Method for efficiently producing cellulase

A cellulase and high-efficiency technology, applied in the field of high-efficiency production of cellulase, to achieve the effects of improved degradation performance, increased activity, and improved fermentation level

Inactive Publication Date: 2010-06-16
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The invention provides a method for producing cellulase with high efficiency, and solves the problem of simply using cellulose solid material or soluble sugar as a carbon source and inducer in the production of cellulase through a unique medium formula and fed-batch controlled fermentation technology. The existing problem is to make the growth of the strain and the formation process of the target metabolite reach a certain coordination and balance, and effectively improve the fermentation level of cellulase

Method used

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  • Method for efficiently producing cellulase
  • Method for efficiently producing cellulase
  • Method for efficiently producing cellulase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Inject the liquid bacteria into 50M at a ratio of 10% (V / V) 3 Fermentation tank, initial pH 4.8, stirring speed 150rpm, 0-72 hours: fermentation temperature 32°C, ventilation 1.0vvm; after 72 hours: fermentation temperature 28°C, ventilation 0.6vvm. The fermentation medium formula is as follows:

[0030] Soluble carbon source (87.5% of which is glucose, 12.5% ​​is sophorose) 15g / L, insoluble carbon source (microcrystalline cellulose) 30g / L, magnesium sulfate 0.8g / L, potassium dihydrogen phosphate 6g / L, soil temperature 802g / L, yeast powder 15g / L, peptone 10g / L, ammonium sulfate 2.5g / L, calcium chloride 0.5g / L, FeSO 4 ·7H 2 O5mg / L, ZnSO 4 ·7H 2 O 1.4mg / L, MnSO 4 ·H 2 O 1.6mg / L, CoCl 2 ·6H 2 O 3.7mg / L, H 3 BO 3 0.4mg / L, ammonium molybdate 1.6mg / L.

[0031]Start fed-batch culture after 72 hours of fermentation, the flow rate is 3.5L / h m 3 fermentation broth. When the pH value of the fermentation broth rose to 4.8, start feeding fresh medium; when the pH value...

Embodiment 2

[0037] Inject the liquid bacteria into 50M at a ratio of 10% (V / V) 3 Fermentation tank, initial pH 4.8, stirring speed 150rpm, 0-90 hours: fermentation temperature 31°C, ventilation 1.2vvm; after 90 hours: fermentation temperature 29°C, ventilation 0.8vvm. The fermentation medium formula is as follows:

[0038] Soluble carbon source (93.5% of which is glucose, 6.5% is sophorose) 15g / L, insoluble carbon source (microcrystalline cellulose) 40g / L, magnesium sulfate 1.0g / L, potassium dihydrogen phosphate 8g / L, soil temperature 804g / L, yeast powder 20g / L, peptone 15g / L, ammonium sulfate 3.5g / L, calcium chloride 0.7g / L, FeSO 4 ·7H 2 O7mg / L, ZnSO 4 ·7H 2 O 1.6mg / L, MnSO 4 ·H 2 O 1.8mg / L, CoCl 2 ·6H 2 O 3.4mg / L, H 3 BO 3 0.5mg / L, ammonium molybdate 1.2mg / L.

[0039] Start fed-batch culture after 90 hours of fermentation, the flow rate is 1.5L / h m 3 fermentation broth. When the pH value of the fermentation broth rose to 4.8, start feeding fresh medium; when the pH value o...

Embodiment 3

[0044] Using xylose residue (the cellulose waste residue obtained from xylose after hydrolyzing corn cob cores with 10g / L sulfuric acid at 110°C for 6 hours) as raw material, cellulase preparations from different sources were added respectively, and at 50°C and pH 4.8 The enzymolysis reaction was carried out under the following conditions, the concentration of xylose residue was 100g / L, and the dosage of cellulase was: 5-30 FPIU (international unit of filter paper enzyme activity) per gram of xylose residue. After 48 hours of reaction, the concentration of reducing sugar in the hydrolyzate was measured. , Calculate the enzymatic hydrolysis yield (Table 1).

[0045] Table 1 Comparison of enzymatic hydrolysis yields of xylose residues from different sources of cellulase preparations

[0046]

[0047] The chemical composition of xylose residue is: 60.9% cellulose, 5.1% hemicellulose, 18.7% lignin, and 15.3% other.

[0048] Enzyme hydrolysis yield (%) = (mass concentration of ...

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Abstract

The invention provides a method for producing cellulase, which comprises the following steps of: inoculating Trichoderma reesei to a fermentation medium; fermenting for 60 to 90 hours, and then starting feed-batch culture; feeding a culture medium when a pH value of a fermentation liquor is more than 4.8 in the process of the feed-batch culture; stopping feeding the culture medium when the pH value of the fermentation liquor is less than 4.5; and stopping fermenting when the total fermentation time reaches 192 to 240 hours. The method integrates an insoluble carbon source and a soluble carbon source to collaboratively induce expression of cellulase genes. The method achieves coordinated balance in the process of growing strains and forming a target metabolic product through the feed-batch culture, solves the problems existing in the process for producing the cellulase by merely taking cellulose or soluble sugar as an inducer, effectively improves fermentation level of the cellulase; and the obtained cellulase has high degradation performance to cellulose substrates.

Description

technical field [0001] The invention relates to the technical fields of microbial fermentation engineering and biochemical industry, in particular to a method for efficiently producing cellulase. Background technique [0002] Cellulose is the most abundant renewable resource in the world. Cellulase is used to hydrolyze it into glucose, and further ferment to produce ethanol, organic acid, single-cell protein, etc., which is helpful to solve the current problems of food and energy shortages and environmental pollution faced by human beings. , to promote sustainable development is of great significance. [0003] The main problem currently faced by the enzymatic hydrolysis process of cellulose is the large amount of cellulase used and the high cost. Cellulase is an induced enzyme, which is generally synthesized by Trichoderma reesei using cellulose as a carbon source and inducer. Since cellulose is a water-insoluble high-molecular solid substance, adding it to a fermenter wil...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12R1/885
Inventor 夏黎明赵晶余燕春
Owner ZHEJIANG UNIV
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