTGCCGGACTCGAAATGAGCGAGCAACG

[0073] ATTCCTTTGGGTGGTGCGGTGTCCCAATGATAAGATCGCCAATGCCACCTTCTTCA

[0074] ATGTCCAGGATAGCACCAACCCTCTGGAGTTCCTTCCTAAAGGATTCCTCGAACGT

[0075] ACTAAAGGATTCGGCCTTGTGCTGCCCAACTGGGCCCCTCAGGCTCGTATTCTGTC

[0076] TCATGAATCTACTGGTGGTTTTCTGACACACTGTGGATGGAACTCCACGCTTGAAA

[0077] GCGTTGTCCATGGAGTCCCTCTTATTGCCTGGCCCCTGTATGCCGAGCAAAAGATG

[0078] AACGCTGTGATGCTTTCGGAAGACATCAAGGTTGCACTCCGTCCCAAAGTCAACG

[0079] AGGAAAACGGTATTGTGGGACGGCTGGAGATTGCTAAGGTGGTCAAGGGTCTGAT

[0080] GGAAGGTGAAGAGGGTAAGGGAGTCAGAAGCCGGATGCGgGACCTTAAAGACGC

[0081] TGCTGCCAAAGTGCTGTCTGAGGATGGCAGCTCCACGAAAGCCCTTGCTGAGCTTGCTACAAAACTGCGGAAGAAGTGTCAAATGATTGACGTCGCCAATCATTAA (SEQ ID NO: 3).

[0082] The nucleotide sequence of the present application can be obtained by polymerase chain reaction (PCR) amplification, recombination or artificial synthesis. Once the nucleotide sequence is obtained, the amino acid sequence can be obtained in large quantities by recombination. The obtained nucleotide sequence is usually cloned into a vector and then introduced into a genetically engineered bacterium, and then the nucleotide sequence is isolated from the proliferated host cell by a conventional method.

[0083] In addition, the nucleotide sequence can be synthesized by a publicly known artificial chemical synthesis method.

[0084] The third aspect of the present application provides a recombinant vector containing the above-described gene.

[0085] In the present application, the "vector" used in the recombinant vector can be selected from various vectors known in the art, such as various plasmids, cosmids, bacteriophages and retroviruses available on the market, and the preferred expression vector of the present application is pET28a plasmid. The recombinant vector can be constructed by using various endonucleases capable of having cleavage sites in the multiple cloning sites of the vector (such as Nco I and Xho I for pET28a) to obtain linear plasmids, and then connecting the gene fragments cleaved by the same endonucleases to obtain the recombinant plasmid. In the present application, the pET28a is preferably cleaved by Nco I and Xho I, and then connected with the gene fragments to construct the recombinant vector pET28a-slyUGT. The recombinant vector pET28a-slyUGT is used as a template to obtain the slyUGT mutant plasmid pET28a-slyUGTmut by using the slyUGTmut-F / slyUGTmut-R loop P.

[0086] The fourth aspect of the present application provides a recombinant strain containing the above-mentioned gene or the above-mentioned recombinant vector.

[0087] In the present application, the recombinant vector can be transformed, transduced or transfected into the host cell (strain) by the conventional methods in the art, such as chemical transformation by calcium chloride method, high-voltage electric shock transformation. The host cell can be a prokaryotic cell or a eukaryotic cell, preferably Escherichia coli and / or Bacillus subtilis, and more preferably the host cell is Escherichia coli, such as Escherichia coli BL21 (DE3).

[0088] The fifth aspect of the present application provides a preparation method of the glucose transferase mutant, which comprises inoculating the above-mentioned recombinant strain into a fermentation medium for fermentation.

[0089] In the present application, the fermentation conditions of the recombinant strain are not particularly limited, as long as the recombinant strain can be proliferated in large quantities through the fermentation process. Preferably, the fermentation process comprises: first, seed culture of the recombinant strain to obtain a seed liquid, and then inoculating the seed liquid into a fermentation medium containing kanamycin to culture the bacterial concentration OD 600 to 0.6-0.8, and then adding isopropyl-β-D-thiogalactoside (IPTG) inducer with a final concentration of 0.1-0.2 mM to culture at a temperature of 20-35 °C for 10-15 h to obtain a fermentation broth.

[0090] According to the present application, preferably, the content of kanamycin in the fermentation medium is 40-60 mg / L.

[0091] Further preferably, the method for preparing the seed liquid comprises picking a single colony of the recombinant strain and inoculating it into a seed culture medium containing kanamycin for seed culture, to obtain the seed liquid. In the present application, the single colony of the recombinant strain can be selected from a freshly prepared recombinant strain or a cryopreserved recombinant strain (e.g., a synthetic lipid recombinant strain cryopreserved in a glycerol cryopreservation tube in a -80°C refrigerator).

[0092] The method for seed culture is not particularly limited in the present application, as long as the method can activate and proliferate the recombinant strain. Preferably, the content of kanamycin in the seed culture medium is 40-60 mg / L; and the temperature, pH, rotation speed, time, etc. used for seed culture can be the conventional settings in the art. Preferably, the conditions for seed culture include a temperature of 30-45°C, a rotation speed of 150-250 rpm, and a time of 8-12 h.

[0093] The seed culture medium and the fermentation medium are not particularly limited in the present application, and can be the conventional culture media used in the art. Preferably, the seed culture medium and the fermentation medium are LB liquid culture medium (tryptone 8-12 g / L, yeast extract 4-6 g / L, NaCl 8-12 g / L).

[0094] The sixth aspect of the present application provides an enzyme preparation comprising the glucosyltransferase mutant prepared by the above method.

[0095] In the present application, the glucosyltransferase mutant can be prepared into a corresponding enzyme preparation, which can exist in a solid, semi-solid or liquid form. The enzyme preparation can contain excipients or additives used for preparing the enzyme preparation, which can be selected by those skilled in the art as needed, and will not be described here.

[0096] In the present application, the fermentation broth obtained by the above preparation method can be directly used as an enzyme preparation, or the fermentation broth can be separated and purified to obtain a glucosyltransferase mutant as an enzyme preparation. The separation and purification can be performed by conventional enzyme separation methods in the art. That is, the glucosyltransferase mutant can be used in the form of whole cells of the recombinant strain, or in the form of a crude enzyme or a purified enzyme separated from the cells of the recombinant strain. If necessary, the glucosyltransferase mutant of the present application can also be prepared into an immobilized enzyme or an immobilized cell using known immobilization techniques in the art.

[0097] The glucose transferase mutant provided by the present application has high specificity for the substrate p-hydroxybenzyl alcohol, which significantly improves the conversion rate of p-hydroxybenzyl alcohol into gastrodin. The seventh aspect of the present application provides the use of at least one of the above-mentioned glucose transferase mutant, the above-mentioned gene, the above-mentioned recombinant vector, the above-mentioned recombinant strain, the glucose transferase mutant prepared by the above-mentioned method and the above-mentioned enzyme preparation in the preparation of gastrodin.

[0098] The eighth aspect of the present application provides a method for preparing gastrodin, which comprises the following steps: contacting at least one of the above-mentioned glucose transferase mutant, the above-mentioned gene, the above-mentioned recombinant vector, the above-mentioned recombinant strain, the glucose transferase mutant prepared by the above-mentioned method and the above-mentioned enzyme preparation with p-hydroxybenzyl alcohol and uridine diphosphate glucose (UDPG). The glucose transferase mutant is used to catalyze the glycosylation of the substrate p-hydroxybenzyl alcohol to generate gastrodin, which has good catalytic synthesis effect and high conversion rate.

[0099] According to the present application, preferably, the weight ratio of the p-hydroxybenzyl alcohol to the uridine diphosphate glucose is 1:1.5-2.5; the contacting conditions include: the temperature is 25-35℃, and the time is 4-8h. Exemplarily, the p-hydroxybenzyl alcohol, the uridine diphosphate glucose and the fermentation broth of the above-mentioned recombinant strain can be mixed to make the substrate contact with the glucose transferase mutant to perform the reaction of catalytic synthesis of gastrodin; the substrate concentration of the p-hydroxybenzyl alcohol can be 150-250mg / L, and the substrate concentration of the uridine diphosphate glucose can be 350-450mg / L relative to the fermentation broth.

[0100] The present application will be described in detail by the following examples.

[0101] In the following examples, the E. coli BL21(DE3) was purchased from Baobioengineering (Dalian) Co., Ltd., and the number was 9126; the pET28a was purchased from Shengong Bioengineering (Shanghai) Co., Ltd., and the number was B540183; the p-hydroxybenzyl alcohol standard was purchased from Shanghai McLean Biochemical Technology Co., Ltd., and the product number was H811051-25g; the gastrodin standard was purchased from Shanghai McLean Biochemical Technology Co., Ltd., and the product number was G828303-5g; the T4 DNA ligase, the restriction endonuclease Nco I and the restriction endonuclease Xho I were all purchased from Fermentens Co.; the rest of the reagents and raw materials were all conventional commercial products.

[0102] LB liquid medium: tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, pH adjusted to 7.0, sterilized by steam under high pressure for 21min, ready for use;

[0103] LB agar plate medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar 12 g / L, pH adjusted to 7.0, sterilized by steam under high pressure for 21 min, ready for use.

[0104] Preparation of glycosyltransferase mutant of Example 1

[0105] The specific process of point mutation of R site in the amino acid sequence of the slyUGT original enzyme (amino acid sequence as shown in SEQ ID NO: 1, nucleotide sequence as shown in SEQ ID NO: 4) and adding 6 histidines at the amino terminal (N terminal) thereof is as follows:

[0106] The codon-optimized tomato-derived slyUGT gene fragment synthesized by the biological company (nucleotide sequence as shown in SEQ ID NO: 4) was used as a template, the primer slyUGT-F / slyUGT-R (nucleotide sequences as shown in SEQ ID NO: 5 and SEQ ID NO: 6) was used to amplify the target gene fragment with homologous arms, and the pET28a plasmid vector was linearized using NcoI and Xho I restriction endonucleases, and the target gene fragment was inserted into the linearized vector using the rapid cloning technology to obtain the recombinant plasmid pET28a-slyUGT (nucleotide sequence as shown in SEQ ID NO: 7); The target gene fragment was inserted into the linearized vector using the rapid cloning technology to obtain the recombinant plasmid pET28a-slyUGT (nucleotide sequence as shown in SEQ ID NO: 7);

[0107] Construction of slyUGT mutant (slyUGTmut) expression plasmid pET28a-slyUGTmut: the plasmid pET28a-slyUGT was used as a template, and the primer slyUGT mut-F / slyUGT mut-R (nucleotide sequences as shown in SEQ ID NO: 8 and SEQ ID NO: 9) was used to amplify the PCR reaction product by loop p;

[0108] After the PCR reaction product was analyzed by 0.9% agarose gel electrophoresis and the PCR was positive, 20 μL of the PCR solution was taken, 1 μL of Dpn I was added, and the enzyme was cut at 37°C for 3 h to remove the template plasmid, inactivated at 65°C for 10 min, transformed into E. coli BL21(DE3) competent cells, spread on LB agar plates containing kanamycin (final concentration of 50 μg / mL), and cultured at 37°C overnight. Colonies were randomly picked and the plasmid was extracted after culture and sequenced by GenScript Biotech (Shanghai) Co., Ltd. The sequencing results showed that the positive clone E. coli BL21(DE3) / pET28a-slyUGTmut was obtained (the nucleotide sequence of pET28a-slyUGTmut is shown as SEQ ID NO: 10; the amino acid sequence of slyUGTmut is shown as SEQ ID NO: 2, and the nucleotide sequence is shown as SEQ ID NO: 3).

[0109] Example 2 Induced expression of the glycosyltransferase mutant

[0110] The recombinant strain E. coli BL21(DE3) / pET28a-slyUGTmut obtained in Example 1 was inoculated into LB liquid medium containing kanamycin (final concentration of 50 mg / L) and cultured at a temperature of 37°C and a rotation speed of 200 rpm for 10 h to obtain a seed solution. The seed solution was inoculated into fresh LB liquid medium containing kanamycin (final concentration of 50 mg / L) at a volume concentration of 1%, and cultured at a temperature of 37°C and a rotation speed of 200 rpm until the cell concentration OD 600 was 0.6-0.8. Then, isopropyl-β-D-thiogalactoside (IPTG) was added to the culture solution at a final concentration of 0.15 mM, and the culture was continued at a temperature of 28°C and a rotation speed of 200 rpm for 12 h to obtain the E. coli BL21(DE3) / pET28a-slyUGTmut fermentation broth, which can be used for the bio-catalytic preparation of gastrodin.

[0111] Example 3 Catalytic preparation of gastrodin by the glycosyltransferase mutant

[0112] The p-hydroxybenzyl alcohol and the UDPG mixture (providing an activated glucose donor) were added to the fermentation broth obtained in Example 2 so that the final concentration of p-hydroxybenzyl alcohol was 200 mg / L and the final concentration of UDPG was 400 mg / L. The reaction was carried out at a temperature of 30°C for 6 h to obtain a reaction solution. 1 mL of the reaction solution was taken as a sample to detect the yield of gastrodin and p-hydroxybenzyl alcohol.

[0113] According to the characteristics of the molecular structure of gastrodin having an absorption peak at a certain ultraviolet wavelength, the yield of gastrodin is detected by using a high performance liquid chromatograph-ultraviolet detector: the sample needs to be treated before determination, first centrifuged at 12000 rpm for 10 min, then 1 mL of supernatant is taken, and then the supernatant is filtered through a water system filter membrane (pore size is 0.22 μm) to remove impurities; the detection conditions are: Shimadzu LC-40, UV-VIS detector, ZORBAX Eclipse Plus C18 chromatographic column (4.6*100 mm, 3.5 μm, Agilent); the mobile phase is pure water (90%) and pure methanol (10%); the flow rate is 0.7 mL / min; the ultraviolet detector, the detection wavelength is 225 nm; the injection amount is set to 10 μL; the column temperature is set to 35 °C.

[0114] The standard curve is obtained by using the response values corresponding to the 5 concentration gradients of gastrodin and p-hydroxybenzyl alcohol respectively, and the value of the correlation coefficient R 2 should be greater than 0.99, and the results are shown in Figure 1 and Figure 2 The retention time (min) of the standard sample p-hydroxybenzyl alcohol and gastrodin is about 19.698 min and 10.966 min respectively; then the contents of the two substances in the sample are determined according to the standard curve.

[0115] The LC detection results of the reaction liquid in Example 3 as a sample are shown in Figure 3 , gastrodin is eluted at 11.004 min, and the yield of gastrodin is 469.17 mg / L.

[0116] Comparative Example 1

[0117] The UGT original enzyme with the nucleotide sequence as shown in SEQ ID NO: 4 is transformed into E. coli BL21(DE3) competent cells by using the method steps provided in Example 1 and Example 2, to obtain a recombinant strain E. coli BL21(DE3) / pET28a-slyUGT, and the E. coli BL21(DE3) / pET28a-slyUGT fermentation broth is obtained by fermentation culture.

[0118] The p-hydroxybenzyl alcohol and the UDPG mixture (providing an activated glucose group donor) are added to the fermentation broth obtained in Comparative Example 1, so that the final concentration of p-hydroxybenzyl alcohol is 200 mg / L and the final concentration of UDPG is 400 mg / L, and the reaction is carried out at a temperature of 30 °C for 6 h to obtain a reaction liquid; 1 mL of the reaction liquid is taken as a sample to detect the yield of gastrodin and p-hydroxybenzyl alcohol, and the LC detection results are shown in Figure 4 , p-hydroxybenzyl alcohol is eluted at 19.670 min, gastrodin is eluted at 10.980 min and the yield is low, only 358.14 mg / L.

[0119] Compared with Comparative Example 1, the E. coli BL21(DE3) / pET28a-slyUGTmut fermentation broth in Example 3 has better catalytic effect on the synthesis of gastrodin from the substrate p-hydroxybenzyl alcohol, and the yield of gastrodin is increased by 31% compared with the original enzyme.

[0120] k of UGT mutant in Example 4 m Determination

[0121] The E. coli BL21(DE3) / pET28a-slyUGTmut fermentation broth obtained in Example 2 was centrifuged at 4°C and 8000 rpm for 5 min, and the bacterial cells were collected. The bacterial cells were suspended in 7 mL of PBS buffer, and the ultrasonic disrupter was used for crushing for 15 min, and ice was replaced every 5 min during the crushing to keep low temperature. Then, the supernatant of the crushing liquid was collected by centrifugation at 4°C and 8000 rpm for 5 min, and the UGT mutant crude enzyme liquid was obtained.

[0122] The catalytic property of glucosyltransferase is that the color of p-nitrophenol will change from yellow to colorless after glycosylation. Based on this property, the k of glucosyltransferase is detected by the reaction. m Firstly, different concentrations of p-nitrophenol solution were prepared, and the fluorescence intensity at OD 405 was detected, and the standard curve of p-nitrophenol concentration and OD 405 was drawn; then, the reaction liquid with gradient substrate concentration was prepared using 4 mM p-nitrophenol mother liquor (mixed with 8 mM UDPG), 0.2 mM PBS buffer, UGT mutant crude enzyme liquid and water, and the reagents were added as shown in Table 1 for testing.

[0123] Table 1

[0124]

[0125] According to the detection data of the test in Table 1, the absorbance curve of the gradient substrate concentration was drawn, and the intercept of the obtained straight line was 1 / Vmax, and the slope was k m / V max The k m value of the UGT mutant crude enzyme liquid was determined to be 1.68, and the V max was 151.52 μm / L / min.

[0126] Comparative Example 2

[0127] The UGT original enzyme crude enzyme liquid was obtained by using the same method in Example 4 to process the E. coli BL21(DE3) / pET28a-slyUGT fermentation broth, and the k m value and V maxThe final results show that the k m value of the UGT original enzyme is 7.39, and the V max value is 120.48 μm / L / min.

[0128] As can be seen from the data of Example 4 and Comparative Example 2, under the same conditions, the substrate specificity of the UGT mutant is better than that of the UGT original enzyme, the k m value is 22.73% of the UGT original enzyme, and the V max value is increased by 25.76% compared with the UGT original enzyme.

[0129] The preferred embodiments of the present application are described in detail above, but the present application is not limited thereto. Within the technical concept of the present application, various simple modifications can be made to the technical solutions of the present application, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be considered as disclosed by the present application and fall within the protection scope of the present application.

Claims

1. A glucosyltransferase mutant, characterized in that, The glucosyltransferase mutant is formed by point mutation at the R position of the amino acid sequence shown in SEQ ID NO: 1, and by adding 4-8 histidines to its N-terminus; the R position is the 15th position, and 6 histidines are added to the N-terminus of the amino acid sequence shown in SEQ ID NO: 1; the glucosyltransferase mutant is an enzyme with the amino acid sequence shown in SEQ ID NO:

2.

2. A gene encoding a glucosyltransferase mutant, characterized in that, The gene is a nucleotide sequence encoding the glucosyltransferase mutant of claim 1; The gene encodes the nucleotide sequence of an enzyme with the amino acid sequence shown in SEQ ID NO:

2.

3. The gene according to claim 2, characterized in that, The gene is the nucleotide sequence shown in SEQ ID NO:

3.

4. A recombinant vector, characterized in that, The recombinant vector contains the gene as described in claim 2 or 3.

5. The recombinant vector according to claim 4, characterized in that, The expression vector for the recombinant vector is the pET28a plasmid.

6. A recombinant bacterial strain, characterized in that, The recombinant strain contains the gene described in claim 2 or 3 or the recombinant vector described in claim 4 or 5.

7. The recombinant strain according to claim 6, characterized in that, The recombinant strain is either Escherichia coli or Bacillus subtilis.

8. The recombinant strain according to claim 7, characterized in that, The recombinant strain is Escherichia coli.

9. A method for preparing a glucosyltransferase mutant, characterized in that, The preparation method includes: inoculating the recombinant strain according to any one of claims 6 to 8 into a fermentation medium for fermentation.

10. An enzyme preparation, characterized in that, The enzyme preparation comprises the glucosyltransferase mutant obtained by the method of claim 9.

11. The use of at least one of the following in the preparation of gastrodin: the glucosyltransferase mutant of claim 1, the gene of claim 2 or 3, the recombinant vector of claim 4 or 5, the recombinant strain of any one of claims 6 to 8, the glucosyltransferase mutant prepared by the method of claim 9, and the enzyme preparation of claim 10.

12. A method for preparing gastrodin, characterized in that, The method includes the following steps: contacting at least one of the glucosyltransferase mutant of claim 1, the glucosyltransferase mutant prepared by the method of claim 9, and the enzyme preparation of claim 10 with p-hydroxybenzyl alcohol and uridine diphosphate glucose.

13. The method according to claim 12, characterized in that, The weight ratio of p-hydroxybenzyl alcohol to uridine diphosphate glucose is 1:1.5-2.5; the contact conditions include a temperature of 25-35°C and a time of 4-8 hours.

Images