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Beta-glucosidase and its coding gene and application

A glucosidase and glucosidase encoding technology, which is applied to beta-glucosidase and its encoding gene and application fields, can solve the problem of low cellulase enzyme activity, high production cost, mismatch of cellulase action temperature and fermentation temperature, etc. question

Inactive Publication Date: 2008-01-09
GUANGXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Using lignocellulose as raw material and using cellulase to hydrolyze cellulose to produce ethanol, the earliest process route is Saccharification followed by fermentation (Separate Hydrolysis and Fermentation, SHF) process, first pretreating lignocellulose, and then using cellulase to hydrolyze the fiber The main disadvantage of this process is that in the presence of glucose produced by hydrolysis, β-glucosidase stops hydrolyzing cellobiose, and cellobiose accumulates leading to the termination of cellulose degradation
The main problems of this process are: the enzyme activity of cellulase is low and the production cost is high; the action temperature of cellulase does not match the fermentation temperature of common fermenting microorganism yeast

Method used

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  • Beta-glucosidase and its coding gene and application
  • Beta-glucosidase and its coding gene and application
  • Beta-glucosidase and its coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1. Obtaining β-glucosidase Umcel3G and its coding gene

[0052] 1. Construction of a metagenomic library of uncultured microorganisms in the rumen of buffalo

[0053]From a slaughterhouse in Nanning, fresh and relatively dry rumen contents of 5 buffaloes were collected and stored at -20°C in the laboratory for later use.

[0054] Weigh 10g of buffalo rumen contents from each sample and mix them together, suspend them in 100ml of 0.18M potassium phosphate buffer (pH 7.2), mix well, and put them in a Beckman Coulter Avanti JE centrifuge JA-10 rotor. Centrifuge the top with 2000g centrifugal force for 10 minutes, pour off the supernatant, and use 100ml extraction buffer (100mM sodium phosphate pH8.0; 100mM Tirs-HCl pH8.0; 100mM EDTA pH8.0; 1.5M NaCl; 1% CTAB; 2% SDS) was suspended, the sample was fully stirred with a mixer, and the sample was repeatedly frozen and thawed in liquid nitrogen and 65°C water bath three times, then lysozyme (20 mg / ml, dissolved in TE soluti...

Embodiment 2

[0063] Example 2. Location of β-glucosidase gene

[0064] The subcloning method was used to locate the β-glucosidase gene on pGLU64. Firstly, pGLU64 was digested with restriction enzyme XhoI, followed by self-ligation, transformation, activity detection, and plasmid analysis. The target gene was located on the 14kb XhoI fragment. Then it was digested with BamHI, and finally the target gene was located on the 5.0kb BamHI fragment, and the BamHI fragment was sent to Dalian Bao Bioengineering Company to perform two-way double-stranded sequencing of the gene using the dideoxynucleotide method.

[0065] The sequence was spliced ​​with the software DNAStar (DNASTAR company, version 5), and the software Blast (http: / / www.ncbi.gov) on NCBI (NationalCenter for Biotechnology Information, http: / / www.ncbi.nlm.nih.gov) was used. nlm.nih.gov / BLAST) to analyze the sequence to obtain the cellulose coding gene, which has the DNA sequence of sequence table 1, named umcel3G. In the sequence table, t...

Embodiment 3

[0067] Example 3. Expression of umcel3G in E. coli

[0068] 1. Construction of umcel3G gene expression plasmid

[0069]In order to express umcel3G in the pET system, all sequences of umcel3G gene except for the signal peptide and stop codon (ie from the 661-2811 positions of the 5'end of sequence 1) were artificially synthesized and inserted into the vector pET-30a(+) (purchased from The recombinant expression vector pET30-umcel3G was obtained between the KpnI and XhoI sites of Novagen. The start and stop codons are provided by the expression vector pET30a(+). There is a His tag (6×His Tag) provided by the expression vector at the N end of the expression product.

[0070] The pET30-umcel3G was transformed into the host E.coli EPI100. The plasmid of the transformant was extracted and analyzed with KpnI and XhoI. The results showed that the recombinant plasmid pET30-umcel3G was digested with KpnI and XhoI and released a 5.4kb vector band and a foreign insert of the expected size. Fi...

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Abstract

A beta-glucosidese, its encoding gene and use are disclosed. It consists of (a) or (b) protein, (a) protein is made of amino-acid sequence in sequence 3; (b) protein is derived by (a) and substituted and / or lost and / or added by one or several amino acid in sequence 3 and has beta-glucosidese. The process is carried out by: taking cellulose as material and synchronized mashing and common fermenting. It has better enzyme activity and can be used to produce alcohol.

Description

Technical field [0001] The present invention relates to a new beta-glucosidase from uncultured microorganisms in the rumen of buffalo and its coding gene and application. Background technique [0002] Cellulose is mainly the most abundant renewable biomass resource on the earth synthesized by plants using carbon dioxide and water under the action of solar energy through photosynthesis. Cellulose is a polymer composed of multiple glucose residues connected by β-1,4-glycoside chains, and its basic repeating unit is cellobiose. The basic structure of natural cellulose is made up of bundles of microfibrils composed of fibrils. Fibrils are composed of 15-40 long chains of cellulose molecules composed of crystalline and non-crystalline regions. The crystalline part of cellulose is formed by folding and arranging cellulose molecules very neatly. In natural cellulose, lignin and hemicellulose form a firm binding layer, which closely surrounds the cellulose. Cellulase is a general term fo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12N15/56C12N15/63C12N1/21C12P7/06C12S3/04
CPCY02E50/17Y02E50/10
Inventor 冯家勋郭鸿封毅莫新春段承杰唐纪良
Owner GUANGXI UNIV
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