A gene encoding β-glucosidase and its expression vector and protein

A technology of glucosidase and gene, which is applied in the field of coding β-glucosidase gene and its expression vector and protein, can solve the problems of low yield, impure culture of microorganisms, limit the development and utilization of microbial resources, etc., and achieve strong biological activity Effect

Active Publication Date: 2020-08-04
HUNAN BUTIAN PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in nature, more than 99% of microorganisms cannot be purely cultured, which seriously limits the development and utilization of microbial resources.
And the microorganisms that produce β-glucosidase mainly include Aspergillus, Trichoderma, yeast and bacteria, but the yield is low, and it is not easy to produce on a large scale.
At present, most of the studies on cellulase-producing strains focus on Trichoderma strains with complete cellulase systems and high enzyme activity, such as Trichoderma viride and Trichoderma reesei. Mycotoxins

Method used

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  • A gene encoding β-glucosidase and its expression vector and protein
  • A gene encoding β-glucosidase and its expression vector and protein
  • A gene encoding β-glucosidase and its expression vector and protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] This embodiment provides an optimized artificially synthesized β-glucosidase gene, the specific sequence of which is shown in SEQ ID NO: 1 in the sequence listing, and the protein sequence corresponding to the gene is shown in SEQ ID NO in the sequence listing: 2. The synthetic sequence has no sequence similarity of 50% in the NCBI database. It is based on the characteristics of E. coli expression such as codon bias, avoiding complex DNA structures, ensuring reasonable GC content, and suitable enzyme cutting sites It is one of the many sequences synthesized with characteristics such as expression tags and termination signals. The sequences described in the present invention and DNA sequences highly homologous to this sequence have higher soluble target protein activity in Escherichia coli than other sequences. Express.

[0068] The above-mentioned optimized gene shown in sequence SEQ ID NO: 1 was constructed into the Escherichia coli expression vector pET28 to obtain t...

Embodiment 2

[0079] This embodiment provides a method for preparing β-glucosidase protein, which specifically includes the following steps:

[0080] S1: Transform the recombinant vector pET28 / GH1 constructed in Example 1 into Escherichia coli TOP10 strain, and then extract the recombinant vector pET28 / GH1 from TOP10; transfer the recombinant vector pET28 / GH1 into the host cell Escherichia coli by heat shock method Among the expression strains, the transformant of the expression strain of Escherichia coli containing the recombinant vector pET28 / GH1 was obtained by screening with an LB plate containing Kan resistance.

[0081] S2: Expression and extraction of soluble β-glucosidase: the Escherichia coli recombinant transformants of the pET28 / GH1 recombinant vector containing the sequence SEQ ID NO: 1 were cultured in liquid LB medium at 37°C until the OD600 was 0.3, and then Add IPTG with a concentration of 0, 0.1, and 0.5 mM respectively, and induce at 18°C ​​for 12 hours. After the inductio...

Embodiment 3

[0091] In this embodiment, the enzyme activity of the purified soluble β-glucosidase is detected, and the specific steps and results are as follows:

[0092] Using p-nitrophenyl-β-D-glucopyranoside as a substrate and p-nitrophenol as a standard product, the enzyme activity of the β-glucosidase purified and concentrated in step S4 of Example 2 was detected.

[0093] (1) Drawing of standard curve: take 5 μmol / L p-nitrobenzene, dilute it with 20mM sodium dihydrogen phosphate solution of pH 6.0 to 800, 400, 200, 100, 50, 25 and 0nmol / L respectively . Take 100 μl of each of the above dilutions, add them to 96-well microplates, repeat each concentration three times, place them in a full-wavelength microplate reader at room temperature, select the absorbance value at 400 nm, and measure the p-nitro group at each dilution concentration. The absorbance value of benzene, and draw a standard curve, the resulting standard curve equation is: Y=0.0011X+0.0024, the correlation system r=0.99...

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Abstract

The invention relates to a gene encoding beta-glucosidase, at least containing a DNA sheet containing one of that following nucleotide sequence: 1) a nucleotide sequence of SEQ ID NO. 1 in a sequencetable; 2) a nucleotide sequence have more than 90% homology with that nucleotide sequence shown in SEQ ID NO. 1 and encoding the same biological function protein; 3) a nucleotide sequence hybridize tothat nucleotide sequence shown in SEQ ID NO. 1 and encoding a protein of the same biological function. The recombinant vector further constructed according to the gene sequence of the invention can realize beta-glucosidase soluble expression, and simple affinity purification, can be obtained more than 95% purity and higher concentration of the active protein to maintain natural conformation. Thisis conducive to the industrial production of beta-Glucosidase.

Description

technical field [0001] The invention belongs to the technical field of biomolecular cloning, and relates to a gene encoding beta-glucosidase, an expression carrier and protein thereof. Background technique [0002] β-glucosidase (β-D-Glucosidase, EC3.2.1.21), also known as β-D-glucoside glucohydrolase, alias gentiobiase, cellobiase (cellobias, CB or β-G ) and amygdalinase. It belongs to cellulase and is an important component of cellulolytic enzyme system. It can hydrolyze the non-reducing β-D-glucose bond bound to the terminal and release β-D-glucose and corresponding ligands at the same time. β-glucosidase also has transglycosidase activity, the purpose is to synthesize functional oligoglucans, oligomeric maltooligosaccharides, oligomeric cellulooligosaccharides, etc. through the glucosidase with transglycosidase activity, which can be used as functional prebiotics carbohydrate. Therefore, the protein has important application value in food, feeding and health products ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/56C12N9/42C12N15/70C12N1/21C12P7/06C12R1/19
CPCC12N9/2445C12N15/70C12P7/06C12P7/065C12Y302/01021Y02E50/10
Inventor 李洪波吴贤文董海丽胡兴
Owner HUNAN BUTIAN PHARMA
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