Construction of temperature-controlled aspergillus niger genetically engineered bacterium started to express excision enzyme of cellulose

A technology of genetically engineered bacteria and temperature control, which is applied in the field of enzyme engineering, can solve the problems of increasing degradation cost, limiting the use range of cellulase, and high cost of cellulase, so as to be suitable for industrial applications, optimize the composition of enzyme systems, and the composition of enzyme systems reasonable effect

Active Publication Date: 2015-04-29
中农华威生物制药(湖北)有限公司
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AI Technical Summary

Problems solved by technology

Mixing with high exonuclease activity cellulase itself increases the process of mixing and increases the cost of degradation
And the cost of cellulase with high exonuclease activity is also high
Mixed fermentation due to toxins produced by Trichoderma reesei greatly limits the scope of use of cellulase

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] A. Construction of homologous recombination plasmid

[0020] (1) Material: Aspergillus niger ( Aspergillus niger ), Escherichia coli ( E.coli) DH5α, Agrobacterium EHA105 and plasmid pWM1 are all commercial products. Among them, Aspergillus niger was purchased from ATCC with strain number ATCC10582; Agrobacterium EHA105 and plasmid pMW1 were purchased from Biovector.

[0021] (2) Synthesis and restriction digestion of homologous recombination fragments

[0022] Artificially synthesized the first homologous fragment of the heat shock protein gene from Aspergillus niger (the start codon ATG was modified to a non-start codon to ensure expression from the start codon of the cellulose exonuclease gene, the nucleotide sequence is as SEQ ID NO. 2), Trichoderma reesei exonuclease gene fragment (nucleotide sequence shown in SEQ ID NO. 3), and homologous fragment of Aspergillus niger heat shock protein gene (nucleotide sequence shown in SEQ ID NO.4) consisting of a homologous recombi...

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PUM

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Abstract

The invention discloses construction of a temperature-controlled aspergillus niger genetically engineered bacterium started to express an excision enzyme of cellulose and belongs to the field of enzyme engineering. The aspergillus niger genetically engineered bacterium disclosed by the invention is an aspergillus niger strain which is integrated with a gene of the excision enzyme of cellulose in a position of a heat shock protein gene. Construction of the aspergillus niger genetically engineered bacterium comprises the following steps: synthesizing a homologous recombinant fragment shown in a nucleotide sequence such as SEQ ID NO. 1; enzyme-digesting the recombinant fragment and a pWM1 plasmid by using Sa1I and then connecting transformed DH5alpha to obtain a homologous recombinant plasmid; transforming agrobacterium by using the homologous recombinant plasmid and obtaining the target strain by agrobacterium tumefaciens-mediated transformation. According to the invention, heat shock expression of the excision enzyme of cellulose is realized, so that the enzyme constitution of the cellulose of aspergillus niger is reasonable, so that the catalytic capacity of the cellulose is enhanced; the strain constructed does not generate toxins and is high in safety and suitable for industrial application.

Description

Technical field [0001] The present invention belongs to the field of enzyme engineering, and specifically relates to a genetically engineered Aspergillus niger bacterium with temperature control to start the expression of cellulose exonuclease and its construction method. Background technique [0002] Cellulose is the most abundant renewable resource on the earth. The cellulose produced by photosynthesis on the earth reaches about 10 billion tons every year. The use of cellulose to produce bioenergy is the key to alleviating the energy crisis and realizing the sustainable development of mankind. [0003] The key to cellulose utilization is to degrade cellulose into fermentable sugar-glucose. The degradation of cellulose requires the joint action of exonuclease, endonuclease and glucosidase. Among them, exonuclease degrades the crystal structure of cellulose, which is the rate-limiting step for cellulase to degrade cellulose, and endonuclease degrades long fragments of cellulose in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/15C12N15/74C12R1/685
CPCC12N9/2437C12Y302/01091
Inventor 薛栋升
Owner 中农华威生物制药(湖北)有限公司
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