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Hypocrea for producing mesophile ethanol-tolerant beta-glucosidase highly and application of hypocrea

A technology of glucosidase and Hypocrea, applied in the directions of enzymes, fungi, microorganism-based methods, etc., can solve the problem of lack of commercial β-glucosidase, achieve the elimination of end product inhibition, reduce production costs, eliminate Inhibitory effect of cellobiose

Active Publication Date: 2012-07-11
GUANGZHOU INST OF ENERGY CONVERSION - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The lack of an effective commercial β-glucosidase is a key factor

Method used

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  • Hypocrea for producing mesophile ethanol-tolerant beta-glucosidase highly and application of hypocrea
  • Hypocrea for producing mesophile ethanol-tolerant beta-glucosidase highly and application of hypocrea
  • Hypocrea for producing mesophile ethanol-tolerant beta-glucosidase highly and application of hypocrea

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1: the screening of bacterial strain

[0024] Sampling dead branches and rotten leaves, soil, fungal fruiting bodies and other materials. Directly placed in the Congo red primary screening medium, the formula of the medium is: each liter of medium contains KH 2 PO 4 0.5g, (NH 4 ) 2 SO 4 2.0g, MgSO 4 ·7H 2 O 0.25g, cellulose powder (microcrystalline fiber) 2.0g, Congo red 0.2g, agar 18-20g, the balance is water, natural pH. Cultivate at 30°C for 2-5 days, pick a single colony with a large hydrolysis transparent zone and purify it on PDA medium, and store the purified strain on a PDA slant.

[0025] Re-screening for β-glucosidase activity of the strains: inoculate the purified strains preserved on the PDA slant into the same amount of re-screening fermentation medium, culture at 30°C, 120 rpm, for 48 hours. Freeze and centrifuge to take the supernatant, which is the crude enzyme solution, and use the standard pNPG method to measure the β-glucosidase a...

Embodiment 2

[0027] Embodiment 2: the preparation of β-glucosidase preparation and its enzyme activity, enzymatic properties assay

[0028] 1. The preparation of β-glucosidase preparation: Hypocrea sp. W63 is activated, and through seed culture, the seed culture medium is that every liter of culture medium contains 200g of potato dipping juice, 20g of glucose, and the balance is water, 115 Sterilize at ℃ for 30min. 30° C., 120 rpm, and culture for 3 days to obtain a seed culture solution. The seed culture solution is inoculated in the fermentation medium with the inoculum size of 5% (v / v), and the formula of this fermentation medium is that every liter of medium contains KH 2 PO 4 2g, (NH 4 ) 2 SO 4 2.5g, MgSO 4 ·7H 2 O 0.3g, CaCl 2 0.3g, rice straw 50g, trace element solution 200μL (CuSO 4 0.1g, MnSO 4 0.1g, FeSO 4 ·7H 2 O 0.1g, CoCl 2 0.1g, dilute to 100mL, water 100mL). 30°C, 120rpm, ferment for 5 days, then collect the fermentation broth, refrigerate and centrifuge a...

Embodiment 3

[0033] Embodiment 3: β-glucosidase is applied to simultaneous saccharification and fermentation

[0034] 1. Substrate: air-exploded straw is crushed to 60 mesh, and dried at 105°C to constant weight.

[0035] 2. Yeast was activated by culturing in YPD liquid medium at 30°C for 24 hours.

[0036] 3. Add cellulase 30 FPU / g substrate enzyme amount to the reaction system, and pre-hydrolyze at 50°C for 24 hours.

[0037] 4. Add the β-glucosidase preparation 30FPU / g substrate of Example 2 to the reaction system, inoculate the activated yeast into the reaction system at an inoculum size of 5% (v / v), ferment at 37°C, and When the yeast is inoculated, it is counted as 0h, and samples are taken at fixed points at 0, 4, 8, 12, 24, 48, 96, and 120h, and the content of ethanol and reducing sugar is detected by HPLC.

[0038] The above-mentioned reaction system is: 200mL of reaction solution in a 500mL shake flask, containing 20g of gas-exploded stalks, inorganic salt components ((NH 4 )...

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Abstract

The invention discloses hypocrea for producing mesophile ethanol-tolerant beta-glucosidase highly and application of the hypocrea. The hypocrea is conserved in China General Microbiological Culture Collection Center (CGMCC) on September 1st, 2011, and the conservation No. is CGMCC No. 5209. The hypocrea can produce novel beta-glucosidase, wherein the enzyme activity of the beta-glucosidase is up to 482.1U / ml, the pH value of the optimum reaction is 4.8, the optimum reaction temperature is 70 DEG C, and the enzyme activity is high in stability at the temperature of 50 DEG C, so the beta-glucosidase is suitable for pyrohydrolysis; ethanol of which the concentration is 10 percent has the maximum promotion effect on the enzyme activity, and the enzyme activity of the beta-glucosidase is improved by nearly 1 time; and the ethanol of which the concentration is less than 30 percent in a reaction system does not inhibit the enzyme activity, and when biomass raw materials are hydrolyzed, the inhibition of cellobiose can be eliminated effectively, so that the yield of the ethanol is improved, and the inhibition of terminal products is eliminated. The production cost of cellulosic ethanol can be reduced, and the industrialized progress of the cellulosic ethanol is accelerated.

Description

technical field [0001] The invention belongs to the field of microbial fermentation and its enzyme engineering application, in particular to a high-yielding mesophilic ethanol-resistant β-glucosidase (Hypocrea sp.) W63 and the produced β-glucosidase, and the β-glucosidase -Application of glucosidase in simultaneous saccharification and fermentation. Background technique [0002] β-glucosidase, the English name β-glucosidase, referred to as BG, is an enzyme that can catalyze the hydrolysis of the glycosidic bond between the aryl or hydrocarbon group and the sugar group, thereby releasing glucose. β-glucosidase is widely distributed in nature and is found in almost all organisms. It has different properties due to different sources. It has important physiological functions in the degradation of glycogen in humans and the carbohydrate metabolism of animals, plants and microorganisms. . [0003] In the process of cellulase hydrolysis of cellulose, the enzymatic action of cellu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/14C12N9/42C12P7/10C12R1/645
CPCY02E50/16Y02E50/10
Inventor 梁翠谊许敬亮袁振宏庄新姝徐惠娟张宇
Owner GUANGZHOU INST OF ENERGY CONVERSION - CHINESE ACAD OF SCI
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