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Cellulosomal enzyme preparations for catalyzing lignocellulosic saccharification

A technology for lignocellulose and cellulosomes, applied in the field of enzyme preparations and cellulosome enzyme preparations, can solve the problems of high cost and cumbersome process, improve synergy, reduce consumption, reduce production cost and process cumbersomeness sexual effect

Active Publication Date: 2021-04-13
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 cellulosome 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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  • Cellulosomal enzyme preparations for catalyzing lignocellulosic saccharification
  • Cellulosomal enzyme preparations for catalyzing lignocellulosic saccharification
  • Cellulosomal enzyme preparations for catalyzing lignocellulosic saccharification

Examples

Experimental program
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Effect test

Embodiment 1

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

[0040] The cellulase Cel9K (exocellulase, encoded by the nucleic acid sequence 2113813 to 2111293 in the genome CP002416.1) in the cellulosome of Clostridium thermocellum and the docking module were selected as the targeted knock-in site point. First, the gene encoding β-1,4-glucosidase BglA (GENBANK sequence number is AFO70070.1) was used as the target sequence, and cloned into the homologous recombination plasmid pHK-HR( figure 2 ), construct the homologous recombination plasmid pHK-HR-BglA. The upstream homology arm HR-up sequence is the nucleic acid sequence from 2111347 to 2112870 in the Clostridium thermocellum DSM1313 genome (the sequence number in the NCBI database is CP002416.1), and the downstream homology arm HR-down is the 2109848 to 2111354 nucleic acid sequence in the DSM1313 genome, The middle homology arm HR-short is the nucleic acid sequence from 2111347 to 21...

Embodiment 2

[0045] Embodiment 2: By the method of direct connection, construct the cellulosomal enzyme preparation based on Clostridium thermocellum cellulite

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

Embodiment 3

[0047] Embodiment 3: By the method of direct connection, construct the cellulosomal enzyme preparation based on Clostridium thermocellum cellulosome

[0048] The gene encoding xylanase XynB (SEQ ID NO: 2) was used as the target sequence, and 5 of the cellulosic exonuclease Cel48S (encoded by the nucleic acid sequence from 3228088 to 3230229 in genome CP002416.1) of Clostridium thermocellum was selected. The 'end was used as the targeted knock-in site, and cloned into the homologous recombination plasmid pHK-HR( figure 2 ), construct the homologous recombination plasmid pHK-HR-xynB. The upstream homology arm HR-up is the nucleotide 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 nucleotide sequence from 3229699 to 3230199 in the DSM1313 genome, and the middle The homology arm HR-short is the nucleic acid sequence from 3230200 to 3230500 in th...

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Abstract

Aiming at the problems of cellulosomes in lignocellulose saccharification in the prior art, the present invention provides a cellulosome enzyme preparation for catalyzing lignocellulose saccharification. The cellulosomal enzyme preparation is a protein complex obtained by binding non-cellulosomal proteins in cellulosomes through the interaction of non-cellulosomal proteins with components in cellulosomes; it is then used for saccharification , that is, the saccharification process does not involve the participation of bacterial cells. The cellulosome enzyme preparation of the present invention not only maintains its stability and activity, but also greatly improves its synergy with other enzymes in the system, thereby reducing the consumption of enzymes, reducing production costs and processes of cumbersomeness.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to an enzyme preparation, in particular to a cellulosome 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 first ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N9/24C12N9/26C12N9/50C12P19/14C12P19/00
CPCC07K14/00C07K2319/00C12N9/2411C12N9/248C12N9/50C12P19/00C12P19/14C12Y302/01004
Inventor 崔球刘亚君李仁民刘世岳祁宽冯银刚
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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