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Beta-glucosaccharase, beta-glucosaccharase mutant and application

A technology of glucosidase and recombinant carrier, applied to mutants of β-glucosidase, in the application field of cellulose degradation, can solve the problems of low efficiency, high cost, limited application of natural cellulose substrates, etc. The effect of improving the overall hydrolysis rate, high expression, and improving transformation efficiency

Active Publication Date: 2015-04-29
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
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Effect test

Embodiment 1

[0044] 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:5.

Embodiment 2

[0046] Using the molecular simulation software Discovery studio, the β-glucosidase of Penicillium piceum H16 and the β-glucosidase of Aspergillus niger CGMCC 3.316 with excellent thermostability were modeled to construct a 3D model . Use the following methods to select mutation sites a. Compare the 3D structures of the β-glucosidases of Penicillium juniperus and Aspergillus niger CGMCC3.316 to observe the difference sites; b. Use Discovery studio to perform virtual mutations and calculate mutations Able to judge the feasibility of the mutation scheme. 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: 5) 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.

[0047] The expression vector mentioned...

Embodiment 3

[0068] Expression and protein purification of β-glucosidase gene and β-glucosidase mutant gene engineering bacteria of the present invention

[0069] The preserved engineering strain E.coli BL21(DE3) / pET28a(+)-bgl1 (amino acid sequence SEQ ID NO:1, nucleotide sequence SEQ ID NO:5), E.coli BL21(DE3) / pET28a(+ )-bgl1 / Q341W (amino acid sequence SEQ ID NO:2, nucleotide sequence SEQ ID NO:6), E.coli BL21(DE3) / pET28a(+)-bgl1 / N335W (amino acid sequence SEQ ID NO:3, Nucleotide sequence SEQ ID NO:7), and E.coli BL21(DE3) / pET28a(+)-bgl1 / S336F (amino acid sequence SEQ ID NO:4, nucleotide sequence SEQ ID NO:8) etc. glycerol Bacteria were 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 a UNICO UV2102 ultraviolet-visible spectrophotometer, LB medium for culture The...

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Abstract

The invention discloses a composition, containing an enzyme, wherein the enzyme has the activity of glucosaccharase and provides a degradation function in cellulose degradation, and the enzyme is the following protein (a) or (b): (a) protein with the amino acid sequence as shown in SEQ ID No: 1, (b) protein with one or more amino acids substituted, lost or added in the amino acid sequence in (a), having the activity of glucosaccharase and derived from (a). The invention also discloses a DNA molecule for encoding the enzyme, a recombinant vector containing the DNA molecule and a regulation sequence connected with the DNA molecule and used for expressing, and a host cell containing the DNA molecule or the recombinant vector. According to the beta-glucosaccharase mutant having the amino acid sequences as shown in SEQ ID No: 2, 3 and 4 provided by the invention, compared with wild beta-glucosaccharase, the thermostability is improved, and the integral hydrolysis rate of compound enzyme liquid in enzyme system compound can be improved.

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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IPC IPC(8): C12N9/42C12N15/56C12N15/10C12N15/70C12N1/21C12R1/80
CPCC12N9/2445C12Y302/01021
Inventor 宗志友张东远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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