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Method for constructing exoglucanase Cel6A and Cel7A in heterologous expression in pichia pastoris

A technology of exoglucanase and Pichia pastoris, which is applied in the field of genetic engineering, can solve the problems of low efficiency and lack of enzymatic hydrolysis of cellulase system, and achieve the potential of improving industrial production, increasing the success rate and reducing errors. Effect

Inactive Publication Date: 2019-05-24
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] As far as Trichoderma reesei, the most widely used cellulase-producing fungus, lacks exonuclease and monoglucosidase in its cellulase system, resulting in low enzymatic hydrolysis efficiency of its cellulase system

Method used

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  • Method for constructing exoglucanase Cel6A and Cel7A in heterologous expression in pichia pastoris
  • Method for constructing exoglucanase Cel6A and Cel7A in heterologous expression in pichia pastoris
  • Method for constructing exoglucanase Cel6A and Cel7A in heterologous expression in pichia pastoris

Examples

Experimental program
Comparison scheme
Effect test

Embodiment example 1

[0028] Example 1 Cloning of the target gene

[0029] 1. Take the Trichoderma reesei QM9414 spore suspension stored in a glycerol tube out of the -80°C refrigerator and place it in ice (or 4°C refrigerator) to thaw until the spore suspension is completely thawed. The spore suspension was shaken and mixed evenly, a small amount of bacterial liquid was dipped in the inoculation ring, and streaked on the PDA medium plate for inoculation. After sealing the plate with parafilm, it was cultured in a 28°C incubator. Culture for about 5-7 days until the surface of the medium is covered with green spores. Remove the plate from the incubator and place it in a 4°C refrigerator for later use.

[0030] 2. Inoculate the fresh spores on the cellulase solid induction culture, cover the surface with sterilized cellophane, and let it stand for culture until the mycelium grows out. The hyphae were scraped from the cellophane and treated with a liquid nitrogen freezer grinder. The UNIQ-10 colum...

Embodiment example 2

[0032] Example 2 Construction of Ppic9k-Cel6A, Cel7A expression vector

[0033] 1. First put the Buffer in ice, and after it is fully melted and mixed evenly, prepare a double-enzyme digestion reaction system. After the preparation is completed, mix it evenly, place it in a 37°C water bath, and perform enzyme digestion for 30 minutes. After the end of the digestion, use a purification and recovery kit to recover the digestion product to remove the residual enzyme and oligonucleotide in the digestion product, so as not to affect the subsequent connection of the plasmid and the target gene.

[0034] 2. Using the 5'-end EcoRI as the restriction site and the 3'-end Not I as the restriction site, insert it into the downstream of the promoter AOX1 of the Ppic9k plasmid vector to construct the expression vector Ppic9k-Cel6A, Cel7A, see the expression vector map image 3 . Colony PCR validation see figure 2 . The expression vector was verified by single enzyme digestion to confirm...

Embodiment example 3

[0035] Example 3 Linearization of expression vectors Ppic9k-Cel6A, Cel7A and electrotransformation

[0036] 1. Take SaII as the linearization site of the Ppic9k-Cel6A and Cel7A expression vectors to realize the linearization of the expression vectors, and verify the linearized plasmids by nucleic acid electrophoresis, see Figure 4 . Transformed into Pichia pastoris GS115 by electroporation, and plated to obtain transformants.

[0037] 2. Use SDS pyrolysis method to crudely extract the genome of the recombinant yeast transformant obtained from 1, and verify its correctness and phenotype by PCR. See the genome PCR nucleic acid map. Figure 5 .Use antibiotic concentration gradient to screen multi-copy transformants, and screen out the colonies that can grow normally on high-concentration resistant plates.

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Abstract

The invention aims to construct recombinant pichia pastoris with heterologous expression of exoglucanase Cel6A and Cel7A. A method includes steps: (1) trichoderma reesei RNA and CDNA acquisition, to be more specific, adopting a solid induction medium for acquiring mycelia, and adopting a kit for extracting total RNA and cDNA; (2) target gene cloning, to be more specific, adopting a reasonable primer for PCR amplification to obtain a target gene; (3) expression vector construction, to be more specific, taking Ppic9k as a carrier, inserting the target gene into a downstream position of a promoter AOX1, wherein 5' and 3' terminated enzyme cutting sites are EcoRI and NotI respectively; (4) recombinant strain acquisition, to be more specific, taking SalI as a linearization site to realize expression vector linearization, and electrically converting to obtain recombinant yeast; (5) induced enzyme production of a recombinant strain, namely shaking for fermentation, and adopting appropriate methanol for inducing enzyme production; (6) recombinant protein activity detection, namely detecting recombinant protein expression quantity and activity through SDS-PAGE and Congo red-CMC. The recombinant strain constructed by the method is capable of efficiently expressing proteins with his tags, and an efficient single enzyme component can be obtained.

Description

technical field [0001] The invention relates to the construction of a recombinant Pichia pastoris system for heterologously expressing exoglucanases Cel6A and Cel7A, and belongs to the technical field of genetic engineering. Background technique [0002] Lignocellulosic biomass is a cheap and abundant renewable resource on earth, and it comes from a wide range of sources, including common crop straws, tree branches and leaves. If the efficient degradation of lignocellulose can be achieved and high value-added products can be produced through biotransformation, it will reduce the waste of such resources to a certain extent, which has positive significance for the sustainable development of human society. The degradation efficiency of traditional cellulases is low. Traditional cellulases consist of 3 classes, namely beta-glucosidases, endoglucanases and exoglucanases. The three types of enzymes cooperate with each other in the process of cellulose hydrolysis. However, only ...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N15/81C12N9/42C12R1/84
Inventor 钟成舒月力李栋梁
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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