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Novel Trx-beta-glucosidase gene and protein expression, carrier and application thereof

A glucosidase and gene technology, applied in the field of biomolecular cloning, can solve problems such as high production costs and limitations in the application of cellulase, and achieve strong biological activity

Active Publication Date: 2019-02-12
HUNAN BUTIAN PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the high production cost of cellulase currently limits the application of cellulase.

Method used

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  • Novel Trx-beta-glucosidase gene and protein expression, carrier and application thereof
  • Novel Trx-beta-glucosidase gene and protein expression, carrier and application thereof
  • Novel Trx-beta-glucosidase gene and protein expression, carrier and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0071] This embodiment provides an optimized artificially synthesized new β-glucosidase gene β-Glucosidase, the specific sequence is shown in SEQ ID NO.1 in the sequence listing, and the protein sequence corresponding to the gene is shown in the sequence listing Shown in SEQ ID NO.2. The sequence provided by the present invention has no sequence with a similarity of 60% in the NCBI database. It is based on the characteristics of Escherichia coli expression such as codon bias, preventing complex DNA structures from affecting transcription efficiency, ensuring reasonable GC content, Select a DNA sequence among many sequences that are artificially optimized and synthesized with the characteristics of suitable restriction site, ideal expression tag and termination signal. The sequence described in the present invention and the highly homologous DNA sequence have higher expression of soluble target protein in Escherichia coli than other sequences.

[0072] The formation of Poria c...

Embodiment 2

[0085] This embodiment provides a method for preparing Trx-β-glucosidase protein, which specifically includes the following steps:

[0086] S1: Expression and extraction of soluble Trx-β-glucosidase protein: the Escherichia coli recombinant transformant of the pET32 / β-Glucosidase vector containing the sequence SEQ ID NO.1 gene was cultivated to OD600 in liquid LB medium at 37°C Then add IPTG with a concentration of 0, 0.1, and 0.5mM, respectively, and induce at 18°C ​​for 10 hours. After the induction, the collected bacteria are ultrasonically crushed, the crushing power is 300W, the crushing is 2s, and the gap is 8s. After 90 cycles, centrifuge and take The supernatant obtained the recombinant soluble fusion protein Trx-β-glucosidase, and the SDS-PAGE results were as follows figure 2 shown.

[0087] S2: Purification of Trx-β-glucosidase protein: After expanding culture and inducing with 0.1mM IPTG for 10 hours at 18°C, collect the expressing bacterial cells induced by IPTG,...

Embodiment 3

[0095] In this embodiment, the enzyme activity of purified Trx-β-glucosidase is detected, and the specific steps and results are as follows:

[0096] Using p-nitrophenyl-β-D-glucopyranoside as the substrate and p-nitrophenol as the standard, the enzyme activity of the purified Trx-β-glucosidase was detected.

[0097] (1) Drawing of standard curve: take 5 μmol / L p-nitrobenzene, dilute it with 20mM sodium dihydrogen phosphate solution of pH 6.0 to 400, 200, 100, 50, 25, 12.5 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 400nm, and measure the p-nitro group at each dilution concentration. Absorbance value of benzene, and draw a standard curve, the resulting standard curve equation is: Y=0.0011X+0.0021, the correlation system r=0.9995, the test results of the standard are shown in Tab...

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Abstract

The invention relates to a novel Trx-beta-glucosidase gene. The gene is shown as a nucleotide sequence of SEQ ID NO.1 in a sequence table, or a sequence which has more than 90 percent of homology as the nucleotide sequence shown as SEQ ID NO.1 and codes the identical biological functional protein, or a sequence which can be hybridized with the nucleotide sequence shown as SEQ ID NO.1 and codes theidentical biological functional protein. For the nucleotide sequence such as SEQ ID NO.1 in the sequence table, the strain can be expressed by pET32 carriers and escherichia coli; the fusion of expression products and solubilizing factors Trx is realized; a great amount of soluble form of Trx-beta-Glucosidase fusion protein can be obtained; through a simple affinity chromatography purification method, the high-purity and high-activity recombinant Trx-beta-glucosidase can be obtained; the basis is provided for mass industrialized production of beta-glucosidase.

Description

technical field [0001] The invention belongs to the technical field of biomolecular cloning, and relates to a new Trx-beta-glucosidase gene and its protein expression, carrier and application. Background technique [0002] Cellulose is a glucan macromolecular chain composed of D-glucose through β-1,4-glycosidic bonds. As an important component of plant cell walls, it is the most abundant, cheapest and underutilized in the world. Renewable resources. Enzymatic saccharification of cellulosic biomass such as agricultural and forestry waste and some urban industrial waste to produce ethanol and other bio-based products is of great significance for solving problems such as food shortage, energy crisis and environmental pollution. The biorefinery of cellulose resources needs to go through three basic steps: crushing, pretreatment, and enzymatic hydrolysis. Improving the hydrolysis efficiency of cellulase on cellulose raw materials is one of the key technologies to reduce costs an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/70C12N1/21
CPCC12N9/2445C12N15/70C12Y302/01021
Inventor 李洪波米丹张赛名胡兴
Owner HUNAN BUTIAN PHARMA
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