A kind of dehydrogenase mutant l283v/l286v and its preparation method and application

A L286V, mutant technology, applied in the field of biomedicine, can solve the problems of undiscovered enzyme design and transformation, and achieve the effects of increasing substrate diversity, reducing reactant components, and improving dehydrogenase activity

Active Publication Date: 2022-02-22
FOSHAN GOLDEN HEALTH TECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is currently no relevant research on the design and modification of enzymes through protein engineering to achieve the synthesis of S-nornicotine with high optical purity using biological enzyme methods

Method used

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  • A kind of dehydrogenase mutant l283v/l286v and its preparation method and application
  • A kind of dehydrogenase mutant l283v/l286v and its preparation method and application
  • A kind of dehydrogenase mutant l283v/l286v and its preparation method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] The establishment of the tertiary structure model of MesPDH enzyme:

[0038] 1. The analysis was derived from Mesorhizobia ( Mesorhizobium sp. L48C026A00)’s 6-phosphogluconate dehydrogenase MesPDH, using the homology modeling tool MODELLER to carry out homology modeling on MesPDH, obtain the monomer MesPDH enzyme model Ma, evaluate the model, and evaluate all amino acids in the reasonable Lagrangian conformation diagram region, the proportion of amino acids falling in the optimal region is as high as 97.3%, the model is reasonable, and the model Ma is as follows figure 1 as shown in a.

[0039] 2. Use the online homology modeling tool Swiss-model to carry out homology modeling on MesPDH, obtain the dimer MesPDH enzyme model Mb, evaluate the model, and evaluate all amino acids in the Lagrange conformation diagram. The proportion of amino acids in the region is as high as 94.6%, the model is reasonable, and the model Mb is as follows figure 1 as shown in b.

[0040] 3...

Embodiment 2

[0042] Molecular docking simulation predicts the binding conformation of each substrate and enzyme and selects the site:

[0043] 1. Molecular docking simulation

[0044] The molecular simulation software Discovery Studio was used to locate the active pocket centers of the three modeling models, and the molecular docking software AutodockVina was used to dock the MesPDH enzyme with Mesmin, glucose, gluconic acid, NAD+, and NADH, and the protein conformation analysis tool PyMOL , for each docking complex analyzed.

[0045] 2. Analysis of docking results

[0046] Such as figure 2 As shown, the MesPDH enzyme model Ma, Mb and Mc were docked with the four reactant molecules respectively, and it was found that the positions of myosmine, glucose and gluconic acid all fell in similar positions, and NAD+ and NADH were located in another pocket position , from the model Mb, it is speculated that the MesPDH enzyme is most likely to exert its enzyme activity in the form of a dimer, wh...

Embodiment 3

[0050] Obtaining of recombinant wild enzyme strain BL21(DE3) / pET-32a-MesPDH:

[0051] The whole gene was synthesized and optimized by Escherichia coli codon preference to obtain the MesPDH enzyme gene, its nucleotide sequence is as shown in SEQ ID NO: 4, which was connected to the pET-32a plasmid, and the obtained recombinant plasmid was named pET-32a-MesPDH , the plasmid was transformed into Escherichia coli BL21 (DE3), and the recombinant strain was named BL21 (DE3) / pET-32a-MesPDH. The amino acid sequence of the wild type MesPDH enzyme expressed by the recombinant wild enzyme strain is shown in SEQ ID NO:3.

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Abstract

A dehydrogenase mutant L283V / L286V and its preparation method and application relate to the field of biomedicine technology; the amino acid sequence of the mutant L283V / L286V is as shown in SEQ ID NO: 1; the amino acid sequence is as shown in SEQ ID NO: 1 The leucine at the 283rd position and the 286th position of the dehydrogenase indicated by ID NO:3 is mutated to valine at the same time. The dehydrogenase mutant L283V / L286V exhibited high selectivity in catalyzing the reduction of mesminin to S-nornicotine in the whole cell system, and had relatively high dehydrogenase and imine reductase activities at the same time, and the enzyme reduced Short time, high conversion rate. The product S-nornicotine obtained from the reaction has extremely high optical purity, which reduces the difficulty of subsequent purification work.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a dehydrogenase mutant L283V / L286V and a preparation method and application thereof. Background technique [0002] Nicotine, commonly known as nicotine, is an alkaloid contained in the Solanaceae plant tobacco, accounting for more than 95% of all alkaloids in tobacco, and its chemical name is 1-methyl (2-pyridyl) pyrrolidine, Molecular formula is C 10 h 14 N 2 , is an organic dibasic weak base with strong basicity, which can form crystallized single or double salts with various inorganic acids or organic acids, and mostly exists in the form of organic salts in tobacco leaves. [0003] Nicotine has a wide range of uses. Nicotine series pesticides are botanical insecticides. They are widely used in food, oil, vegetables, fruits, pasture and other crops because of their characteristics of steam fumigation, stomach poisoning, contact killing and rapid degradation without resid...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/10C12N15/70C12P41/00C12P17/16
CPCC12N9/0008C12N15/1031C12N15/70C12P41/002C12P17/165C12Y102/04001C12Q2531/113C12N9/0006C12N9/0004C12P17/12
Inventor 黄佳俊李荣旭江锐冰吴子蓥胡浩轩白少钰周金林卢宇靖
Owner FOSHAN GOLDEN HEALTH TECH CO LTD
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