A new pectinase gene and its protein expression, carrier and application

A pectinase and gene technology, applied in application, genetic engineering, plant genetic improvement and other directions, can solve the problems of low expression and few reports, and achieve the effect of strong biological activity, cost reduction, and mass production.

Active Publication Date: 2022-05-17
HUAIHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are still few reports on the development of high-activity pectinases
In the previous research of the inventor, a new type of highly active pectinase derived from Poria cocos was found, but the expression level of the enzyme in Poria cocos was very low, so the new recombinant enzyme was efficiently expressed by using an exogenous gene expression system Protein is the only way to develop the pectinase

Method used

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  • A new pectinase gene and its protein expression, carrier and application
  • A new pectinase gene and its protein expression, carrier and application
  • A new pectinase gene and its protein expression, carrier and application

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Embodiment 2

[0040] This embodiment provides an optimized artificially synthesized new pectinase gene, 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 SEQ ID NO: 2 in the sequence listing shown. 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.

[0041] The formation of Poria cocos is that the...

Embodiment 3

[0054] The present embodiment provides a kind of method for preparing pectinase protein, specifically comprises the following steps:

[0055] S1: Expression and extraction of soluble pectinase protein: the Escherichia coli recombinant transformant containing the pET32 / pectinase vector containing the sequence SEQ ID NO: 1 gene was cultivated in liquid LB medium at 37°C until the OD600 was 0.6, and then Add IPTG with a concentration of 0, 0.1, and 0.5 mM 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, the gap is 8s, and after 90 cycles, the supernatant is obtained by centrifugation. Obtain the soluble fusion protein pectinase of reorganization, SDS-PAGE result is as follows image 3 As shown, there was no significant difference in the expression of the target protein when induced by IPTG with a final concentration of 0, 0.1, and 0.5 mM. In order to save costs,...

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Abstract

The present invention relates to a new pectinase gene, which is shown in the nucleotide sequence of SEQ ID NO.1 in the sequence table; or has more than 90% of the nucleotide sequence shown in SEQ ID NO.1 A sequence that is homologous and encodes the same biological function protein; or a sequence that can hybridize with the nucleotide sequence shown in SEQ ID NO.1 and encodes the same biological function protein. The nucleotide sequence of SEQ ID NO.1 in the present invention can use the pET32 vector and Escherichia coli expression strain to realize the fusion of the expression product and the solubilizing factor Trx, and obtain a large amount of Trx pectinase fusion protein in soluble form , and purified by nickel affinity chromatography and DEAE anion exchange method to obtain high-purity and high-activity recombinant pectinase protein, which provides the basis for mass production of recombinant pectinase.

Description

technical field [0001] The invention belongs to the technical field of biomolecular cloning, and relates to a new pectinase gene and its protein expression, carrier and application. Background technique [0002] Pectin is a water-soluble gel-forming substance extracted from carrots for the first time, which is mainly a straight-chain and neutral sugar (rhamnos A polymer compound composed of side chains formed from sugar, arabinose, galactose and xylose). Pectinase refers to the general term of various enzymes that can catalyze the decomposition of pectin, which widely exist in plant fruits. At present, the research on pectinase has been developed to the molecular level, from the screening and isolation in nature to the combination of mutagenesis and genetic engineering. The development of separation and purification methods has also promoted the research on the characteristics of pectinase. The rapid development of my country's fruit planting and fruit processing industry...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/60C12N15/56C12N15/55C12N9/88C12N9/26C12N9/18C12N15/70C12N1/21C12R1/19
CPCC12N9/88C12N9/2408C12N9/18C12Y301/01011C12Y302/01015C12Y402/02002C12Y302/01067C12N15/70
Inventor 李洪波岳芬芳米丹何伶靖李露露
Owner HUAIHUA UNIV
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