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Glycanase and application thereof in biosynthesis of ligustrazine

A technology of glycanase and ligustrazine, which is applied in the field of glycanase and its application in the biosynthesis of 2,3,5,6-tetramethylpyrazine, can solve the problems of low yield and high cost, and achieve high yield High, reduce preparation cost, low price effect

Active Publication Date: 2021-07-20
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

What this method adopts is pure enzyme reaction, and output is low, and cost is high

Method used

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  • Glycanase and application thereof in biosynthesis of ligustrazine
  • Glycanase and application thereof in biosynthesis of ligustrazine
  • Glycanase and application thereof in biosynthesis of ligustrazine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Embodiment 1, construct the whole cell catalytic system of acetoin synthesis

[0056] 1.fls gene acquisition

[0057] FLS is an artificially designed enzyme whose gene sequence is SEQ ID NO.1, which is obtained through gene synthesis.

[0058] 2. Construction of expression vector

[0059] The fls gene shown in SEQ ID NO.1 is replaced with pET-28a vector (Novagen, Kan + ) cutting the DNA fragment between NdeI and XhoI to obtain a recombinant plasmid named pET-28a-FLS.

[0060] 3. Gene Expression

[0061] In order to detect the activity of FLS enzyme in vitro, the enzyme was expressed and purified in Escherichia coli.

[0062] (1) The E. coli expression recombinant plasmid pET-28a-FLS was transferred into E. coli BL21(DE3) to obtain the recombinant bacteria. Positive clones were screened using kanamycin-resistant plates (Kan + , 0.1mg mL -1 ), cultured overnight at 37°C to obtain recombinant bacteria including the FLS coding sequence;

[0063] (2) Pick a single cl...

Embodiment 2

[0081] Example 2. Codon optimization improves FLS expression

[0082] 1. Build a strain library

[0083] Through primer design, replace the original codon with a degenerate codon of 12 amino acids at the 5' end of FLS, and the primers are as follows:

[0084] Forward primer:

[0085] 5'-CATATGATGgcnATGathacngggngggngarctrgtngtncgnacnctnATTAAAGCTG-3' (SEQ ID NO.6)

[0086] Reverse primer:

[0087] 5'-CCATGCAGGCCAAACAGATGTTCTACGCCAGCTTTAAT-3' (SEQ ID NO.7)

[0088] Using the plasmid pET-28a-FLS as a template, obtain the target band by PCR, then digest the template with DMT enzyme, purify the PCR product, and finally transform the purified product into BL21(DE3), and spread it on a medium containing 0.1mg mL -1 LB plates of kanamycin were cultured at 37°C.

[0089] (2) SDS-PAGE screening: Transfer the colonies on the plate to a medium containing 0.1mg mL -1 Kanamycin in 5mL LB medium, cultured at 37°C to OD 600 When is 0.6, add 0.5mmol L -1 IPTG induces protein expression. ...

Embodiment 3

[0091] Embodiment 3, the application of FLS mutant in the biosynthesis of acetoin

[0092] 1. FLS directed evolution

[0093] The catalytic activity of FLS to acetaldehyde is 92.3M -1 ·s -1 (The reaction conditions are: 50mM acetaldehyde, 30°C, 50mM phosphate, 5mM MgSO 4 , pH7.0. The reaction kinetics of microplate reader test), the result is as follows Figure 6 As shown in A. In order to further improve the catalytic efficiency of FLS, we used the method of directed evolution to mutate the histidine (His) at position 21 of the amino acid sequence (SEQ ID NO.2) of the FLS 77 mutant to glutamine (Gln) , so as to obtain the mutant FLS 77:H21Q (SEQ ID NO.5), its kcat / Km reaches 188M -1 ·s -1 , the catalytic activity to acetaldehyde is twice that of FLS, the results are as follows Figure 6 Shown in B.

[0094] 2. Whole-cell catalytic activity of FLS-77:H21Q

[0095] Cell dry weight is 5g L -1 The mutant FLS-77:H21Q was subjected to whole-cell reaction with 600mM aceta...

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Abstract

The invention belongs to the field of biosynthesis, and particularly relates to glycanase and application thereof in biosynthesis of ligustrazine. According to the glycanase, degenerate codons are adopted to replace part of nucleotide in the glyanase, codon optimization is achieved, and the catalytic activity of the glycanase is improved. The invention also provides a preparation method of acetoin, the acetoin is prepared by using microbial cells expressing the glycanase for whole-cell catalysis, and ligustrazine can be further prepared. The method disclosed by the invention is high in substrate conversion rate and high in yield, reduces the cost and is suitable for industrial production.

Description

technical field [0001] The invention belongs to the field of biosynthesis, and in particular relates to a glycanase and its application in the biosynthesis of 2,3,5,6-tetramethylpyrazine. Background technique [0002] 2,3,5,6-tetramethylpyrazine (2,3,5,6-tetramethylpyrazine, TMP), also known as ligustrazine, is a nitrogen-containing heterocyclic compound. It is a colorless crystalline compound with a melting point of 80-82°C, easily soluble in organic solvents such as ethanol and propylene glycol, and slightly soluble in ether. Ligustrazine has a special burnt sweet, nutty and roasted taste, and it exists in nuts, cocoa beans, coffee and other popular foods. In addition, it is also an important component in liquor, which not only helps to enhance the aroma of the liquor, but also has health benefits to a certain extent. Ligustrazine is a recognized safe substance and is a food additive allowed by national standards, with a maximum concentration of 10mg / kg. [0003] In add...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12N15/56C12P7/26C12P17/12
CPCC12N9/2402C12P7/26C12P17/12Y02P20/54
Inventor 江会锋郭丹刘玉万彭凯卢丽娜
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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