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Organic solvent resisting basified protease producing strain, gene and application thereof

A technology resistant to organic solvents and proteases, applied in applications, genetic engineering, plant genetic improvement, etc., can solve the problems of protease loss of activity and low stability, and achieve strong solvent tolerance, high specific activity, and a wide range of pH Effect

Inactive Publication Date: 2008-07-09
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The optimal reaction pH of the published solvent-tolerant protease is 8.0-8.5, and the protease loses its activity when the pH of the reaction system is >10; and the tolerance of this type of protease to various solvents (hydrophilic and hydrophobic) It shows that its relevant tolerance concentration is about 25% (v / v), and its stability is relatively low
There is no report of Bacillus licheniformis producing organic solvent-resistant protease

Method used

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  • Organic solvent resisting basified protease producing strain, gene and application thereof
  • Organic solvent resisting basified protease producing strain, gene and application thereof
  • Organic solvent resisting basified protease producing strain, gene and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0033] This experiment illustrates the screening procedure for natural strains producing organosolv-resistant proteases.

[0034] Organic solvent-resistant extremophiles were screened from oily soil samples and other samples using different concentrations of cyclohexane, toluene, acetone and other organic solvents as the selection pressure. Using SMA medium, the specific formula is: skimmed milk powder 12g / L, nutrient agar 13.8g / L, inoculate organic solvent-resistant extremophiles on SMA plates, and obtain strains that can produce high-activity protease according to the ratio of colony to transparent circle size. Eight strains of organic solvent-resistant extremophiles with higher protease activity were screened by this method.

[0035] In order to further test the solvent tolerance of the secreted proteases, the protease activity of the eight strains and the organic solvent resistance of the proteases produced were comprehensively tested. Inoculate the producing bacteria wit...

Embodiment 2

[0038] This experiment illustrates the biological properties of organic solvent-resistant extremophile Bacillus licheniformis YP1A.

[0039] Physiological and biochemical properties

[0040] Gram stain observation of the bacteria indicated that the strain was G + Bacillus, with spores, observed by transmission electron microscope showed that the strain had pericytic flagella, the size of which was 0.5μm×2~3μm. After growing in broth medium for 14 hours, the colony size is 1.5-2 mm, the growth temperature range is 25-42 °C, the optimum temperature is 35 °C, the growth pH is 7-12, and the optimum pH is 8.5. Its physiological and biochemical characteristics It is shown that the utilization of citric acid, D-xylose and mannitol is positive for the contact enzyme reaction, and the utilization of D-arabinose, α-D-lactose and D-galactose is negative, and it grows under aerobic conditions. Part of the physiological and biochemical identification experimental characteristics of the b...

Embodiment 3

[0046] This experiment illustrates the purification procedure for organosolv-resistant proteases.

[0047] First, precipitation of ammonium sulfate resistant to organic solvent protease was carried out. After the strain was cultured in the enzyme production medium for 48 hours, the supernatant enzyme solution was taken by centrifugation. The crude enzyme solution was placed in an ice bath, and ammonium sulfate was slowly added to 80 ° % Saturation, let stand overnight at 4°C. Then centrifuge at 13,000 rpm for 30 min, and add the enzyme solution obtained by 80% ammonium sulfate precipitation to a solution containing 1 mol (NH 4 ) 2 SO 4 , 50mmol / L Glycine-NaOH buffer A (pH9.0) equilibrated Phenyl sepharose CL-4B chromatography column, using A, B buffer mixture for stage elution (buffer B is 50mmol / L Glycine-NaOH, pH9.0). Protease activity was present in fractions collected at 50% B. with SP Sepharose Tm For FF strong cation exchange chromatography, the pH of the upper co...

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Abstract

The invention discloses an organic solvent fastness alkaline proteinase generation strain and the gene and an application for catalyzing peptide to synthesize and resolve racemic amine and amino acid in the organic phase. The bacterial categorization naming is Bacillus licheniformis YP1A whose preservation register number is CCTCC No: M 207021 as Gram-positive bacillus and can tolerate certain density of a plurality of organic solvents. The invention separates and clones to obtain the coding gene of the proteinase generation strain, which is provided with nucleic acid sequence which is represented as SEQ ID NO: 1 and amino acid sequence as SEQ ID NO: 2. The organic solvent fastness alkaline proteinase is high in specific activity, strong in solvent tolerance, wide in action pH scale, strong in thermostable and strong alkalinity tolerance and the like. The proteinase can be applied in the application such as peptide synthesis in the organic phase, racemic amine and amino acid resolution and the like.

Description

technical field [0001] The invention relates to an organic solvent-resistant alkaline protease producing bacterium, its organic solvent-resistant alkaline protease gene, and its application in organic phases to catalyze peptide synthesis, split racemic amines and amino acids, belonging to microbiology and enzymology field. Background technique [0002] Since protease was tested as an additive ingredient of detergent in 1914, it has attracted widespread attention due to its important commercial use. Today, the output of protease accounts for more than 40% of the enzyme market, and is widely used in detergents, food, pharmaceuticals, leather, diagnostic reagents, sewage treatment and other fields. Moreover, according to statistics, after 2005, the amount of protease has increased significantly. Proteases widely exist in all organisms such as animals, plants, fungi, and prokaryotes, among which proteases derived from microorganisms account for more than 2 / 3 of the total produ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/56C12N15/57C12P21/06C12P41/00C12R1/10
Inventor 何冰芳欧阳平凯李霜柏中中姚忠
Owner NANJING UNIV OF TECH
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