Coding gene of cellulose of glycosyl hydrolase family 5 and its application

A technology of cellulase and gene, which is applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of low efficiency, high cost, and high price

Inactive Publication Date: 2005-06-08
GUANGXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the wide use of cellulase and the need to use cellulase with different properties for different purposes, and because of the low efficiency and high price of cellulase, the cost of producing fuel alcohol from cellulose is too high to be truly To achieve industrialization, therefore, new cellulase enzymes are required

Method used

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  • Coding gene of cellulose of glycosyl hydrolase family 5 and its application
  • Coding gene of cellulose of glycosyl hydrolase family 5 and its application
  • Coding gene of cellulose of glycosyl hydrolase family 5 and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1. Composting of compost

[0019] Composting is carried out in a self-made 1m x 1m x 1.2m cement trough with built-in ventilation ducts. The pipe is connected to a blower outside the tank. The compost formula is: straw, 50kg; cow manure, 50kg; pig manure, 10kg; chicken manure, 10kg; waste newspaper, 5kg; rural compost, 80kg; forest soil, 2.5kg; green turf, 5kg; orchard soil, 2.5kg ; Vegetable garden soil, 5kg; Bamboo forest soil, 5kg; Yeast powder, 0.065kg; Ammonium sulfate, 1kg; Urea, 0.411kg; Water, 67kg. Cut the straw into a length of about 5cm, weigh each raw material according to the above formula, mix all formula materials except the straw evenly, and then mix it with the straw straw, add an appropriate amount of water in the process to make the final water content of the compost Controlled at around 59%. After mixing evenly, fill the compost fermentation tank. The air supply method adopts the forced ventilation of the blower, and the air is supplie...

Embodiment 2

[0020] Example 2. Construction of a genomic library of uncultivated microorganisms in compost

[0021] Get 50g compost soil, suspend in the 0.18M potassium phosphate buffer solution (pH7.2) of 100ml, after thorough mixing, on the JA-10 rotor of Beckman Coulter Avanti J-E centrifuge (purchased from Beckman Coulter Company, catalog number 369003) Centrifuge at 600g for 10 minutes, collect the supernatant, add 40ml of PVPP (polyvinylpolypyrrolidone, polyvinylpolypyrrolidone) solution (PVPP solution: every 100mg of PVPP (purchased from Sigma, catalog number P-6755) and 1ml of 0.18M potassium phosphate buffer (pH7.2) and mix well), shake for 30 seconds, then add 200 μl of 3M CaCl2 solution, shake for 30 seconds, centrifuge at 600 g for 5 minutes, and collect the supernatant in another centrifuge tube. Then use the same centrifuge and rotor to centrifuge for 15 minutes to collect the bacterial cells in the supernatant. Fully suspend the collected cells in 1ml TE (10mM Tris / HCl, pH8...

Embodiment 3

[0023] Example 3. Screening of clones expressing cellulase activity from a genomic library of composted uncultured microorganisms

[0024]The transformants obtained on the LA plate containing ampicillin (about 200 colonies per plate) were respectively copied to 0.5% carboxylmethylcellulose (carboxylmethylcellulose, CMC) (purchased from Sigma Company, catalog number C) by plate copy method. -5678) LA plate and LA plate containing ampicillin (100 μg / mL), place the plate upside down in a 37°C incubator and culture for 24 hours, then store the LA plate containing ampicillin full of colonies in a 4°C refrigerator 1. Stain the LA plate containing carboxymethylcellulose covered with colonies with 0.5% Congo red solution for 15 minutes, decolorize with 1M NaCl solution for 15 minutes, and then detect whether there is a hydrolysis circle around the colonies (see image 3 ), as a result, 4 clones with hydrolysis circles around the colonies were screened. The present invention only invol...

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Abstract

A gene umcel5A coding cellulase features that it contains the nucleoside sequence shown by SEQ ID No.2 or its homologous sequences. The cellulase coded by said gene and its application in degradating cellulose are also disclosed.

Description

technical field [0001] The present invention relates to a novel cellulase gene encoding glycosyl hydrolase family 5, in particular to a novel cellulase gene encoding glycosyl hydrolase family 5 cloned from compost uncultivated microorganisms. The protein encoded by the gene can be used for the degradation of cellulose. Background technique [0002] Cellulose is mainly the most abundant renewable biomass (biomass) resource on earth synthesized by plants using carbon dioxide and water through photosynthesis under the action of solar energy. According to reports, the global annual production of cellulose through photosynthesis is as high as 1.55×10 9 tons, 89% of which have not been utilized by humans (Dunlap C, Chiang G C. Utilization and recycle of agriculture wastes and residues. Shuler M L. Boca Raton, Florida. USA: CRC Press Inc. 1980.19). Cellulose is a polymer composed of multiple glucose residues linked by β-1,4-glycosidic chains, and its basic repeating unit is cello...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/63C12P19/00C12S3/04
Inventor 冯家勋段承杰庞浩靳振江张鹏封毅许跃强唐纪良
Owner GUANGXI UNIV
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