Beta-glucosaccharase, beta-glucosaccharase mutant and application

A technology of glucosidase and recombinant carrier, applied to the mutant of β-glucosidase, in the application field of cellulose degradation, can solve the problems of low efficiency, high cost and limited application of natural cellulose substrates, and achieve Improve the overall hydrolysis rate, good effect, reduce the effect of enzyme dosage

Active Publication Date: 2015-04-22
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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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 limited.

Method used

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  • Beta-glucosaccharase, beta-glucosaccharase mutant and application
  • Beta-glucosaccharase, beta-glucosaccharase mutant and application
  • Beta-glucosaccharase, beta-glucosaccharase mutant and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Firstly, the entire genome of Penicillium piceum H16 was sequenced, and the library was constructed with the β-glucosidase protein sequences produced by all fungal genera through bioinformatics methods, and blastx was used to search the whole genome of Penicillium piceum. and classified to obtain the sequences of multiple extracellularly secreted β-glucosidases, such as Figure 1(a) and 1(b) As shown, the reverse transcription PCR (RT-PCR) method was used to analyze the expression levels of these extracellular secreted proteins, and it was found that one of the genes, G8221, had the highest amount, so that the target sequence was the cell with the highest expression level in Penicillium juniper H16. Exocrine β-glucosidase, its amino acid sequence is shown in SEQ ID NO:1, and its DNA molecular sequence is shown in SEQ ID NO:8.

Embodiment 2

[0050] Using the molecular simulation software Discovery studio, the β-glucosidase of Penicillium piceum H16 and the β-glucosidase of Aspergillus niger CGMCC3.316 with excellent thermal stability were modeled to construct a 3D Model. Use the following method to select the mutation site: a. mutate the glycine closest to the loop region of β-glucosidase into proline; 3D structure comparison to observe the difference sites. Mutation primers were designed according to the above mutation scheme, and the recombinant plasmid containing the wild-type β-glucosidase DNA sequence (SEQ ID NO: 8) was used as a template for mutation. Using molecular simulation software to rationally design β-glucosidase and select mutation sites can effectively save the time for mutation site screening and improve mutation efficiency.

[0051] The expression vector mentioned in the above construction method refers to pET15, pET22 or pET28 and the like.

[0052] 1. Construction of pET28a(+)-bgl1: a recomb...

Embodiment 3

[0079] Expression and protein purification of β-glucosidase gene and β-glucosidase mutant gene engineering bacteria of the present invention

[0080]The preserved engineering strain E.coli BL21(DE3) / pET28a(+)-bgl1 (amino acid sequence SEQ ID NO:1, nucleotide sequence SEQ ID NO:8), E.coli BL21(DE3) / pET28a(+ )-bgl1 / Q325R (amino acid sequence SEQ ID NO:2, nucleotide sequence SEQ ID NO:9), E.coli BL21(DE3) / pET28a(+)-bgl1 / T444K (amino acid sequence SEQ ID NO:3, Nucleotide sequence SEQ ID NO:10), E.coli BL21(DE3) / pET28a(+)-bgl1 / D618Q (amino acid sequence SEQ ID NO:4, nucleotide sequence SEQ ID NO:11), E.coli BL21(DE3) / pET28a(+)-bgl1 / E629Q (amino acid sequence SEQ ID NO:5, nucleotide sequence SEQ ID NO:12), E.coli BL21(DE3) / pET28a(+)-bgl1 / G100F amino acid Sequence SEQ ID NO:6, nucleotide sequence SEQ ID NO:13), E.coli BL21(DE3) / pET28a(+)-bgl1 / G172F amino acid sequence SEQ ID NO:7, nucleotide sequence SEQ ID NO: The glycerol bacteria of 14) were inoculated in 20 mL liquid LB medium ...

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Abstract

The invention discloses a composition which comprises an enzyme. The enzyme has the activity of glucosaccharase, and provides a degrading function in cellulose degradation. The enzyme is a protein shown in (a) or (b), wherein (a), the amino acid sequence of the is shown as SEQ ID NO:1; (b), one or several amino acid are displaced, removed out or added into the amino acid sequence in the (a), and the protein is derived from the (a) and has the activity of the glucosaccharase. DNA molecules of the enzyme are coded. Carriers are recombined and comprise the DNA molecules and adjusting sequences connected with the DNA molecules and used for conducting expressions. Host cells comprise the DNA molecules or the recombined carriers. Compared with the beta-glucosaccharase (with the amino acid sequence of SEQ ID NO:1), a beta-glucosaccharase mutant with the amino acid sequences of SEQ ID NO:6 and SEQ ID NO:7 has the beneficial effects that the heat stability is improved; and the hydrolysis rate of compound enzyme liquid can be improved in enzyme system compounding.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to a kind of β-glucosidase, and a mutant of β-glucosidase and some mutants of β-glucosidase with improved heat resistance obtained by gene site-directed mutation, and Use of β-glucosidase and β-glucosidase mutants in cellulose degradation. 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 to produce glucose, and then to ferment and produce 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 imbala...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/70C12N1/21C12R1/19
CPCC12N9/2445C12Y302/01021
Inventor 张东远宗志友高乐王国坤李晨崔超陈树林
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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