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Method for promoting cellulolytic enzymes synthesis of trichoderma reesei by using dilute sulfuric acid treated pulp as carbon source

A technology for synthesizing cellulose and Trichoderma reesei, which is applied in the field of microbial cultivation in biochemistry, can solve the problems of difficulty in supplying lignocellulose raw materials at the same time, lack of cellulose utilization, and low cellulase activity, so as to benefit microorganisms The effect of using and promoting the growth of bacterial cells

Active Publication Date: 2008-08-27
CHINA PETROLEUM & CHEM CORP
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] General lignocellulosic raw materials (such as pulp) are difficult to meet the requirements of providing both components
Monosaccharides such as glucose are often added in production to promote the growth of bacterial cells, but bacterial cells grown with glucose as a carbon source lack the ability to utilize cellulose, resulting in low cellulase activity
[0008] So far, no lower cost, equivalent or better alternative carbon source to promote the synthesis of cellulase by Trichoderma reesei has been found

Method used

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  • Method for promoting cellulolytic enzymes synthesis of trichoderma reesei by using dilute sulfuric acid treated pulp as carbon source
  • Method for promoting cellulolytic enzymes synthesis of trichoderma reesei by using dilute sulfuric acid treated pulp as carbon source
  • Method for promoting cellulolytic enzymes synthesis of trichoderma reesei by using dilute sulfuric acid treated pulp as carbon source

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Cellulase Production by Trichoderma reesei Using Commercial Pure Cellulose as Carbon Source

[0022] Enzyme production medium: commercial pure cellulose (delignified cellulose, Solkc Flock200) 12g / L, glucose 1g / L, peptone 1g / L, ammonium sulfate 2.2g / L, urea 0.5g / L, chloride Calcium 0.3g / L, magnesium sulfate 0.3g / L, ferrous sulfate 0.005g / L, manganese sulfate 0.0016g / L, sulfuric acid 0.0014g / L, cobalt chloride 0.0037g / L, Tween 802 drops / 50mL. The medium was adjusted to pH 4.5-5.0 with citric acid buffer.

[0023] Enzyme production conditions: Trichoderma reesei mycelium is inoculated into the enzyme production medium, and the inoculum amount of Trichoderma reesei mycelium accounts for 10% of the volume of the medium. The culture temperature is 26-32°C, and the rotation speed is 150-250r / min. Samples were taken every other day, centrifuged at 3000r / min, and the supernatant was taken to measure the pH value, filter paper enzyme activity and protein concentration respecti...

Embodiment 2

[0027] Dilute sulfuric acid treatment of pulp

[0028] Crush ordinary commercial pulp into particles with a diameter of about 1 mm, add sulfuric acid solution with a concentration of 0.75-4.0% according to the ratio of solid-liquid ratio of 1:10, soak for 1-5 hours, and then hydrolyze at 110-130 ° C for 40-80 minutes.

[0029] The pulp treated with dilute sulfuric acid is washed with water until neutral, and suction filtered until the moisture content is about 50-80%.

[0030] Carry out electron microscope (SEM) analysis to paper pulp and dilute sulfuric acid treated pulp, electron microscope equipment and operating steps are as follows:

[0031] Equipment: Philips quanta200 electron microscope, Hitachi HCP-2 critical point dryer, Hitachi E-1010 ion sputtering apparatus.

[0032] Dilute sulfuric acid treated pulp sample preparation steps: fixation, gradient dehydration, critical point drying, sticking, conductive treatment.

[0033] Electron microscopy analysis showed that, ...

Embodiment 3

[0036] Trichoderma reesei cellulase production medium was prepared with ordinary pulp and pulp treated with dilute sulfuric acid as carbon source.

[0037] Ordinary pulp and dilute sulfuric acid treated pulp are used in a ratio of 3:1 to 2 to replace the pure cellulose used in Example 1, and the rest are the same as Example 1, and the enzyme production conditions are also the same as Example 1.

[0038] attached by Figure 4 It can be seen that using pulp and dilute sulfuric acid treated pulp as carbon source, the highest enzyme activity of filter paper is 3.12IU / mL, and the enzyme protein concentration is 1.34g / L. Commercial pure cellulose (delignified cellulose, Solkc·Flock200) is a good carbon source for inducing cellulase synthesis, but it is expensive and can only be used for laboratory research. In Example 1, 12 g / L of commercially pure cellulose was used as the carbon source, and the maximum enzyme activity of the filter paper was 2.23 IU / mL. In this example, a mixtur...

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Abstract

The invention relates to a method for taking paper pulp as raw materials and as a carbon source which is needed for synthesizing cellulose enzyme by trichoderma reesei after the paper pulp is processed with dilute sulfuric acid. Compared with a method for taking pure cellulose as the carbon source, the filter paper enzyme activity can be increased by 40% through using the method for preparing the cellulose enzyme, and the problems that somatic cell is lack of the ability to utilize cellulose and the activity of the cellulose enzyme is not high when the paper pulp and dextrose are taken as the carbon source to prepare culture medium are solved.

Description

1. Technical field [0001] The invention belongs to the technical field of microbial cultivation in biochemistry, and particularly relates to a method for treating pulp with dilute sulfuric acid as a carbon source to promote the synthesis of cellulase by Trichoderma reesei. 2. Background technology [0002] Prior art: cellulase is a general term for a group of enzymes used to degrade cellulose, the main components are endoglucanase (endoglucanase), exoglucanase (exoglucanase) and β-glucose Glucosidase (β-glucosidase). The mechanism of cellulase degrading cellulose is: endoglucanase randomly cuts cellulose macromolecules into short-chain cellulose, and exoglucanase cuts short-chain cellulose ends to form cellobiose or oligosaccharides, β - Glucosidase acts on cellobiose and oligosaccharides to form glucose. The strains that can be used to produce cellulase mainly include Trichoderma, Aspergillus and Penicillium. One of the more studied strains is Trichoderma reesei. [000...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42D21C9/00C12R1/885
Inventor 余世袁勇强饶庆隆
Owner CHINA PETROLEUM & CHEM CORP
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