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f85-20 protein and its coding gene and its application as β-glucosidase

A technology of glucosidase and protein, applied in application, glycosylase, genetic engineering, etc., can solve problems that limit the efficient development of the biofuel industry

Active Publication Date: 2017-04-05
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This limits the efficient development of the biofuel industry and is also an important factor restricting the reduction of production costs of biofuels
In order to achieve higher heat resistance and thermal stability of enzymes, many scholars screen new enzymes from thermophilic microorganisms, but they are rarely applicable in industrial conditions

Method used

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  • f85-20 protein and its coding gene and its application as β-glucosidase
  • f85-20 protein and its coding gene and its application as β-glucosidase
  • f85-20 protein and its coding gene and its application as β-glucosidase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1. Cloning of F85-20 gene

[0041] In the previous research, the Fosmid vector was used to construct the metagenomic library of the dry fermentation sludge system. The ORF is predicted by sequencing and softberry, as well as the NCBI and Pfam database annotations. The F85-20 gene is obtained. The nucleotide sequence of the gene is the 19th to 1404th nucleotides from the 5'end of sequence 1 in the sequence list, and the encoded protein is named F85-20, and its amino acid sequence is the sequence in the sequence list 2 From the N'-terminal amino acids 1-462. After comparison, the F85-20 protein has the highest 60% similarity with β-glucosidase from bacterium UASB270, which is predicted to be β-glucosidase.

Embodiment 2

[0042] Example 2. Application of F85-20 as β-glucosidase

[0043] 1. Construction of recombinant vector

[0044] Using artificially synthesized sequence 1 as a template, using F85-20F and F85-20R as primers, and using TAKARA's PrimeStar high-fidelity enzyme for PCR amplification, a 1413 bp PCR product was obtained, and its nucleotide sequence was sequence 1.

[0045] F85-20F: 5’-CATG CCATGG AGATACAAATGAATAAACAT-3’ contains Nco I restriction site

[0046] F85-20R:5’-CCC AAGCTT GGCATAAAGGGCCTCC-3’ contains Hind III restriction site

[0047] The above PCR amplification system is as follows:

[0048]

[0049]

[0050] The PCR program is as follows:

[0051] 94℃ 1min pre-denaturation

[0052] 98℃ 10s

[0053] 68℃ 90s

[0054] Back to 2, 24 cycles

[0055] 72℃ 5min

[0056] End

[0057] The PCR product was double digested with Nco I and Hind III, and the digested product was ligated with the pET-28a vector (Novagen pET-28a DNA Cat. No. 69864-3) that had undergone the same digestion to obtain a rec...

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Abstract

The invention discloses an F85-20 protein, a coding gene thereof and an application of the F85-20 protein as beta-glucosidase. The invention provides the protein shown in the following 1) or 2): 1) a protein formed by amino acid residues shown in a sequence 2 in a sequence table; 2) a protein which is obtained by carrying out substitution and / or deletion and / or addition of one or more amino acid residues on an amino acid residue sequence of the sequence 2 in the sequence table, has the same functions as the protein shown in 1) and is derived from the protein shown in 1). Experiments prove that new beta-glucosidase F85-20 can still retain 80% of activity after being incubated at 50 DEG C for two hours under the conditions that p-nitrophenol glucoside (pNPG) is taken as the substrate and the pH value is 6.0, and the optimum temperature is 65 DEG C; new beta-glucosidase F85-20 can retain more than 70% of residual activity in a pH value range of 4.0-7.5 after being incubated in buffer solutions with different pH values for 24 hours at 4 DEG C.

Description

Technical field [0001] The present invention relates to the field of biotechnology, in particular to a F85-20 protein and its coding gene and its application as a β-glucosidase. Background technique [0002] According to statistics, every year, plants in the world synthesize 5 billion tons of fibers through photosynthesis. Cellulose is an important component of plant cell walls and the most abundant renewable resource in nature. Cellulase is a type of enzyme that can hydrolyze cellulose into oligoglucose and glucose. Many natural microorganisms contain cellulase. Because cellulase can be widely used in textile, paper, food and biofuel industries, it has attracted more and more scholars' research interest in recent years. However, in the process of cellulase hydrolysis, a large amount of cellobiose and oligosaccharides produced will strongly inhibit the activity of cellulase, resulting in a sharp drop in the efficiency of cellulose hydrolysis and a reduction in overall productio...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/70C12N1/21
CPCC12N9/2445C12Y302/01021
Inventor 吴晓磊王蒙来国莉聂勇耿爽
Owner PEKING UNIV
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