Organic solvent resistant protease producing strain, gene of organic solvent resistant protease produced thereby and application of organic solvent resistant protease

A technology resistant to organic solvents and proteases, applied in applications, genetic engineering, plant genetic improvement, etc., can solve the problems of strong substrate selection specificity, low assay tolerance concentration, low enzyme yield, etc., to achieve high yield, The effect of high specific activity and huge application potential

Inactive Publication Date: 2011-04-20
NANJING UNIV OF TECH
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  • Abstract
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AI Technical Summary

Problems solved by technology

[0005] At present, most of the reported organic solvent-resistant protease-producing bacteria are Pseudomonas (Pseudomonas), but most of the reported protease-related solvent determination tolerance concentration Low (about 25%, v / v), and there are disadvantages such as low enzyme yield, difficult purification, strong substrate selection specificity, etc., thus affecting its application in industrial catalysis

Method used

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  • Organic solvent resistant protease producing strain, gene of organic solvent resistant protease produced thereby and application of organic solvent resistant protease
  • Organic solvent resistant protease producing strain, gene of organic solvent resistant protease produced thereby and application of organic solvent resistant protease
  • Organic solvent resistant protease producing strain, gene of organic solvent resistant protease produced thereby and application of organic solvent resistant protease

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Effect test

Embodiment 1

[0026] Use different concentrations of organic solvents such as cyclohexane and toluene as screening pressure to obtain organic solvent-resistant extremophiles from oily soil samples: use milk agar plate medium, the specific formula is (g / L): tryptone 5, yeast powder 3. Skim milk powder 25, agar 12. The screened organic solvent-resistant microorganisms were inoculated on milk agar plates, and the strains with high protease production were initially screened according to the ratio of the colony to the size of the transparent circle. This method screened 13 strains of extremophile resistant to organic solvents with high protease production.

[0027] In order to further test the solvent tolerance of the protease obtained from the preliminary screening, the protease production ability of the 13 strains and the organic solvent resistance of the protease produced were comprehensively tested. Inoculate the protease-producing bacterial strain obtained by preliminary screening into th...

Embodiment 2

[0032] Biological properties of the strain WQ9-2: Gram staining showed that the strain was a Gram-positive strain with spores and grew under aerobic conditions. After growing in broth medium for 24 hours, the colony size and diameter are 3mm-5mm, the growth temperature range is 20°C-40°C, the optimum growth temperature is 35°C, the growth pH range is 6.0-11.0, and the optimum growth pH is 8.0 , its physiological and biochemical characteristics are negative in oxidase reaction, positive in the utilization of glucose, maltose, sucrose and D-fructose, and negative in the utilization of D-xylose and lactose.

[0033] Species identification of strain WQ9-2: The BIOLOG automatic bacterial identification instrument and 16SrDNA sequence analysis showed that the strain belonged to the genus Bacillus cereus, and it was named Bacillus cereus WQ9-2.

[0034] Study on fermentation conditions of Bacillus cereus WQ9-2 protease production: through single factor experiment and response surface...

Embodiment 3

[0037] After culturing Bacillus cereus WQ9-2 in the enzyme-producing medium for 48 hours, the fermentation broth was centrifuged at 10,000 rpm and 4°C for 10 minutes, and the supernatant was taken as the crude enzyme solution; the crude enzyme solution was placed in an ice bath and collected by ethanol precipitation 45%-75% (v / v) part of the precipitated protein was then dissolved with 0.05M Tris-HCl buffer solution of pH 8.5, and centrifuged at 10,000 rpm at 4°C for 10 min to remove insoluble miscellaneous proteins. The enzyme solution obtained from the above treatment was purified with a DEAE-Sepharose FF ion-exchange chromatography column, and eluted with 0.05M Tris-HCl (pH 8.5) buffer solution to collect protease activity peaks. By SDS-PAGE electrophoresis ( figure 1 ), found that the two-step purified organic solvent-resistant protease (named as organic solvent-resistant WQ9-2 protease) had reached electrophoretic purity, and the molecular weight of the protease subunit w...

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Abstract

The invention relates to an organic solvent resistant protease producing strain, a gene of organic solvent resistant protease and an application of the organic solvent resistant protease, in particular relating to the organic solvent resistant protease producing strain as well as the gene of the organic solvent resistant protease thereby and the application of the organic solvent resistance protease in catalytic synthesis of small peptides in nonaqueous phases, belonging to the fields of microbiology and enzymology. Bacterial strains are named as Bacilluscereus WQ9-2 by classification, are gram-positive strains and can resist a plurality of organic solvents with a certain concentration. The protease-coding gene which is separated and cloned on organic solvent resistant protease produced by the producing strain is provided with a nucleotide sequence shown as SEQ ID NO:1, and an amino acid sequence shown as SEQ ID NO:2. The protease has the characteristics of high yield, high specific activity, wide and strong tolerance and the like. The protease has industrial application value for the catalytic synthesis of the small peptides in the nonaqueous phases.

Description

technical field [0001] The invention relates to an organic solvent-resistant protease-producing bacterium, the gene of the organic-solvent-resistant protease produced therein and its application in non-aqueous phase to catalyze the synthesis of small peptides, belonging to the fields of microbiology and enzymology. Background technique [0002] Protease refers to a class of enzymes that can catalyze the hydrolysis of peptide bonds. Due to its important commercial value, it has attracted widespread attention and has gradually become one of the three major industrial enzymes. Today, the production of protease has occupied more than 40% of the commercial enzyme market, and it is widely used in detergent, food, medicine, leather, organic synthesis, waste treatment and other fields. Although proteases exist in almost all organisms, since the proteases produced by microorganisms are mainly extracellular enzymes, they are more suitable for industrialization compared with proteases ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/54C12N15/57C12P21/06C12R1/085
Inventor 何冰芳欧阳平凯许家兴孙洪林吴斌
Owner NANJING UNIV OF TECH
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