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Method for constructing heterologous expression endoglucanases EG II, EG IV and EG V in pichia pastoris

A technology of endoglucanase and Pichia pastoris, which is applied in the field of genetic engineering, can solve the problems of low enzymatic hydrolysis efficiency and lack of cellulase system, so as to improve industrial production potential, improve expression efficiency, and induce good effect Effect

Inactive Publication Date: 2019-07-05
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 heterologous expression endoglucanases EG II, EG IV and EG V in pichia pastoris
  • Method for constructing heterologous expression endoglucanases EG II, EG IV and EG V in pichia pastoris
  • Method for constructing heterologous expression endoglucanases EG II, EG IV and EG V in pichia pastoris

Examples

Experimental program
Comparison scheme
Effect test

Embodiment example 1

[0026] Implementation Case 1 Cloning of target gene

[0027] 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 completely melts. Shake the spore suspension to mix evenly, dip the inoculation loop with a small amount of bacterial solution, and streak on the PDA medium plate for inoculation. After the plate was sealed with parafilm, it was placed in a 28°C incubator for cultivation. Cultivate for about 5-7 days until the surface of the medium is covered with green spores. The plates were taken out of the incubator and placed in a 4°C refrigerator for later use.

[0028] 2. Inoculate fresh spores on cellulase solid induction culture, spread sterilized cellophane on the surface, and let the culture stand until the hyphae grow out. The hyphae were scraped off from the cellophane and treated with a liquid nitrogen freezer grinder. RNA was extra...

Embodiment example 2

[0030] Implementation case 2 Construction of Ppic9k-EGⅡ, EGⅣ, EGⅤ expression vectors

[0031] 1. Put the Buffer in ice first, after it is fully melted and mixed evenly, prepare a double enzyme digestion reaction system, mix evenly after the preparation is complete, put it in a 37°C water bath, and digest for 1 hour. After digestion, use a purification recovery kit to recover the digested product to remove residual enzymes and oligonucleotides in the digested product, so as not to affect the subsequent connection of the plasmid and the target gene.

[0032] 2. With EcoRI at the 5' end and NotI at the 3' end as the restriction site, insert the downstream of the Ppic9k plasmid vector promoter AOX1 to construct the expression vector Ppic9k-EGⅡ, EGⅣ, EGⅤ. See the expression vector map image 3 . Colony PCR verification see figure 2 . The expression vectors were verified by single enzyme digestion to confirm that the expression vectors Ppic9k-EGⅡ, EGⅣ and EGⅤ were successfully c...

Embodiment example 3

[0033] Implementation Case 3 Linearized expression vectors Ppic9k-EGⅡ, EGⅣ, EGⅤ and performed electrotransformation

[0034] 1. Use bglⅠ as the linearization site of the Ppic9k-EGⅡ, EGⅣ, EGⅤ expression vector to realize the linearization of the expression vector, and verify the linearized plasmid by nucleic acid electrophoresis, see Figure 4 . Transformed into Pichia pastoris GS115 by electrotransformation, and plated to obtain transformants.

[0035] 2. Use the SDS pyrolysis method to roughly extract the genome of the recombinant yeast transformant obtained in 1, and verify its correctness and phenotype by PCR. The nucleic acid map of the genome PCR is shown in Figure 5 .Use the antibiotic concentration gradient to screen multi-copy transformants, and screen out the colonies that can grow normally on high-concentration resistance plates.

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Abstract

The invention aims to construct recombinant pichia pastoris for heterologous expression of EG II, EG IV and EG V proteins. A method comprises the following specific steps: (1) obtaining of trichodermareesei RNA and CDNA: obtaining mycelia by using a solid induction culture medium, and extracting total RNA and cDNA by using a kit; (2) cloning of a target gene: amplifying a target gene by a reasonable primer PCR; (3) constructing an expression vector: taking Ppic9k as a vector, inserting the target gene into the downstream of a promoter AOX1, wherein the 5 ' and 3 ' end enzyme digestion sites are EcoR I and Not I respectively; (4) obtaining of a recombinant strain: taking bgl I as a linearization site of an expression vector to realize linearization and electrotransformation of the expression vector to obtain recombinant yeast; (5) induction and enzyme production of the recombinant strain: carrying out shaking flask fermentation, and inducing enzyme production by using appropriate methanol; and (6) recombinant protein activity detection: detecting the expression and activity of recombinant protein by SDS-PAGE and Congo red-CMC. The recombinant strain constructed by the method can efficiently express the protein with a his label, and a high-efficiency 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 endoglucanases EGII, EGIV and EGV, and belongs to the technical field of genetic engineering. Background technique [0002] Lignocellulosic biomass is a cheap and abundant renewable resource on the earth, and it comes from a wide range of sources, including common crop straws, tree branches and leaves, etc. If the efficient degradation of lignocellulose can be achieved and high value-added products can be produced through biotransformation, this will reduce the waste of such resources to a certain extent, which is of positive significance to the sustainable development of human society. The degradation efficiency of traditional cellulase is low. Traditional cellulases consist of 3 classes, namely β-glucosidases, endoglucanases and exoglucanases. The three types of enzymes cooperate with each other in the process of cellulose hydrolysis. Ho...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N9/42
CPCC12N9/2437C12N15/815C12Y302/01004
Inventor 钟成舒月力李栋梁
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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