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Xylose isomerase mutant, DNA molecule coding xylose isomerase, recombination strain with introduced DNA molecule and application thereof

A technology of xylose isomerase and DNA molecules, which is applied in the fields of protein engineering and genetic engineering, can solve the problems that cannot meet the actual needs, and achieve the effect of wide application value and high-efficiency sugar-alcohol conversion fermentation

Active Publication Date: 2019-03-15
JILIN COFCO BIOCHEM +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, how to effectively obtain xylose isomerase with improved catalytic activity and apply it in industrial production is still rarely reported in this field, which cannot meet the actual needs

Method used

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  • Xylose isomerase mutant, DNA molecule coding xylose isomerase, recombination strain with introduced DNA molecule and application thereof
  • Xylose isomerase mutant, DNA molecule coding xylose isomerase, recombination strain with introduced DNA molecule and application thereof
  • Xylose isomerase mutant, DNA molecule coding xylose isomerase, recombination strain with introduced DNA molecule and application thereof

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Experimental program
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Embodiment 1

[0056] Example 1: Preparation and Activity Determination of Xylose Isomerase Mutants

[0057]The xylose isomerase mutant described in the present invention is preferably obtained by the following method, but for those skilled in the art, the specific parameters in each operation can be adjusted appropriately according to actual needs, which is a matter for those skilled in the art to read It can be easily accomplished on the basis of the present invention.

[0058] 1. Obtaining the gene fragment encoding xylose isomerase mutant

[0059] Hereinafter, the xylose isomerase mutant Ala144Thr is taken as an example to describe in detail.

[0060] Invitrogen was entrusted to synthesize the gene fragment (SEQ ID No.14) encoding natural xylose isomerase, and EcoR I and Not I restriction sites were introduced at both ends of it for the purpose of cloning. After double digestion with EcoR I enzyme and Not I enzyme, it was cloned into the multiple cloning site of the pPIC9K vector that ...

Embodiment 2

[0067] The construction of embodiment 2 recombinant Saccharomyces cerevisiae strains

[0068] 1. Construction of the recombinant plasmid containing the XI gene expression cassette and the XK gene expression cassette

[0069] 1.1 Construction of plasmid containing XI gene expression cassette or XK gene expression cassette

[0070] Using the Golden Gate method, the XI gene (SEQ ID No.7 or SEQ ID No.14) and the TEF2p promoter (SEQ ID No.15) and CYC1t terminator (SEQ ID No.16) were assembled on the pUC19 vector to construct is the XI gene expression cassette.

[0071] Using the Golden Gate method, the XK gene (gene number 398366094), PGK1p promoter (SEQ ID No. 17) and PGK1t terminator (SEQ ID No. 18) were assembled on the pUC19 vector to construct an XK gene expression cassette.

[0072] 1.2 Construction of recombinant plasmids containing XI gene expression cassette and XK gene expression cassette

[0073] Using the Golden Gate method, the constructed XI gene expression cassett...

Embodiment 3

[0082] Adaptive evolution of embodiment 3 recombinant Saccharomyces cerevisiae strains in xylose medium

[0083] The selected strains were subjected to adaptive evolution in the YEPX medium, and the specific method was as follows: pick a single colony on the YEPX plate into a test tube containing 5ml YEPX, cultivate overnight at 30°C and 200rpm, and use the cultured bacterial solution with The absorbance at 600 nm was measured with a spectrophotometer. Take 200 μl of the overnight culture liquid and inoculate it into a test tube containing fresh YEPX medium, measure the absorbance at 600 nm after overnight culture, and repeat the operation until the cell density of the recombinant Saccharomyces cerevisiae strain in the xylose medium is stable. During the adaptive evolution process, the xylose utilization rate and sugar alcohol conversion rate of the recombinant Saccharomyces cerevisiae strain were detected.

[0084] The recombinant Saccharomyces cerevisiae strains that have e...

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Abstract

The invention relates to a xylose isomerase mutant with improved catalytic activity, a DNA molecule coding the xylose isomerase, a recombination strain containing the DNA molecule and application thereof. By mutating amino acid residues at specific sites in the natural xylose isomerase, the xylose isomerase mutant with improved catalytic activity can be obtained, and the obtained xylose isomerasemutant has high thermal stability; furthermore, recombinant yeasts containing the xylose isomerase mutant established by a synthetic biological method can be effectively applied to C5 sugar and C6 sugar co-fermentation for producing ethanol, and the fermentation efficiency and ethanol yield are obviously improved. Therefore, the xylose isomerase mutant and the recombination strain disclosed by theinvention have wide application value in the fields of chemical engineering, energy, foods, medicines and the like, and sugar-alcohol conversion fermentation can be efficiently performed.

Description

technical field [0001] The invention relates to the fields of protein engineering and genetic engineering. Specifically, the present invention relates to a xylose isomerase mutant with improved catalytic activity, a DNA molecule encoding the enzyme, a recombinant strain introduced into the DNA molecule, and applications thereof. Background technique [0002] Xylose isomerase (Xylose isomerase, XI; EC 5.3.1.5) can catalyze the conversion of five-carbon sugar D-xylose into D-xylulose, which plays an important role in the process of sugar metabolism in microorganisms and has extremely broad industrial application value. In particular, in natural microorganisms, there are two pathways for metabolizing xylose to xylulose, including a pathway via xylose isomerase and a pathway via xylose reductase and xylitol dehydrogenase. In bacteria (such as Actinoplanes missouriensis, Bacillus coagulans, Streptomyces rubiginosus, Arthrobacter sp. and Escherichia coli )), a few fungi (such a...

Claims

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Application Information

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IPC IPC(8): C12N9/92C12N15/61C12N1/19C12P7/06C12R1/865C12R1/84
CPCC12N9/92C12P7/06C12Y503/01005Y02E50/10
Inventor 佟毅张子剑贾力耕沈乃东何太波宋思琦苏立国张媛王靖张广昊吴延东李义袁敬伟
Owner JILIN COFCO BIOCHEM
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