Method for preparation of co-expressed recombinase with genetic engineering technology

A genetic engineering and co-expression technology applied in the field of dual-enzyme co-expression

Inactive Publication Date: 2014-04-09
SHANXI TIANJIAO FOOD +2
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no publication of the co-expression of glucose isomerase and D-psicose 3-epimerase to obtain the two e

Method used

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  • Method for preparation of co-expressed recombinase with genetic engineering technology
  • Method for preparation of co-expressed recombinase with genetic engineering technology
  • Method for preparation of co-expressed recombinase with genetic engineering technology

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Embodiment Construction

[0040] The present invention will be further described below in conjunction with specific examples.

[0041] The invention adopts bacillus as the enzyme source of the glucose isomerase, and Escherichia coli as the host bacterium of the nucleotide sequence of the recombination enzyme.

[0042] The concrete flow process that the first three steps of the present invention adopts genetic engineering technology to prepare co-expression recombinant enzyme is:

[0043] Strain screening GI site-directed mutagenesis carrier selection Get target gene Construction of recombinant expression plasmids Induced expression of target protein SDS-PAGE Analysis and Identification Analysis of enzymatic properties.

[0044] 1. Source of enzyme gene:

[0045] The strain with the highest glucose isomerase activity was screened out through primary screening and re-screening, molecular identification and enzymatic property research. Using BLAST software in NCBI to analyze the 16SrDNA...

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Abstract

The invention belongs to the technical field of co-expression of dienzyme, and in particular relates to co-expression of site-specific mutational glucose isomerase and D-allulose-3-epimerase. For overcoming the shortcomings of the prior art, the invention provides a method for performing site-specific mutagenesis to the 146th amino acid of the glucose isomerase and performing co-expression with the glucose isomerase and the D-allulose-3-epimerase. Specifically, the method comprises the following steps: respectively performing PCR (Polymerase Chain Reaction) amplification, purification and cloning to the nucleotide sequence of the glucose isomerase after site-specific mutagenesis and the nucleotide sequence of the D-allulose-3-epimerase; respectively digesting the cloned bienzyme nucleotide sequence with a pCDFDuet-1 carrier; purifying and connecting the digestion product to build a recombinant expression vector; inducing expression to obtain recombinant bacteria; collecting the recombinant bacteria and treating the recombinant bacteria by ultrasonication to obtain a crude enzyme liquid containing bienzyme.

Description

technical field [0001] The invention belongs to the technical field of double-enzyme co-expression, and specifically relates to the co-expression of site-directed mutation glucose isomerase and D-psicose 3-epimerase. Background technique [0002] Xylose isomerase (Xylose isomerase, Xiase, EC 5.3.1.5), also known as glucose isomerase (glucose isomerase, GIase), can catalyze the isomerization reaction of D-glucose to D-fructose outside the cell, and is a food In industrial production, it is one of the key enzymes for the preparation of fructose syrup from starch. [0003] D-psicose 3-epimerase (D-psicose 3-epimerase, abbreviated as DPE) is currently a major enzyme that can realize the biotransformation between D-fructose and D-psicose. Ketose 3-epimerase family, the optimal substrate is D-psicose. Therefore, applying genetic engineering technology to prepare recombinant enzymes of glucose isomerase and D-psicose 3-epimerase can effectively convert D-glucose into D-fructose, ...

Claims

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Application Information

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IPC IPC(8): C12N9/90C12N9/92C12N15/61C12N15/70
CPCC12N9/90C12N9/92C12N15/70C12Y503/01005
Inventor 王晓艳门燕冯俊敏孙媛霞张佩舜朱玥明康振奎巩晋龙郑丽萍王小鹏李奠础
Owner SHANXI TIANJIAO FOOD
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