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Whole-bacterium enzymic preparation for catalyzing saccharification of lignocellulose

A technology of lignocellulose and whole-bacteria enzymes, applied in the biological field, can solve the problems of high cost and cumbersome process, and achieve the effects of improving synergy, reducing consumption, maintaining stability and activity

Active Publication Date: 2018-12-11
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The whole bacteria enzyme preparation solves the problems of high cost and cumbersome process caused by the consumption of enzymes in the saccharification process

Method used

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  • Whole-bacterium enzymic preparation for catalyzing saccharification of lignocellulose
  • Whole-bacterium enzymic preparation for catalyzing saccharification of lignocellulose
  • Whole-bacterium enzymic preparation for catalyzing saccharification of lignocellulose

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Construction of Clostridium thermocellum expressing exocrine β-1,4 glucosidase

[0040] The cellulase Cel9K (exocellulase, encoded by nucleotide sequences 2113813 to 2111293 in genome CP002416.1) in the cellulosome of Clostridium thermocellum was selected as a targeted knock-in site between the enzymatic catalytic domain and the docking module point. First, the β-1,4-glucosidase BglA (Genbank sequence number is AFO70070.1) encoding gene was used as the target sequence, and the restriction sites of MluI and EagI were used to clone into the homologous recombination plasmid pHK-HR ( figure 2 ), the homologous recombination plasmid pHK-HR-BglA was constructed. The HR-up sequence of the upstream homology arm is the nucleic acid sequence of 2111347 to 2112870 in the genome of Clostridium thermocellum DSM1313 (the sequence number in the NCBI database is CP002416.1), and the HR-down of the downstream homology arm is the nucleic acid sequence of 2109848 to 2111354 i...

Embodiment 2

[0045] Example 2: Construction of a whole bacterial enzyme preparation based on Clostridium thermocellum cellosomes by direct linking

[0046] Using the method of overlap extension polymerase chain reaction, xylanase XynC (SEQ ID NO: 1) was combined with the sequence of the type II adhesion module CohIIct (SEQ ID NO: 10) or the type I docking module DocIct of Clostridium thermocellum The sequences of XynC (SEQ ID NO: 9) were directly linked, wherein the sequence of CohIIct or DocIct was linked to the 3' end of the XynC sequence, thereby obtaining the XynC-CohIIct and XynC-DocIct sequences. Reuse the BamHI and XbaI restriction sites, and clone the connected recombinant sequence as the target sequence into the expression plasmid pHK ( figure 1 )superior. Since pHK carries the promoter and signal peptide sequence of cellulase Cel48S derived from Clostridium thermocellum (SEQ ID NO: 11), the expressed target gene can be excreted outside the cell. The constructed plasmid was tran...

Embodiment 3

[0047] Example 3: Construction of a whole bacterial enzyme preparation based on Clostridium thermocellum cellosomes by direct linking

[0048] The xylanase XynB (SEQ ID NO: 2) encoding gene was used as the target sequence, and the cellulosic exonuclease Cel48S (encoded by the 3228088 to 3230229 nucleic acid sequences in the genome CP002416.1) of the Clostridium thermocellum cellosome was selected. The ' end was used as a targeted knock-in site, and the restriction sites of MluI and EagI were used to clone into the homologous recombination plasmid pHK-HR ( figure 2 ), the homologous recombination plasmid pHK-HR-xynB was constructed. The upstream homology arm HR-up is the nucleic acid sequence from 3230200 to 3230700 in the genome of Clostridium thermocellum DSM1313 (the sequence number in the NCBI database is CP002416.1), the downstream homology arm HR-down is the nucleic acid sequence from 3229699 to 3230199 in the DSM1313 genome, and the middle The homology arm HR-short is ...

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Abstract

The invention provides a whole-bacterium enzymic preparation for catalyzing saccharification of lignocellulose aiming at the problem of cellulosome in lignocellulosic saccharification in the prior art. The whole-bacterium enzymic preparation for catalyzing the saccharification of lignocellulose is a bacterial strain obtained after modifying cellulosome by binding cellulosome proteins in a cellulosome complex through the mutual action of non-cellulosome proteins and components in the cellulosome; then the bacterial strain is used for saccharifying, namely, bacterial cells are involved in the saccharification process. The whole-bacterium enzymic preparation provided by the invention not only achieves the maintenance of stability and activity, but also greatly enhances the synergistic effectwith other enzymes in the system, thereby reducing the enzyme consumption, and reducing the production cost and the trivialness of the process.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to an enzyme preparation, in particular to a whole-bacteria enzyme preparation for catalyzing lignocellulose saccharification. Background technique [0002] Lignocellulose is a kind of renewable material with abundant supply and environmental friendliness. It is the only resource that can be regenerated on a large scale and completely replace fossil energy. Vigorously developing the utilization of lignocellulose raw materials in energy and other fields is the key to speeding up the development of circular economy and ensuring An important strategic task for national energy security and carbon emission reduction. However, the biggest bottleneck of lignocellulose conversion is the refractory degradation of the cellulose crystallization region, resulting in low enzymatic hydrolysis efficiency and high cost. Therefore, to achieve efficient utilization of lignocellulose, it is necessary to fir...

Claims

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Application Information

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IPC IPC(8): C12N9/24C12N9/50C12N9/26C12N9/42C12N9/18C12N9/80C12P19/14
CPCC12N9/18C12N9/2402C12N9/2411C12N9/2437C12N9/248C12N9/50C12N9/80C12P19/14C12Y301/01011C12Y302/01004C12Y302/01015C12Y402/02002
Inventor 崔球刘亚君刘世岳李仁民祁宽冯银刚
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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