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6-phosphoric acid-beta-glucosidase and coding gene and application thereof

A technology of glucosidase and phosphoric acid, applied in application, glycosylase, genetic engineering, etc., can solve problems such as less research

Active Publication Date: 2019-03-01
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few studies on 6-phospho-β-glucosidase

Method used

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  • 6-phosphoric acid-beta-glucosidase and coding gene and application thereof
  • 6-phosphoric acid-beta-glucosidase and coding gene and application thereof
  • 6-phosphoric acid-beta-glucosidase and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Amplification of the thermophilic Bacillus CCTCC No: M2011468 6-phospho-β-glucosidase gene pbgl

[0023] Genomic DNA of thermophilic Bacillus CCTCC No: M2011468 was prepared by conventional methods, and the process can be referred to the instructions of the Takara MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0 kit.

[0024] Design primers to introduce restriction enzyme cutting sites that can be inserted into the EcoRI and KpnI of the plasmid pETDuet-1. The primer sequences are as follows:

[0025] Upstream primer 5'-caccacagccaggatcc gaattc ggtgtgccatttgggacaaat-3' (underline represents EcoRI)

[0026] Downstream primer 5'-cggtttctttaccagactcgag ggtacc ctatgaaaaaatcaatt-3' (underline represents KpnI)

[0027] Using the extracted genome of thermophilic Bacillus CCTCC No: M2011468 as a template, the above primers were used to amplify to obtain the 6-phospho-β-glucosidase gene sequence.

Embodiment 2

[0028] Example 2: Alignment and analysis of the pbgl sequence of the thermophilic bacillus CCTCC No: M2011468 6-phospho-β-glucosidase gene pbgl

[0029] The product obtained by PCR amplification in Example 1 was purified using a Promega purification kit, and sent to a sequencing company for sequencing. Sequencing results showed that the nucleotide sequence of pbgl was 1095bp, as shown in sequence 1 in the sequence listing. The gene encodes a protein with a size of 364 amino acids, as shown in sequence 2 in the sequence listing, with a molecular weight of about 41.7 kDa and an isoelectric point of 6.342. Through NCBI Blast comparison analysis, the protein belongs to DUF871 superfamily (DUF, Domain of UnknownFunction, domain of unknown function), for example, the results are shown in the attached image 3 shown. Most of the reported 6-phospho-β-glucosidases belong to the GH1 or GH4 family of glycoside hydrolases, and their amino acid sequences were compared with the reported g...

Embodiment 3

[0030] Embodiment 3: Construction of the recombinant expression bacterial strain comprising 6-phosphate-beta-glucosidase gene pbgl

[0031] 1. Construction of recombinant plasmid comprising 6-phospho-β-glucosidase gene pbgl

[0032]The product obtained by PCR amplification in Example 1 was purified using a Promega purification kit. The pETDuet-1 plasmid was double digested with restriction endonucleases EcoRI and KpnI, and purified using the Promega purification kit. The above two purified products were ligated at 50°C for 15 minutes using the full gold pEASY-Uni Seamless Cloning and Assembly Kit, and the ligated products were heat-shocked to transform Escherichia coli Trans-T1 competent cells, and coated with ampicillin-resistant plates to screen for positive recombinants strains, and use the universal primers pETUpstream Primer and T7TerminatorPrimer suitable for pETDuet-1 to sequence to determine the positive recombinant strains.

[0033] 2. Construction of a recombinant ...

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Abstract

The invention discloses a 6-phosphoric acid-beta-glucosidase Pbgl and a coding gene and application thereof. The amino acid sequence of the 6-phosphoric acid-beta-glucosidase is shown in SEQ ID NO.2,or, amino acid residue in the amino acid sequence shown in the SEQ ID NO.2 is subjected to any one or more modification of substitution, deletion and addition to obtain amino acid sequence with 6-phosphoric acid-beta-glucosidase activity. According to the 6-phosphoric acid-beta-glucosidase Pbgl and the coding gene and application thereof, clone is carried out on bacillus thermophilus CCTCC No: M2011468 to obtain a new 6-phosphoric acid-beta-glucosidase gene pbgl, the new 6-phosphoric acid-beta-glucosidase gene pbgl can be expressed in host cells to produce the 6-phosphoric acid-beta-glucosidase for degradation of cellulose.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a 6-phospho-β-glucosidase and its coding gene and application. Background technique [0002] In order to solve the global energy crisis and environmental degradation that mankind is currently facing, the development of science and technology has gradually turned to the research, development and utilization of renewable biomass resources. Lignocellulose is the most abundant renewable biomass resource on earth synthesized by plants using carbon dioxide and water through photosynthesis under the action of sunlight. Lignocellulose is mainly composed of polysaccharides such as cellulose or hemicellulose and lignin. The polysaccharides are hydrolyzed into small molecular sugars under the action of cellulase, hemicellulase, and glycosidase, which can be utilized by microorganisms to realize the production of biofuels or bio-based chemicals. [0003] Cellulose is a polymer of glucose linke...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/70C12N1/21C12P19/14C12P19/02
CPCC12N9/2434C12N15/70C12P19/02C12P19/14C12Y302/01086
Inventor 欧阳嘉邹丽花郑兆娟欧阳水平徐海军
Owner NANJING FORESTRY UNIV
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