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Method for constructing yeast expression vector and preparing cellobiohydrolase by yeast expression vector and application thereof

A yeast expression vector and cellulase technology, applied in the field of genetic engineering, can solve problems such as wasting resources, polluting the environment, and harming animals, and achieve the effects of improving utilization rate, high activity, and increasing utilization channels

Inactive Publication Date: 2019-07-19
JIANGXI AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] However, in production, these straws are not widely used in animal husbandry, and most of them are burned directly (Cao Guoliang et al., Estimates of emissions from open burning of farmland straw in China, Science Bulletin, 2007 (15): 1826-183 ) or decomposed and returned to the field, not only wasting resources, but also seriously polluting the environment
The main reason is that the lignocellulose in the straw has a complex structure and cannot be effectively utilized by ruminants such as cattle and sheep, which reduces its nutritional value.
[0004] In order to improve the utilization rate of these straw feeds, in the past, methods such as acidification and alkalization were used to treat rape straw, but the residual acid and alkali would cause harm to the animals themselves and pollute the environment (Zhang Wenjie et al., Research on Straw Treatment Methods Progress, Chinese Animal Husbandry and Veterinary Medicine, 2011(07): 30-33)

Method used

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  • Method for constructing yeast expression vector and preparing cellobiohydrolase by yeast expression vector and application thereof
  • Method for constructing yeast expression vector and preparing cellobiohydrolase by yeast expression vector and application thereof
  • Method for constructing yeast expression vector and preparing cellobiohydrolase by yeast expression vector and application thereof

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Experimental program
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Effect test

Embodiment 1

[0037] Optimization of exo-cellulase gene fragments: According to the codon preference of Pichia pastoris and the characteristics of pPICZαA vector, the gene sequence of Lentinula edodes cellulase CEL7A (cel7A) mRNA, complete cds , GenBank: AF411250.1, https: / / www.ncbi.nlm.nih.gov / nuccore / AF411250.1 / ), optimize and add restriction endonuclease EcoR I and Xba I sites at both ends of the fragment And synthesize a new gene sequence such as SEQ ID NO.1.

[0038] Construction of the recombinant expression vector pPICZαA-Cel7A: Use EcoR I and Xba I restriction endonucleases to perform double enzyme digestion on the gene sequence such as the exo-cellulase gene fragment of SEQ ID NO.1 and the expression plasmid pPICZαA, respectively. The system is: 5 μL of 10×QuickCut buffer, 1 μL of EcoR I, 1 μL of Xba I, 8 μL of plasmid, 35 μL of sterilized water, and incubate at 37°C for 15 minutes. Gel recovery of exo-cellulase gene fragment; purify digested pPICZαA plasmid; take 4 μl of exo-cell...

Embodiment 2

[0040] Transform Pichia pastoris: use the restriction endonuclease Sac I to linearize the recombinant expression vector pPICZαA-Cel7A, the enzyme digestion system is: 10×QuickCut buffer 5 μL, Sac I 1 μL, plasmid 8 μL, sterile water 36 μL, 37 Incubate at ℃ for 15min. Use the PCR product purification kit to purify the digested product; use the electroporation instrument to transfer the linearized expression vector pPICZαA-Cel7A into Pichia pastoris X33 competent cells, and add sorbitol 0.5-1mol / L, bleomycin Culture in 0.1-1 μL / mLYPD medium (1% yeast extract, 2% peptone, 2% glucose), use pPICZαA vector primer: α-factor primer: 5′-TACTATTGCCAGCATTGCTGC-3′;

[0041] 3'AOX1 primer: 5'-GCAAATGGCATTCTGACATCC-3' by PCR (94°C for 1 minute pre-denaturation, 98°C for 10 seconds, 55°C for 15 seconds, 68°C for 1 minute, cycle 30 times, 72°C for 5 minutes), Screen positive clones. Pick 1 white colony and add it to 2 ml of YPD medium, shake overnight at 30°C. Transfer the whole bacterial s...

Embodiment 3

[0046] Characteristic identification of exo-cellulase: the reaction system of 5-10 micrograms of exo-cellulase prepared in Example 2, 1% microcrystalline cellulose, and 100 mM citric acid buffer (pH 3.0-7.0) was placed in Incubate in a water bath at 40° C. with shaking for 1 h, measure the concentration of reducing sugar, and determine the dependence of exo-cellulase on pH. The results showed that the exo-cellulase had the strongest activity at pH 5.0 ( figure 2 ). Similarly, the reaction system was placed in a buffer solution with a pH of 5.0, shaken and incubated at 20-80°C for 1 hour, and the reducing sugar concentration was measured to determine the temperature dependence of the exo-cellulase. The results show that the optimum temperature of the exo-cellulase in the present invention is 60°C ( image 3 ).

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Abstract

The present invention discloses a method for constructing a yeast expression vector and preparing cellobiohydrolase by the yeast expression vector and an application thereof. A modern molecular technology successfully optimizes a gene sequence encoding the shii-take cellobiohydrolase, constructs the expression vector pPICZ alpha A-Cel7A of the cellobiohydrolase by expressed by yeasts in vitro, amdthe vector and pichia pastoris are utilized to prepare the cellobiohydrolase and thus provide technical supports for an application of the cellobiohydrolase in production. The recombinant pichia pastoris obtained by the method is high in enzyme yield, high in activity and excellent in effects.

Description

technical field [0001] The present invention relates to the technical field of genetic engineering, and more specifically relates to a method and application of constructing a yeast expression vector and expressing exo-cellulase. Background technique [0002] At present, my country's agricultural field produces a large amount of crop straw every year, with rice straw, corn straw, and wheat straw as typical representatives. These straws contain a large amount of carbohydrates such as cellulose and hemicellulose, and are important feed sources for ruminants (Zhao Mengmeng et al., Composition Analysis of Several Crop Straws, Materials Herald, 2011(16): 122-125). [0003] However, in production, these straws are not widely used in animal husbandry, and most of them are burned directly (Cao Guoliang et al., Estimates of emissions from open burning of farmland straw in China, Science Bulletin, 2007 (15): 1826-183 ) or decomposing and returning to the field, not only wasting resou...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N15/66C12N9/42A23K20/189
CPCC12N15/815C12N15/66C12N9/2437A23K20/189
Inventor 赵向辉刘婵娟瞿明仁潘珂
Owner JIANGXI AGRICULTURAL UNIVERSITY
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