A kind of highly active composite cellulase and its preparation and application method

A technology of compound cellulase and application method, which is applied in the application field of enzymatic hydrolysis and saccharification of lignocellulose, can solve the problem of low activity of BG type cellulase, achieve low-cost enzymatic hydrolysis, and improve the effect of enzymatic hydrolysis yield

Active Publication Date: 2017-07-14
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

The currently recognized bacterial species that can produce highly active EG and CHB cellulase is Trichoderma reesei, while the BG cellulase activity produced by this strain is relatively low

Method used

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  • A kind of highly active composite cellulase and its preparation and application method
  • A kind of highly active composite cellulase and its preparation and application method
  • A kind of highly active composite cellulase and its preparation and application method

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Experimental program
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Embodiment 1

[0025] Embodiment 1: The present invention provides the optimization process of the production process of β-glucosidase by Aspergillus niger C112 by single factor and orthogonal test, specifically as follows.

[0026]Seed culture of Aspergillus niger C112: Pick two rings of spores from the Aspergillus niger C112 potato solid plate medium preserved in the laboratory, inoculate in a 250ml shaker flask containing 50ml potato medium, and cultivate at 28°C and 200r / min 2 to 2.5 days as the inoculum. Aspergillus niger C112 seed medium: potato 300g / L, glucose 20g / L, pH 6.80.

[0027] Enzyme-producing culture of Aspergillus niger C112: Inoculate the activated Aspergillus niger liquid with 6% (v / v) inoculum in a 250ml shake flask with 50ml enzyme-producing medium, at 28°C, under the condition of 200r / min Cultured for 5 days. All shake flask experiments were performed in triplicate. Centrifuge at 4000r / min for 10min, take the supernatant (crude enzyme solution) to measure its β-gluco...

Embodiment 2

[0052] Trichoderma reesei RUT C30 (Trichoderma reesei RUT C30) used in the present invention was purchased from the American Type Culture Collection (ATCC), number: ATCC 56765, and its cellulase production method, see reference 3.

[0053] The preparation of the high-activity composite cellulase of the present invention is specifically as follows: the cellulase is produced by mutagenesis-selected Aspergillus niger C112 and Trichoderma reesei Ru-C30 (ATCC 56765) purchased from ATCC of the United States. The cellulase β-glucosidase activity produced by Aspergillus niger C112 is 15.496U / mL, and the enzyme activity of the filter paper is 0.215IU / mL. - Glucosidase activity is 20.491U / mL, filter paper enzyme activity is 0.93IU / mL. The cellulase β-glucosidase activity produced by Trichoderma reesei RUT C30 was 1.352U / mL, and the filter paper enzyme activity was 6.212IU / mL. After being concentrated by ultrafiltration concentrator UOF 48 (Tianjin Motianmo Technology Co., Ltd.), the β-...

Embodiment 3

[0055] The present invention adopts dilute phosphoric acid combined with steam explosion to pretreat poplar, specifically as follows: the pulverized poplar wood chips (1-2mm in diameter, 2-15mm in length) and dilute phosphoric acid (0.05%-2.0%) are mixed according to the solid-to-liquid ratio of 1:1 -1:10 pre-impregnation (1h-12h) followed by steam explosion (steam explosion machine QBS-80, developed by Hebi Zhengdao Heavy Machinery Factory), steam explosion conditions: steam explosion pressure is 0.5-2.5Mpa, pressure holding time is 90 -220s. The resulting steam explosion product is added with an appropriate amount of 0.05 mol / L, pH 4.8 citric acid buffer, and then adjusted to 4.5-6.0 with 1 mol / L ammonia water for detoxification, and then enzymatically hydrolyzed and saccharified.

[0056] The present invention utilizes composite cellulase to enzymatically hydrolyze and saccharify poplar wood, which is preferably specifically as follows: air-dried poplar wood chips, the diam...

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Abstract

The invention discloses a high-activity compound cellulase and preparation and application methods thereof. The preparation method comprises the steps: liquid fermentation is carried out by using aspergillus niger C112 to produce beta-glucosidase, wherein under certain culture conditions, the enzyme activity of the obtained beta-glucosidase can reach 15.496 IU / ml; the beta-glucosidase produced by the aspergillus niger C112 and cellulase produced by trichoderma reesei RUTC30 are subjected to enzyme compound, and the high-activity compound cellulose is prepared. When the beta-glucosidase / filter paper enzyme ratio is 1.536, enzymolysis saccharification and dilute phosphoric acid and steam explosion pretreatment on aspen wood are carried out, the content of the obtained highest reducing sugar is 61.858 g / L, and the reducing sugar yield is 81.63%.

Description

technical field [0001] The invention belongs to the technical field of enzyme engineering, and specifically relates to the preparation of a high-activity composite cellulase by means of biotechnology, and relates to the application of the composite cellulase in enzymatic hydrolysis and saccharification of lignocellulose. Background technique [0002] Lignocellulose is the most abundant and cheapest renewable resource in nature, accounting for about 40% of the total biomass of the earth. The degradation of natural cellulose into usable sugars through biotransformation technology is of great significance for alleviating the earth's energy crisis and environmental pollution. Cellulase refers to the general term for a class of multi-component complex enzyme systems that can degrade cellulose, mainly including three enzyme components: endo-β-glucanase (exdo-1, 4-β-D- glucanase, EG), exo-β-glucanase (exo-1,4-β-D-glucanase, CHB) and β-glucosidase (β-glucosidase, BG). The mechanis...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/42C12P19/14C12R1/685C12R1/885
CPCC12N9/2437C12N9/2445C12P19/14C12P2201/00C12Y302/01021
Inventor 陈介南詹鹏王芳张林王挥刘进陈立君
Owner CENTRAL SOUTH UNIVERSITY OF FORESTRY AND TECHNOLOGY
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