Method for preparing carboxypeptidase from aspergillus usamii

A technology of Aspergillus usami and carboxypeptidase is applied in the field of solid-state fermentation of Aspergillus usami E001 strain to produce carboxypeptidase and carboxypeptidase. and other problems, to achieve the effect of low production cost, simple equipment and low investment

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 usamii E001 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, 13mL of tap water, 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, 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 30 min, and centrifuge (4°C, 4000 rpm, 10 min). The supernatant is the crude enzyme solution.

[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, 2.5mmol / L) solution into a 5mL centrif...

Embodiment 2

[0029] 1. Strain: Aspergillus usamii E001 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 E001 bran koji seeds after cooling, and culture at 30°C for 96 hours.

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

Embodiment 3

[0038] 1. Strain: Aspergillus niger E001 strain.

[0039] 2. Solid-state fermentation medium: 10g of fermentation base material (9g of bran, 1g of rapeseed meal) in a 250mL Erlenmeyer flask, (NH 4 ) 2 SO 4 100mg, MgSO 4 10mg, CaCl 2 10mg, KH 2 PO 4 50mg, 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 pipette 1.8mL substrate (Z-Phe-Tyr, 2...

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PUM

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Abstract

The invention relates to a method for preparing carboxypeptidase from aspergillus usamii and belongs to the technical field of biological engineering. In the method, agricultural and sideline products such as brans, bean cake powder, corn meal and the like are used as fermentation base materials; and the carboxypeptidase is prepared by suitably changing the ratio of a carbon source to a nitrogen source in a culture medium and a crude enzyme extraction time and utilizing the aspergillus usamii E001 to carry out solid fermentation. The invention provides a solid fermentation culture medium formula and a culturing condition for experimental production in a laboratory. The activity of the carboxypeptidase for maturely fermenting yeast materials reaches 3,452.4 to 5,321.6U per gram of brans. The method has the characteristics of simple equipment, low investment, high response, low production cost, environment friendliness, higher activity of the carboxypeptidase and the like, is beneficial to development and utilization of the carboxypeptidase and has higher economic and practical values.

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 usamii (Aspergillus usamii) E001 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 acids at the carboxyl end of the chain to achieve the pu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/48C12R1/66
Inventor 吴静闵柔邬敏辰李剑芳陈伟唐存多
Owner JIANGNAN UNIV
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