Ketoreductase dna molecule, recombinant vector and bacterial strain and application

A technology of DNA molecules and recombinant vectors, applied in the field of genetic engineering, can solve the problems of difficult industrialization, low specific enzyme activity, unstable expression, etc., achieve suitable scale production, large expression of enzyme activity, and avoid flammable and flammable The effect of explosive chemicals

Active Publication Date: 2021-02-02
安琪酶制剂(宜昌)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] In summary, for the prior art, the main problems are: the specific enzyme activity of the ketoreductase genetically engineered bacteria is low, the expression is unstable, and it is difficult to realize industrialization

Method used

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  • Ketoreductase dna molecule, recombinant vector and bacterial strain and application
  • Ketoreductase dna molecule, recombinant vector and bacterial strain and application
  • Ketoreductase dna molecule, recombinant vector and bacterial strain and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Embodiment one provides a kind of step of constructing mutant genetically engineered bacteria of the present invention, as follows:

[0060] (1) Screening of mutant genes

[0061] The 4H8N, 3WG6, and K amino acid sequences of ketoreductases from three different sources of wild fungus, Candida lipolytica, Candida portuguese, and Candida magnolia, were compared for homology comparison and analysis. The analysis of the hydrogen bond in the functional region, the binding site between the coenzyme and the ketoreductase, and the subunit interaction determined that the ketoreductase K sequence SEQ ID NO.3 of Candida magnolia was used as a template.

[0062] Wherein, the sequence of SEQ ID NO.3 is as follows:

[0063]

[0064]

[0065] Based on the ketoreductase K sequence SEQ ID NO.3, three amino acid mutation sites were selected through computer-aided semi-rational design, namely E102S (gag-tcc), L133I (tta-atc) and E172D (gaa-gat), that is, replace the 102nd glutamic...

Embodiment 2

[0125] Example 2 provides a comparison experiment of the specific enzyme activity of the ketoreductase produced by the strain prepared in Example 1 of the present invention and the ketoreductase produced by the original strain.

[0126] Among them, the specific enzyme activity refers to the unit of activity per mg of enzyme protein, which is the enzyme activity divided by the mass of enzyme protein.

[0127] Ligate the target fragment gene of known sequence and the gene fragment of the ketoreductase mutant with the double combination of amino acid sites in the 102 and 133 regions to the large fragment of the plasmid pBAD respectively, take 10 μL of the ligation reaction and add it to 100 μL of the sensory In the EP tube, mix gently in the EP tube. After 30 minutes of ice bath, heat shock at 42°C for 90 seconds. After taking it out, quickly ice bath for 2 minutes to cool the bacteria. Add 1 mL of antibiotic-free LB liquid medium (containing 10 μL of 10% arabinose inducer), shak...

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Abstract

The invention relates to the field of genetic engineering, in particular to DNA molecules, protein or polypeptide sequences for producing ketoreductase, recombinant vectors, strains and applications. The base sequence of the ketoreductase DNA molecule is shown in SEQ ID NO.1; or it is selected from the gene encoding the following protein (a) or (b): (a) the amino acid shown in SEQ ID NO.2 (b) a polypeptide or protein derived from (a) that has undergone substitution, deletion or addition of one or several amino acids in the amino acid sequence defined in (a) and has ketoreductase activity. In the present invention, the connection of the mutant gene with the plasmid pBAD is more stable, and under the induction of arabinose, the fermentation enzyme activity is higher, which can reach more than 120U / mL, and has the characteristics of high expression, stable expression system, and simple fermentation control. Suitable for large-scale production.

Description

technical field [0001] The present invention relates to the field of genetic engineering, in particular to a method for producing ketoreductase mutants and engineering bacteria, in particular to ketoreductase mutant DNA molecules, protein or polypeptide sequences, recombinant vectors, bacterial strains and applications. Background technique [0002] (1) The three major killers that threaten human health and safety are "hyperglycemia, hyperlipidemia, and hypertension." Among them, hyperlipidemia is the main cause of atherosclerosis and coronary heart disease, hypertension, and cerebrovascular disease. Hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitors lipid-lowering drugs are currently the first choice for lowering blood lipids. Statins limit the metabolism of cholesterol in the body by inhibiting the pathway efficiency of HNG-CoA reductase, and reduce the levels of cholesterol and lipoproteins in plasma, thereby reducing blood lipids. The statin drugs currently...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N9/04C12N15/70C12N1/21C12R1/19
CPCC12N9/0006C12Y101/01002
Inventor 余华顺俞学锋李知洪姚鹃戴秋红龚大春王健李建华邹林汉
Owner 安琪酶制剂(宜昌)有限公司
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