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High-activity composite cellulase and preparation thereof, and application method for same in enzymatic saccharification of wood fiber

A technology of compound cellulase and application method, which is applied in the field of high-activity compound cellulase and its preparation and application in lignocellulosic enzymatic saccharification, can solve the problems of reducing ethanol yield, affecting the efficiency of enzymatic saccharification, fiber two Sugar accumulation and other problems, to achieve the effect of low cost, low cost and high activity

Inactive Publication Date: 2012-11-21
CENTRAL SOUTH UNIVERSITY OF FORESTRY AND TECHNOLOGY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, Trichoderma reesei is recognized as an excellent cellulase-producing strain, but the β-glucosidase activity in the cellulase it produces is low, resulting in the accumulation of cellobiose, which affects enzymatic hydrolysis and saccharification efficiency, thereby reducing the yield of ethanol

Method used

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  • High-activity composite cellulase and preparation thereof, and application method for same in enzymatic saccharification of wood fiber
  • High-activity composite cellulase and preparation thereof, and application method for same in enzymatic saccharification of wood fiber
  • High-activity composite cellulase and preparation thereof, and application method for same in enzymatic saccharification of wood fiber

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Pick a ring of spores from the laboratory-preserved Penicillium javanese ZN-205 potato solid plate medium and inoculate it into a 250ml shaker flask filled with 50ml of seed medium, and culture it on a shaker at 28°C and 200rpm for 2.5-3 days as inoculation things. According to the inoculum amount of 5% (v / v), the above culture was inoculated into a 250ml shake flask containing 50ml of enzyme-producing medium, and cultured at 28°C and 200rpm for 5 days. Centrifuge at 4000rpm for 10min, and the supernatant is the crude enzyme solution. Its β-glucosidase activity was determined to be 1.521IU / ml.

[0033]Penicillium javanica ZN-205 seed medium: glucose 20g / L, peptone 1g / L, Mandels nutrient salt concentrate 100ml / L, Mandels trace element concentrate 1ml / L, 1M citric acid buffer 50ml / L, add water to 1L.

[0034] Penicillium javanica ZN-205 enzyme production medium: microcrystalline cellulose 25g / L, peptone 5g / L, KH 2 PO 4 2g / L, CaCl 2 2H 2 O 0.4g / L, MgSO 4 ·7H 2 O ...

Embodiment 2

[0043] The present invention continues to optimize the β-glucosidase production process of Penicillium javanica ZN-205 in Example 1 by using a single factor experimental design, as follows.

[0044] Single factor test of enzyme production medium: keep other components in the enzyme production liquid medium unchanged, change the four factors of carbon source, nitrogen source, pH value and surfactant (Tween-80) respectively (Table 1), screen The single factor affecting the optimal enzyme-producing medium of Penicillium javanica ZN-205 for producing β-glucosidase was found. The order of the influence of various factors on the activity of β-glucosidase was: carbon source > nitrogen source > surfactant > initial pH. The optimal combination was obtained from the test results (Table 2): the initial pH was 6.0, the mass concentration of nitrogen source was 0.75%, the mass concentration of carbon source was 2.5%, and the addition of Tween-80 was 0.05wt%.

[0045] Table 1 Orthogonal ex...

Embodiment 3

[0061] The preparation method of composite cellulase of the present invention is specifically as follows:

[0062] Using self-selected Penicillium javanica ZN-205 to carry out shake-flask fermentation under the optimal conditions of the above-mentioned Example 2 to produce β-glucosidase, and purchased Trichoderma reesei Ru-C30 (ATCC 56765) to prepare cellulose Enzyme (Ref. 1 for its method). The method for measuring β-glucosidase activity adopts the method provided by the present invention, and the method for measuring filter paper enzyme activity (FPA) adopts the method recommended by Ghose (see reference 2). The filter paper enzyme activity (FPA) of Trichoderma reesei Ru-C30 crude enzyme solution was 6.212IU / ml, and the β-glucosidase activity was 1.352IU / ml; the ultrafiltration concentrator of Tianjin Motianmo Technology Co., Ltd. (UOF 48) After concentration for 20 minutes, the filter paper enzyme activity (FPA) of the enzyme liquid is 46.912IU / ml, the β-glucosidase activi...

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Abstract

The invention discloses a high-activity composite cellulase and preparation thereof, and an application method for the same in enzymatic saccharification of wood fiber. According to the invention, Eupenicillium javanicum ZN-205 is subjected to shake-flask fermentation so as to produce beta-glucosidase; best enzyme production conditions are that an initial pH value is 6, the concentration of peptone is 0.75%, the concentration of microcrystalline cellulose is 2.5%, the addition amount of tween-80 is 0.05%, culture temperature is 28 DEG C, the volume of liquid filled in a 250 ml triangular flask is 100 ml, a revolving speed of a shaker is 175 r / min, inoculum amount is 5% and the highest activity of beta-glucosidase is 2.312 IU / ml; beta-glucosidase prepared from Eupenicillium javanicum ZN-205 and cellulase prepared from Trichoderma reesei Rut C-30 are compounded, and an obtained optimal ratio of beta-glucosidase activity to filter paper activity of 1.4. Utilization of the compounded enzyme for enzymatic saccharification of wood fiber enables high-efficiency and low-cost enzymatic saccharification effects to be achieved.

Description

technical field [0001] The invention relates to a compound enzyme prepared by high-yield fungal cellulase, a preparation method thereof, and its application in enzymatic hydrolysis and saccharification of lignocellulosic fibers. Background technique [0002] Due to the important role of alleviating energy shortage, reducing environmental pollution, promoting rural development, and avoiding "competition with people for food and land with farmers", the production of fuel ethanol by using the abundant lignocellulosic plant resources in nature has attracted more and more attention from the society. And support, cellulosic fuel ethanol has become an important part of renewable energy. The bioconversion process from lignocellulose to ethanol usually includes: ①pretreatment of lignocellulosic raw material; ②production of cellulase; ③hydrolysis and saccharification of cellulose; ④fermentation of cellulose hydrolyzate (hexoses) and ⑤hemicellulose Fermentation of vegetarian hydrolysa...

Claims

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Application Information

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IPC IPC(8): C12N9/00C12N9/42C12P19/14C12R1/645
Inventor 陈介南詹鹏张林王义强陈石兰
Owner CENTRAL SOUTH UNIVERSITY OF FORESTRY AND TECHNOLOGY
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