Method for producing carboxypeptidase by aspergillus niger

A technology of carboxypeptidase and Aspergillus niger, applied in the field of carboxypeptidase production, can solve the problems of limited wide application, high cost of carboxypeptidase production and application, low carboxypeptidase activity, and achieve low investment, low production cost, The effect of simple equipment

Inactive Publication Date: 2011-08-17
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

To sum up, carboxypeptidases widely exist in Aspergillus, but at present, whether it is liquid fermentation or solid state fermentation, the activity of microorganisms to produce carboxypeptidases is low, which leads to high production and application costs of carboxypeptidases, thus limiting the production of carboxypeptidases. Wide application of enzymes

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] 1. Strain: Aspergillus niger LW-1 strain.

[0021] 2. Solid-state fermentation medium: 10g of fermentation base material (10g of bran) in a 250mL Erlenmeyer flask, (NH 4 ) 2 SO 4 50mg, MgSO 4 5mg, CaCl 2 10mg, KH 2 PO 4 30mg, tap water 13mL, natural pH.

[0022] 3. Solid-state fermentation conditions: sterilize at 121°C for 40 minutes, inoculate one fungus E001 bran koji seeds after cooling, and culture at 30°C for 96 hours.

[0023] 4. Crude enzyme solution extraction:

[0024] After solid-state fermentation, 1 g of the culture was taken, and 10 mL of 50 mM sodium citrate buffer (pH 5.0) was added to each Erlenmeyer flask, milled for 10 min, filtered after resting for 30 min, and centrifuged (4°C, 4000 rpm, 10 min) to obtain the above The clear liquid is the crude enzyme liquid.

[0025] 5. Ninhydrin reaction

[0026] Dissolve 0.05mmol Z-Phe-Tyr in 20mL 50mM sodium acetate buffer (pH3.0) as a substrate, quantitatively pipette 1.8mL substrate (Z-Phe-Tyr, 2...

Embodiment 2

[0029] 1. Strain: Aspergillus niger LW-1 strain.

[0030] 2. Solid-state fermentation medium: 10g of fermentation base material (i.e. 8g of bran, 1g of bean cake powder, 1g of rapeseed cake powder) in a 250mL Erlenmeyer flask, (NH 4 ) 2 SO 4 75mg, MgSO 4 7.5 mg, CaCl 2 10mg, KH 2 PO 4 40mg, tap water 13mL, natural pH.

[0031] 3. Solid-state fermentation conditions: sterilize at 121°C for 40 minutes, inoculate one fungus LW-1 bran koji seeds after cooling, and culture at 30°C for 96 hours, during which the koji is turned once at 24 hours and 48 hours respectively.

[0032] 4. Crude enzyme solution extraction:

[0033]After solid-state fermentation, 1 g of the culture was taken, and 10 mL of 50 mM sodium citrate buffer (pH 5.0) was added to each Erlenmeyer flask, milled for 10 min, filtered after resting for 30 min, and centrifuged (4°C, 4000 rpm, 10 min) to obtain the above The clear liquid is the crude enzyme liquid.

[0034] 5. Ninhydrin reaction

[0035] Disso...

Embodiment 3

[0038] 1. Strain: Aspergillus niger LW-1 strain.

[0039] 2. Solid-state fermentation medium: 10g of fermentation base material (i.e. 8g of bran, 1g of bean cake powder, 1g of rapeseed cake powder) in a 250mL Erlenmeyer flask, (NH 4 ) 2 SO 4 75mg, MgSO 4 7.5 mg, CaCl 2 10mg, KH 2 PO 4 40mg, tap water 13mL, natural pH.

[0040] 3. Solid-state fermentation conditions: sterilize at 121°C for 40 minutes, inoculate one fungus E001 bran koji seeds after cooling, and culture at 30°C for 96 hours.

[0041] 4. Crude enzyme solution extraction:

[0042] After solid-state fermentation, take 1 g of the culture, add 10 mL of 50 mM sodium citrate buffer (pH 5.0) to each Erlenmeyer flask, grind for 10 min, filter after resting for 6 h, and centrifuge (4°C, 4000 rpm, 10 min) to obtain the above The clear liquid is the crude enzyme liquid.

[0043] 5. Ninhydrin reaction

[0044] Dissolve 0.05mmol Z-Phe-Tyr in 20mL 50mM sodium acetate buffer (pH3.0) as a substrate, quantitatively ...

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PUM

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Abstract

The invention provides a method for producing carboxypeptidase by aspergillus niger, belonging to the technical field of biological engineering. The method comprises the following steps: taking agricultural and sideline products such as the bran, the bean cake flour, the corn flour and the like as the fermentation base material, optimizing enzyme liquid extraction time by properly changing the proportion of a carbon source and a nitrogen source in a culture medium, and performing the solid fermentation by the aspergillus niger LW-1 strain. The method provides the solid fermentation medium prescription and the culture condition for the small-scale production in the laboratory; and the carboxypeptidase activity of the mature leaven material achieves 3194.4U / g-5584.2U / g. The method has the characteristics of being simple in equipment, small in investment, fast in effect, low in production cost, environment-friendly, higher in carboxypeptidase activity and the like, is good for the development and the application of the carboxypeptidase, and is higher in economic value and practical value.

Description

technical field [0001] The invention relates to a new method for producing carboxypeptidase, belonging to the technical field of bioengineering. Specifically, it refers to a new technology of using Aspergillus niger (Aspergillus niger) LW-1 strain to produce carboxypeptidase by solid-state fermentation. Background technique [0002] Carboxypeptidase (Carboxypeptidase) is a kind of peptide chain terminal hydrolase, which acts on the free carboxyl terminal of the peptide chain to release a single amino acid, which has a wide range of applications in the fields of medicine and food industry. In the field of medicine, it can be used for the production of recombinant human insulin, the prodrug treatment of tumor antibody-directed enzymes, and as a serum marker for diagnosing the severity of acute pancreatitis, etc.; in the food industry, carboxypeptidase can act on bitter peptides and hydrolyze peptides Hydrophobic amino acid at the carboxyl end of the chain to achieve the purpo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/48C12R1/685
Inventor 邬敏辰闵柔吴静李剑芳陈伟唐存多
Owner JIANGNAN UNIV
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