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Cellobiohydrolase mutant and application thereof

A cellobiose, hydrolase technology, applied in the directions of hydrolase, application, glycosylase, etc., can solve the problems of limited application, high cost, low efficiency of natural cellulose substrates, etc., to reduce enzyme dosage and improve conversion. The effect of efficiency

Active Publication Date: 2018-03-09
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the conversion of cellulose to biofuels remains challenging due to the low efficiency and high cost of cellulase enzymes in degrading natural cellulose substrates.
At the same time, due to the specificity of the substrate and the imbalance of the enzyme system of the cellulase fermentation broth, its application is also limited.

Method used

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  • Cellobiohydrolase mutant and application thereof
  • Cellobiohydrolase mutant and application thereof
  • Cellobiohydrolase mutant and application thereof

Examples

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Effect test

Embodiment 1

[0058] The wild-type cellobiohydrolase in this example is derived from Hypocrea jecorina, its amino acid sequence is shown in SEQ ID NO: 1, and its DNA molecular sequence is shown in SEQ ID NO: 2. Gene synthesis. The first and last 6 bases of SEQ ID NO: 2 and SEQ ID NO: 4 are restriction sites for NdeI (5') and XhoI (3').

[0059] The cellobiohydrolase mutant provided by the present invention is obtained by mutating the protein encoded by the DNA molecule shown in SEQ ID NO:2. The recombinant plasmid containing the cellobiohydrolase DNA sequence (SEQ ID NO: 2) was used as a template for mutation to obtain cellobiohydrolase mutants T389K (SEQ ID NO: 3), T389R (SEQ ID NO: 5), T389D (SEQ ID NO: 7), T389E (SEQ ID NO: 9) and T389Q (SEQ ID NO: 11).

[0060] The expression vector used in the above construction method refers to pET15, pET22 or pET28 and the like.

Embodiment 2

[0062] 1. Construction of pET28a(+)-bgl1: a recombinant vector encoding the gene (nucleotide sequence SEQ ID NO:2) of wild-type cellobiohydrolase (amino acid sequence SEQ ID NO:1)

[0063] After the wild-type gene sequence (SEQ ID NO: 2) and an expression vector pET28a of the pET series were digested with NheI and XhoI enzymes, the digested gene fragment and the pET vector were purified and recovered, and the gene fragment was ligated The linker pET28a(+)-bgl1 was obtained on the pET28a vector. Transform the linker pET28a(+)-bgl1 into Escherichia coli E.coli DH5α, verify whether it is a correct gene clone (identical to the nucleotide sequence SEQ ID NO:2) to the obtained transformant sequencing, and select the correct sequence containing After the corresponding bacterial strain E.coli BL21(DE3) / pET28a(+)-bgl1 was cultivated in large quantities, the plasmid was extracted to obtain a large amount of correct recombinant vectors of pET28a(+)-bgl1 (amino acid sequence SEQ ID NO:1, ...

Embodiment 3

[0079] Expression and protein purification of genetic engineering bacteria containing cellobiohydrolase mutation of the present invention

[0080]The cellobiohydrolase mutant (nucleotide sequence SEQ ID NO:3) was inoculated in 20 mL of liquid LB medium containing kanamycin at a volume ratio of 2%, and cultured overnight at 28°C. Inoculate the activated culture solution into 100mL LB liquid medium containing kanamycin, place it at 37°C and 250rpm and cultivate it until OD600=0.6 (with UNICOUV2102 ultraviolet-visible spectrophotometer, the LB medium for culture is Blank control); then add IPTG with a final concentration of 0.5mM for induction, and continue to cultivate for 10 hours at 28°C and 180rpm; collect the bacteria by centrifugation at 4°C and 8000g, and add 0.1 times the volume of bacterial liquid binding buffer (Binding buffer), 350W power, ultrasonic 40min under the condition of ice bath to disrupt the cells, centrifuge at 30000g to collect the supernatant to obtain th...

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Abstract

The invention discloses a cellobiohydrolase mutant. The activity of the cellobiohydrolase mutant provides a degradation function in cellulose degradation, an enzyme is a protein shown in a formula (a)or (b), theronine at a 389th site of an amino acid sequence shown in SEQ ID NO:1 of (a) is substituted by other amino acid, the other amino acid is lysine, arginine, aspartic acid, glutamic acid or glutamine, and an amino acid sequence of (b) in (a) is a protein which is derived by (a), is substituted, deleted or added with one or more amino acids and has cellobiohydrolase activity. The inventionalso discloses a composition, the composition contains one or several enzymes, and the enzymes are cellobiohydrolase mutants. The invention also discloses application of the cellobiohydrolase mutantor composition in cellulose hydrolysis. The cellobiohydrolase mutant disclosed by the invention has cellulose hydrolysis activity, and the mutant with the activity improved is obtained, so that the cellulose hydrolysis effect is improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a cellobiohydrolase mutant, a cellobiohydrolase mutant obtained by gene site-directed mutation, and a cellobiohydrolase mutant in cellulose degradation application. Background technique [0002] Due to the increasingly serious energy crisis, the renewable and environmentally friendly lignocellulosic saccharification process has attracted more and more attention. The use of cellulase to degrade lignocellulose into cellobiose, which is then converted into monosaccharides, and fermentative production of bio-based products including ethanol has important practical significance for social and economic development. However, the conversion of cellulose to biofuels remains challenging due to the low efficiency and high cost of cellulase degradation of natural cellulose substrates. At the same time, due to the specificity of the substrate and the imbalance of the enzyme system o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/70C12N1/21C12P19/14C12P19/02C12P19/12C12R1/19
CPCC12N9/2437C12N15/70C12P19/02C12P19/12C12P19/14C12Y302/01091
Inventor 蔡文生宗志友邵学广
Owner NANKAI UNIV
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