Trichoderma reesei mutant strain for highly yielding neutral cellulase

A neutral cellulase, Trichoderma reesei technology, applied in the direction of enzymes, enzymes, fungi, etc., can solve the problems of unfavorable deep liquid fermentation, short fermentation cycle, long fermentation time, etc., to promote promotion and application, reduce Production cost, obvious effect of amplification

Pending Publication Date: 2018-01-19
QINGDAO VLAND BIOTECH GRP
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AI Technical Summary

Problems solved by technology

Although the fungi producing neutral cellulase have strong enzyme production ability, the fermentation time is often very long, which affects the production intensity and efficiency; and in industry, most filamentous fungi produce enzymes by solid fermentation, which is not conducive to submerged liquid fermentation; For eukaryotes, enzyme-producing genes contain introns, and the enzymes produced often need to be induced, which brings inconvenience to the construction of genetically engineered bacteria
[0006] Cellulase derived fr

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0015] Example 1 Neutral cellulase strain shake flask fermentation and enzyme activity detection

[0016] The neutral cellulase-producing Trichoderma reesei HGD43 (this strain is an engineered strain constructed by the inventor Liu Yanping to express the recombinant neutral cellulase) was inoculated on a fresh PDA plate and cultured at 30°C for 5-7 days. Cut a 2cm×2cm bacterial block and inoculate it into a 50ml liquid shake flask medium (lactose 2%; glucose 1%; corn steep liquor 1.5%; ammonium sulfate 0.9%; potassium dihydrogen phosphate 2%; diammonium hydrogen phosphate 0.4% Magnesium sulfate heptahydrate 0.15%; Citric acid 0.073%; Calcium chloride 0.12%; Ferrous sulfate heptahydrate 0.075%; Zinc sulfate heptahydrate 0.006%; Copper sulfate pentahydrate 0.0012%; Manganese sulfate monohydrate 0.00053%; Boric acid 0.0003 %), cultured at 30°C for 2 days, and then at 25°C for 3 days. After culturing for 5 days, the supernatant obtained by centrifuging the fermentation broth is the ...

Example Embodiment

[0034] Example 2 Mutagenesis Screening and Fermentation Verification

[0035] Determine the lethality rate: inoculate the starting strain of Trichoderma reesei HGD43 on a PDA plate, and culture at 30°C for 5-7 days. When the surface of the colony turns white and a large number of spores are produced, draw 5ml of sterile water to elute to obtain the spore liquid, resuspend in sterile water after centrifugation, and count with a hemocytometer to make the spore concentration about 5×10 7 Pcs / ml. Take a 90mm sterile petri dish into the rotor, add 10ml of the diluted spore suspension, and stir on a magnetic stirrer to make the spore liquid in a uniform state. In a sterile ultra-clean workbench, use an ultraviolet lamp with a power of 9w to irradiate at a vertical distance of 20cm and irradiate them for 60s, 90s, 120s, 150s, and 180s respectively. Dilute the irradiated spore liquid by 10000 times and take 100ul to coat the PDA The plates were cultured at 30°C for 2-3 days and counted....

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Abstract

The invention relates to the technical field of an enzyme preparation, and particularly relates to a trichoderma reesei mutant strain and application thereof. After the mutant strain is subjected to shake-flask fermentation for 5 days, the enzyme activity of the neutral cellulase in fermentation supernatant fluid is 114m/ml, and is improved by 34 percent through being compared with that of an original strain; in the 20L fermentation tank small tests, the enzyme activity of the neutral cellulase in the fermentation supernatant fluid reaches 1335 u/ml; the amplification effect is obvious on theexpression quantity after the comparison with a shake-flask result; and the mutant strain has the advantages of thick and short mycelia, high dissolved oxygen level, low fermentation liquid viscosityand the like in the fermentation process; and the unexpected technical effects are achieved. The trichoderma reesei mutant strain can be widely applied to cellulase production, so that the productioncost of the cellulase can be favorably reduced; and the popularization and the application of the cellulase in relevant fields can be promoted.

Description

technical field [0001] The invention belongs to the technical field of microbial screening, and specifically relates to a mutant strain of Trichoderma reesei with high production of neutral cellulase and its application. technical background [0002] Cellulase is a general term for a group of enzymes that can degrade cellulose and finally generate glucose. It is not a simple enzyme, but a multi-component enzyme system that acts synergistically in the process of degrading cellulose. A class of glycoside hydrolases that hydrolyze β-1,4-glycosidic bonds. According to the optimal pH value of enzyme activity, cellulase can be divided into acid cellulase, alkaline cellulase and neutral cellulase. [0003] The cellulase whose optimum pH is between 6.0-8.0 is called neutral cellulase, which is also a multi-component enzyme system, but the ratio and composition of various enzymes have their particularity. Because the earliest researched cellulase-producing microorganisms were mainl...

Claims

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Application Information

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IPC IPC(8): C12N1/14C12N9/42C12R1/885
Inventor 吴佳鹏刘艳萍黄亦钧王华明
Owner QINGDAO VLAND BIOTECH GRP
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