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Cellulase preparation and application thereof

A cellulase and enzyme preparation technology, applied in the directions of enzymes, enzymes, hydrolase, etc., can solve the problems of high equipment requirements, reduced activity and stability, complex processes, etc., and achieve high fermentable sugar yield and high fermentable sugar. Yield, simple effect of saccharification process

Active Publication Date: 2018-11-23
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since the activity and stability of the added β-1,4-glucosidase will decrease with the progress of the saccharification process, the consumption of the enzyme needs to increase the amount of enzyme added or the number of times added
Adding β-1,4-glucosidase as a free enzyme to the saccharification system will significantly reduce the synergistic effect with other enzymes in the system, which will inevitably lead to the addition of enzymes larger than the demand, resulting in high cost of enzyme preparations
Not only that, this saccharification strategy that relies on the addition of exogenous free enzymes has complex processes, high equipment requirements, and conversion efficiency that cannot meet the requirements of industrial production.

Method used

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  • Cellulase preparation and application thereof
  • Cellulase preparation and application thereof

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Experimental program
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Embodiment 1

[0059] The screening of embodiment 1β-1,4-glucosidase

[0060] For effective feedback inhibition of cellobiose exposure, it is necessary to select β-1,4-glucosidase with high activity, high stability and high glucose tolerance.

[0061] Selection of β-1,4-glucosidases from Pyrolyticum genus, Thermoanaerobacillus, Clostridium and metagenomics were performed in Escherichia coli BL21(DE3) in a conventional manner. Recombinant expression was carried out in , and the protein was purified by affinity chromatography. The purified protein was tested for enzymatic activity using pNPG as a substrate, and the enzymatic properties were analyzed and compared (see Table 1).

[0062] The enzyme activity detection system is 200μL, which includes 50mM sodium acetate and 1mM pNPG, adding enzyme to start the catalytic reaction, adding 1mL sodium carbonate to stop the reaction after 5-10 minutes of reaction, and detecting the visible light absorption at 405nm, and according to the following The...

Embodiment 2

[0067] Construction of a recombinant strain of Clostridium thermocellum expressing β-1,4-glucosidase with type II adhesion module

[0068] Using the method of overlap extension polymerase chain reaction, β-1,4-glucosidase CtBglA (Table 1, SEQ ID NO: 6) is connected with the sequence (SEQ ID NO: 5) of type II cohesion module CohII, wherein The sequence of CohII is linked to the 3' end of the CtBglA sequence. 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: 9) of the cellulase Cel48S derived from Clostridium thermocellum, the expressed target gene can be secreted to the extracellular space. The constructed plasmid was transformed into Clostridium thermocellum DSM1313, thereby obtaining a Clostridium thermocellum recombinant strain DSM1313::pHK-CtBglA-CohII expressing β-1,4-glucosidase with type II cohes...

Embodiment 3

[0074] Construction of Scarless Genetic Operating System of Clostridium thermocellum

[0075] In order to achieve the precise editing of the DNA sequence on the genome of Clostridium thermocellum DSM1313, a Clostridium thermocellum scarless genetic operating system was established, including the mutant strain ΔpyrF of the pyrF knockout Clostridium thermocellum as the chassis cell of the system, and a homologous recombination plasmid pHK-HR( figure 2 ). The homologous recombination plasmid includes two selectable markers and three homology arms. Among them, the screening markers are bidirectional screening marker pyrF and reverse screening marker tdk; the three homology arms include two long homology arms (HR-up, HR-down, about 1200bp), and the middle of the front and rear homology arms Short homology arm (HR-short, about 300bp), wherein the middle short homology arm has the same sequence as the 3' end of the front homology arm to ensure the occurrence of the second homologou...

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Abstract

The invention belongs to the technical field of biology, and in particular relates to a cellulase preparation for catalyzing lignocellulose saccharification, and application of the cellulase preparation. The cellulase preparation provided by the invention contains a cellulosome complex and beta-1, 4-glucosidase at the same time, wherein the beta-1, 4-glucosidase is compounded in the cellulosome complex in a way of interaction of covalent or non-covalent proteins or fusion expression with the cellulosome complex. The cellulase preparation not only can realize high-efficiency hydrolysis of a lignocellulose substrate and has high fermentable sugar yield and saccharification rate, but also has the advantages of being low in production cost, and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a cellulase preparation for catalyzing lignocellulose saccharification and application thereof. Background technique [0002] Lignocellulose is a kind of renewable biomass with abundant supply and environmental friendliness. It is the only resource that can be regenerated on a large scale and can completely replace fossil energy. Vigorously developing the utilization of lignocellulose raw materials in energy and other fields is to accelerate the development of circular economy. An important strategic task to ensure 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 achieve efficient hydrolysis and s...

Claims

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Application Information

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IPC IPC(8): C12N9/42C07K19/00C12P19/14C12P19/02
CPCC07K2319/00C12N9/2445C12P19/02C12P19/14C12Y302/01021
Inventor 崔球刘亚君张杰刘世岳冯银刚
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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