Trichoderma reesei bacterial strain for expressing saccharifying enzyme

A technology of Trichoderma reesei and glucoamylase, which is applied in the direction of enzymes, fungi, and microbial-based methods, can solve the problems of high production cost, inability to tolerate high temperature, and low yield of glucoamylase, so as to reduce production cost and shorten production time. High saccharification time and catalytic efficiency

Active Publication Date: 2014-03-05
QINGDAO VLAND BIOTECH GRP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the currently used glucoamylases generally cannot tolerate high temperature, and the yield of glucoamylase is relatively low, and the production cost is high, thus affecting the wide application of glucoamylase in industry

Method used

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  • Trichoderma reesei bacterial strain for expressing saccharifying enzyme
  • Trichoderma reesei bacterial strain for expressing saccharifying enzyme
  • Trichoderma reesei bacterial strain for expressing saccharifying enzyme

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] The cloning of embodiment 1 glucoamylase gene and the construction of expression vector

[0017] 1.1 Extraction of Trichoderma total genomic DNA

[0018] Trichoderma reesei (Trichoderma reesei) was inoculated with shake flask medium for overnight culture, and an appropriate amount of bacteria was placed in a centrifuge tube, centrifuged at 13000rpm for 5min, and the supernatant was discarded; 400μl of extraction buffer (100mM Tris-HCl, 100mM EDTA, 250mM NaCl, 1%SDS); then add 100mg of quartz sand or glass beads, vibrate vigorously in a bead beater for about 2min; add 200μl 10M NH after 20min in a water bath at 65℃ 4 AC ice bath for 10min; centrifuge at 13000rpm for 10min to take the supernatant, then add 2 times the volume of absolute ethanol, place at -20°C for 30min; centrifuge at 13000rpm for 10min, discard the supernatant, wash twice with 70% ethanol; dry in the air, add appropriate amount of water to dissolve Store at -20°C.

[0019] 1.2 Gene cloning

[0020] Us...

Embodiment 2

[0025] Embodiment 2 transformation and screening

[0026] 2.1 Protoplast preparation

[0027] Inoculate Trichoderma reesei mycelia and grow on PDA plates for 4 days; cut colonies with a diameter of about 3 cm and place them in liquid medium of about 60ml YEG (0.5% yeast powder, 1% glucose, 1% Uridine), 30 Cultivate with shaking at 200rpm overnight; collect the mycelia by filtering with multi-layer gauze; place the mycelium in 10-20ml of lysing enzyme solution (Sigma L1412) for 2-3 hours; take out the enzymatic solution and add 0.7M NaCl solution, Shake gently, pour it on three layers of sterilized lens tissue, collect the filtrate, centrifuge at 3000rpm for 10min; discard the supernatant, add 10-20ml STC solution (20% sucrose, 50mM Tris-Cl, 50mM CaCl 2 ) suspension, 3000rpm, centrifuged for 10min; add appropriate amount of STC suspension (150μl / tube, 10 8 pieces / ml).

[0028] 2.2 Transformation and verification

[0029] Take 2 μg pKDN-GA DNA and add it to 150 μl protoplast...

Embodiment 3

[0031] Embodiment 3 mutagenesis and screening

[0032] Trichoderma reesei HDGA-1 strain was inoculated into PDA medium, cultured at 30°C for 7 days, and a single spore suspension was prepared with physiological saline, and its concentration was controlled at 10 6 A / ml or so. Take 10 mL of the spore suspension and put it in a petri dish with a radius of 9 cm, put it into a dark box with a magnetic stirrer, and irradiate it under a 9W ultraviolet lamp at a distance of 20 cm for 2 min (the ultraviolet lamp should be preheated for 30 min); Bacterial water diluted to 104 、10 5 、10 6 Concentration, respectively take 0.1ml coated PDA plate (in order to prevent back mutation, this operation is carried out under red light); place the coated plate in a constant temperature incubator at 30°C for 3 days in the dark; The changed single colonies were picked and cultured on PDA medium.

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Abstract

The invention aims at providing a trichoderma reesei bacterial strain for producing saccharifying enzyme. The trichoderma reesei bacterial strain is trichoderma reesei HDGAU-1 (Trichoderma reesei HDGAU-1) with a preservation number of CCTCC (China Center for Type Culture Collection) NO: M2013584. The trichoderma reesei bacterial strain disclosed by the invention can efficiently express the saccharifying enzyme, and fermentation enzyme activity is as high as 4000 U/ml which is improved by 54% in comparison with that before mutation, and protein amount exceeds 2.1 g/L which is improved by about 50% in comparison with that before mutation. According to the invention, production cost of the saccharifying enzyme can be greatly lowered by utilizing the trichoderma reesei bacterial strain to produce the saccharifying enzyme, and wide application of the saccharifying enzyme is facilitated. Moreover, the saccharifying enzyme recombinant and expressed by the trichoderma reesei bacterial strain disclosed by the invention has an optimum acting temperature of 70 DEG C and an optimum acting pH of 5.5, has catalytic efficiency higher than that of the common saccharifying enzyme from aspergillus niger at present, is more high temperature resistant, can effectively shorten saccharifying time and can lower production cost.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering microorganism construction and screening, and in particular relates to a Trichoderma reesei strain recombinantly expressing glucoamylase and an application thereof. Background technique [0002] Glucoamylase, the system name is a-1,4-glucan glucohydrolase (a-1,4-glucan-glucohydrolase (EC.3.2.1.3)). Studies have shown that the substrate specificity of glucoamylase is low. It can not only cut the a-1,4 bond from the non-reducing end of the starch chain, but also slowly cut the a-1,6 bond. Therefore, it can quickly cut the glucose units from the non-reducing end of amylose in sequence. When encountering a-1,6 bond splitting, first split the a-1,6 bond, and then a-1,4 Bond splitting, thereby hydrolyzing amylopectin to glucose. Glucoamylase is used in the fermentation of various antibiotics, organic acids, amino acids and vitamins with glucose as the fermentation medium; it is also widely...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/14C12N9/34C12N15/56C12N15/80C12R1/885
Inventor 王华明张慧丹吴佳鹏黄亦钧
Owner QINGDAO VLAND BIOTECH GRP
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