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High yielding cellulase engineering bacterium and application thereof

A technology of cellulase and engineering bacteria, applied in the directions of enzymes, enzymes, fungi, etc., can solve the problems of low cellulase yield, unsuitable for large-scale production and use, etc., and achieve the effect of improving enzyme activity and increasing yield

Inactive Publication Date: 2015-08-12
上海泰坦科技股份有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although the above-mentioned prior art discloses the cultivation methods of some cellulase high-yielding bacterial strains, which can meet certain needs, there are still certain defects in these: the cellulase production of engineering bacteria is relatively low, and it is not suitable for large-scale production in industry use

Method used

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  • High yielding cellulase engineering bacterium and application thereof
  • High yielding cellulase engineering bacterium and application thereof
  • High yielding cellulase engineering bacterium and application thereof

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Effect test

Embodiment 1-3

[0020] Embodiments 1-3 of the present invention relate to the construction of a high-yielding cellulase engineering bacterium, all consisting of the following steps:

[0021] A. Construction of Amylase Gene Knockout Plasmid

[0022] (1) Synthesis of recombinant fragments

[0023] The recombinant fragment consists of the first half of the amylase gene (SEQ ID NO.2), the hygromycin B resistance gene expression unit (SEQ ID NO.3), and the second half of the amylase gene (SEQ ID NO.4). The recombinant fragment (SEQ ID NO.1) was digested with 2 μL of NcoI enzyme solution (purchased from TakaRa) at a certain temperature for 16 hours;

[0024] (2) Extraction and digestion of pCAMBIA1300

[0025] Escherichia coli E.coli DH5α containing the pCAMBIA1300 plasmid was shaken and cultured at 37°C for 12 hours; 1.5 mL of the bacteria was placed in an EP tube, centrifuged at 4000 rpm for 3 min, and the supernatant was discarded; 0.1 mL of solution I (1% glucose, 50 mM EDTA pH 8.0, 25mM Tri...

Embodiment 1

[0061] As mentioned above, the synthesized recombinant fragment was digested with 2 μL of NcoI enzyme solution at 32.8° C. for 16 hours.

Embodiment 2

[0063] As mentioned above, the synthesized recombinant fragment was digested with 2 μL of NcoI enzyme solution at 32.3° C. for 16 hours.

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Abstract

The invention relates to a high yielding cellulase engineering bacterium. The high yielding cellulase engineering bacterium is prepared through the following steps: composing a recombinant fragment by using the first half of amylase gene, a hygromycin B resistant gene expression unit and the latter half of the amylase gene, and carrying out enzyme digestion on the recombinant fragment by using a NcoI enzyme solution at 32.3-33.4DEG C; carrying out enzyme digestion on a pCAMIA1300 plasmid by using a NcoI enzyme; connecting the enzyme digested recombinant fragment with the enzyme digested plasmid through a DNA ligase to obtain an amylase gene knockout plasmid; converting Agrobacterium by using the amylase gene knockout plasmid; and carrying out mixing culture on the amylase gene knockout plasmid-containing Agrobacterium and Aspergillus niger, and carrying out hygromycin B resistance screengin to obtain the Aspergillus niger of the knockout amylase gene. The cellulase engineering bacterium provided by the invention substantially improves the output of cellulase, and is suitable for being used in industrial large-scale production.

Description

technical field [0001] The invention relates to an enzyme-producing engineering bacterium and its application, in particular to a high-production cellulase engineering bacterium and its application. Background technique [0002] Cellulase is a general term for a series of enzymes that can degrade cellulose and generate cellobiose and glucose. According to the different functions of each enzyme, it can be divided into endoglucanase, exoglucanase and β -Three categories of glucosidases, under the synergistic effect, they can completely hydrolyze the cellulose material into glucose, and then ferment to produce ethanol, so as to solve the problems of energy regeneration and environmental pollution. At present, the high cost and low enzyme activity of cellulase seriously restrict its wide application in production. In order to solve this problem, on the one hand, it is necessary to screen cellulase-producing bacteria with high yield and high enzyme activity; Molecular modificat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/80C12N1/15C12N9/42C12R1/685
Inventor 谢应波张庆张华徐肖冰罗桂云
Owner 上海泰坦科技股份有限公司
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