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Liquid-state fermentation production method of cellulase

A cellulase and production method technology, applied in microorganism-based methods, biochemical equipment and methods, enzymes, etc., can solve problems such as difficult large-scale industrial production, easy contamination of miscellaneous bacteria, and difficult extraction, and improve product quality. , The effect of shortening fermentation time and easy operation

Active Publication Date: 2014-02-05
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Solid-state fermentation has low cost, but it is easy to contaminate miscellaneous bacteria, difficult to extract, and difficult to carry out large-scale industrial production

Method used

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  • Liquid-state fermentation production method of cellulase
  • Liquid-state fermentation production method of cellulase
  • Liquid-state fermentation production method of cellulase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Cellulase activity assay

[0026] The enzyme activity involved in the present invention is determined as filter paper enzyme activity.

[0027] The various enzyme activities involved in the present invention are agreed to be defined as: under the condition of 50±0.1°C and pH4.8, 1ml of enzyme solution hydrolyzes the substrate for 1min to produce the amount of reducing sugar equivalent to 1 μmol of glucose, which is 1 enzyme activity unit. Expressed in IU / ml.

[0028] The enzyme activity detection methods are all carried out according to the international method IUPAC.

[0029] Determination method of filter paper enzyme activity: 1ml of crude enzyme solution, under the conditions of 50±0.1°C and pH4.8, the amount of reducing sugar produced by hydrolyzing 50±1mg of filter paper for 1 hour, and the amount of reducing sugar produced was determined by DNS method.

Embodiment 2

[0031] 1) Experimental group: cellulase liquid state fermentation method of the present invention

[0032] Strain activation: Among the cellulase-producing bacteria, representative Trichoderma reesei RUT-C30 and Penicillium juniperi 9-3 were selected as the production bacteria for cellulase liquid fermentation. Store at 4°C for later use;

[0033] Bacteria culture: Wash the slant spores with sterile water, and adjust the spore concentration to 10 on the hemocytometer 7 Individual / ml, inoculate the seed culture medium according to the volume ratio of the inoculation amount of 3:100, and cultivate the strain seed liquid at 28°C for 18-24h;

[0034] Primary fermentation: (1) inoculate the fermentation medium with the seed solution of Penicillium and Trichoderma according to the volume ratio of 1:20, ferment Penicillium at 30°C for 120h, and ferment Trichoderma at 26°C 120h, obtain Penicillium primary fermentation liquid and Trichoderma primary fermentation liquid respectively, ...

Embodiment 3

[0043] Strain activation: Among the cellulase-producing bacteria, select the representative Trichoderma reesei RUT-C30 as the production strain. After passage and activation, store it on a PDA slope at 4°C for later use;

[0044] Bacteria culture: Wash the slant spores with sterile water, and adjust the spore concentration to 10 on the hemocytometer 7 Each / ml, inoculate the seed medium according to the volume ratio of 3:100, and cultivate at 28°C for 18-24h;

[0045] Primary fermentation: Inoculate the cultured strain Trichoderma reesei into the fermentation medium according to the volume ratio of 1:20, ferment for 120 hours at 26°C, centrifuge at 1200 rpm for 5 minutes to obtain a primary fermentation liquid, and centrifuge the primary fermentation liquid Filter through 0.22 μm, 0.45 μm and 0.8 μm filter membranes to remove impurities (residual hyphae, spores, etc.) The fermented liquid that was filtered and only centrifuged was used as a control. The results showed that the...

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Abstract

The invention discloses a liquid-state fermentation production method of cellulose. The method is characterized by comprising a primary fermentation step and a secondary fermentation step. By adopting the method for secondarily fermenting an enzyme liquid, the fermentation process route of the cellulose is optimized, the fermentation time for acquiring identical enzymatic activity of the existing fermentation process can be shortened, so that the product quality of enzymic preparations is improved, the production cost is reduced, and the industrialized production can be realized. Moreover, the production process is simple, the method is simple and convenient to operate, and the technical method is easy to control; the additive is sourced from the filtered fermentation enzymic liquid, so that the additive is unnecessary to buy additionally, and the cost is not increased.

Description

technical field [0001] The invention relates to the field of cellulase fermentation, in particular to a liquid state fermentation production method of cellulase. Background technique [0002] Cellulose is a very abundant renewable resource on earth, it accounts for 35-50% of the dry weight of plants. Using cellulase to degrade cellulose into oligosaccharides, disaccharides or monosaccharides, so as to further produce other bio-based products, such as single-cell protein, alcohol, etc., is very important for solving the current energy crisis, food shortage, environmental pollution and other problems. is of great significance. Cellulase is a mixed enzyme system composed of three components, including endoglucanase (C1 enzyme), exoglucanase (Cx enzyme), and β-glucosidase (BG enzyme). At present, the application of cellulase has been extended to various fields such as medicine, genetics, textile, daily chemical industry, papermaking, environment, food fermentation, feed, indus...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12R1/80C12R1/885
CPCC12N9/2437C12N9/2445C12Y302/01004C12Y302/01021C12Y302/01091
Inventor 张东远张粲李晨贾文娣赫荣琳武改红陈树林
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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