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Gene engineering strain MI033WZ expression M1033 GI mutation enzyme GIG 38P and construction method thereof

A technology of M1033GI, genetically engineered strains, applied in the field of genetic engineering for constructing the strains, can solve the problems such as not given genetically engineered strains, and achieve the effect of avoiding genetic manipulation

Inactive Publication Date: 2003-05-21
ZHONGKE DAYIYUAN BIOLOGICAL TECH ANHUI
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Problems solved by technology

[0003] Chinese patent "SM33GI Mutant Enzyme GIG138P and a Mutation Scheme for Improving GI Thermal Stability" (95112782.9) provides the glucose isomerase SM33GI (also called M1033GI) for No. 7 Streptomyces amylase SM33 (also called M1033) The mutant enzyme GIG138P obtained by mutation transformation and its mutation scheme, but no stable and high-efficiency genetically engineered strains that can be applied to industrial production

Method used

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  • Gene engineering strain MI033WZ expression M1033 GI mutation enzyme GIG 38P and construction method thereof
  • Gene engineering strain MI033WZ expression M1033 GI mutation enzyme GIG 38P and construction method thereof
  • Gene engineering strain MI033WZ expression M1033 GI mutation enzyme GIG 38P and construction method thereof

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Experimental program
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Embodiment Construction

[0033] The specific construction process of M1033WZ is described as follows in conjunction with the accompanying drawings:

[0034] (1) Construction of glucose isomerase gene knockout-deficient strain M1033LJ:

[0035] 1) Construction of gene knockout homologous recombination plasmid pTR

[0036] The pUB1 plasmid contains the complete GI structural gene, promoter and 3' non-regulatory sequence. First, pUB1 was cut with XhoI, and the 4.9Kb large fragment was recovered after Klenow fill-up. At the same time, pIJ702 was digested with BclI, and the 1.1Kb tsr gene fragment was recovered by electrophoresis after filling in bluntly; the recombinant plasmid pTR was obtained by double-blunt end ligation. The 700th position to the 400th position after the 3' end of the GI structural gene in the recombinant plasmid was replaced by the tsr gene. The inserted tsr gene interrupts the GI structural gene on the one hand, and serves as a selectable marker for the recombinant strain after ho...

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Abstract

The present invention relates to a gene engineering strain for expressing mutant enzyme GIG138P of M1033GI and its construction scheme and relates to gene engineering strain for expressing glucose isomerase and its construction method. It is characterized by that it utilizes homologous recombination method of gene knockout and gene substitution to make the gene frame for expressing GI chromosome of No.7 diastasic streptomycete M1033 undergo the process of tricyclic chromosome frame modification to implement chromosome molecule mutation, i.e, the structural gene 138 site can be introduced intoGIG 138P mutant site to construct the gene engineering strain. The gene level, protein level and strain passage tests show that the activity expression of M1033WZ enzyme constructed by said method isstable.

Description

Technical field: [0001] The invention relates to a genetic engineering bacterial strain expressing glucose isomerase and a genetic engineering method for constructing the bacterial strain. Background technique: [0002] Glucose isomerase (GI for short), as a key enzyme for large-scale industrial amylase production of high fructose syrup, has extremely important economic value. The wild-type GI isolated and purified from natural strains has been used in industrial production and has achieved huge economic and social benefits. However, the thermal stability of wild-type GI at higher reaction temperature is not ideal, and the higher the temperature in the isomerization reaction, the more the thermodynamic equilibrium tends to fructose. Therefore, improving the thermal stability of wild-type GI can increase the yield of fructose in the reaction equilibrium process, thereby reducing the input of bacteria, simplifying the process and improving economic benefits. [0003] Chinese...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N9/90C12N15/61C12N15/63C12N15/76
Inventor 徐冲滕脉坤牛立文朱国萍伍传金杨永辉张颖廖军王庆华龚为民朱忠良王玉珍
Owner ZHONGKE DAYIYUAN BIOLOGICAL TECH ANHUI
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