Organic solvent resisting proteinase high-yield bacterium, gene and application of the organic solvent resisting proteinase

A technology resistant to organic solvents and proteases, applied in applications, genetic engineering, plant genetic improvement, etc., can solve the problems of enzyme variability and inactivation, low tolerance concentration, low yield, etc., and achieve easy purification and organic solvent tolerance Strong, high-yield effects

Inactive Publication Date: 2010-07-21
NANJING TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The organic solvents in the three systems all affect the enzyme, and the enzyme is volatile and inactivated
3. Using physical and chemical modification methods such as immobilization, embedding, and macromolecule modification to improve the activity and stability of enzymes in organic media; 4. Using genetic modification to improve the stability of enzymes in organic solvents; however, the above methods can only be used to a certain extent. Improve the activity and stability of enzymes in organic solvents
Most of the organic solvent-resistant protease-producing bacteria reported so far are concentrated in Pseudomonas and Bacillus, and these proteases have obvious tolerance to various organic solvents. The solvent tolerance concentration of the reported protease is low (about 25%-v / v), and the yield is relatively low (all lower than 2000U / mL), so far there is no report of organic solvent-resistant protease-producing bacteria

Method used

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  • Organic solvent resisting proteinase high-yield bacterium, gene and application of the organic solvent resisting proteinase
  • Organic solvent resisting proteinase high-yield bacterium, gene and application of the organic solvent resisting proteinase
  • Organic solvent resisting proteinase high-yield bacterium, gene and application of the organic solvent resisting proteinase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] This experiment illustrates a screening procedure for the production of natural strains resistant to organic solvent proteases.

[0029] Extremophiles resistant to organic solvents were screened from oily soil samples using different concentrations of cyclohexane, toluene, acetone and other organic solvents as screening pressures. The milk agar plate medium was adopted, and the specific formula was: tryptone 5g / L, yeast powder 3g / L, skimmed milk powder 25g / L, agar 12g / L. The screened microorganisms resistant to organic solvents 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 10 strains of extremophile resistant to organic solvents with high protease production.

[0030] In order to further test the solvent tolerance of the secreted protease, the protease production ability of the 10 strains and the organic solvent r...

Embodiment 2

[0033] This experiment illustrates the biological properties of the organic solvent-resistant extremophile Pseudomonas aeruginosa PT121.

[0034] Physiological and biochemical properties

[0035] Gram staining of the strain showed that the strain was a Gram-negative strain without spores. Observation with a transmission electron microscope (Figure 1) showed that the strain was a monoflagellate bacterium with a size of 0.5 μm×1-2 μm. After growing in broth medium for 24 hours, the colony size is 2-3mm, the growth range is 30-40°C, the optimum growth temperature is 37°C, the growth pH is 6-11, and the optimum pH is 8.0. The characteristic is that the results of catalase reaction, oxidase reaction, nitric acid reduction reaction, gelatin reaction, glucose, D-xylose and D-fructose utilization are positive, and the results of lactose, maltose and mannose utilization are negative. grow under oxygen conditions. Some physiological and biochemical characteristics of the bacteria are ...

Embodiment 3

[0041] This experiment illustrates the purification procedure for organosolvent-tolerant proteases.

[0042] First, culture the strain in the enzyme-producing medium for 72 hours, centrifuge at 10,000rmp at 4°C for 15 minutes, take the supernatant as the crude enzyme solution, put the crude enzyme solution in an ice bath, and slowly add NaCl while stirring to a final concentration of 1.6 mol / L. Add the treated supernatant to the Phenyl sepharose Fast Flow chromatography column equilibrated with 1.2mol / L NaCl, 0.05M Tris-HCl buffer (pH8.0), and use 0.05M Tris-HCl (pH8.0 , NaCl content is 1mol / L) buffer solution to carry out elution, collect eluate. The supernatant of the fermentation broth was only separated by one-step chromatographic column, and the analysis by SDS-PAGE (Figure 3) showed that the purified protease had reached electrophoretic purity. This protease subunit has a molecular weight of approximately 33 kDa. The recovery rate and purification multiple of protease...

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Abstract

The invention discloses a tolerance organic solvent protease high yield bacterial strain, protease gene and the protease application in organic phase catalysis peptides synthesis. The bacterial strain sorting naming is Pseudomonas aeruginosa PT121, preserving registration number is CCTCC M 208029, and is gram negative bacterial strain and various organic solvent which can resist certain concentration. The organic solvent-tolerance protease encode gene produced from the bacterium is separated and cloned by the invention which comprises nucleotide sequence showed as SEQ ID NO:1 and amino acid sequence showed as SEQ ID NO:2. The tolerance organic solvent protease has the habitude of high yield, high specific activity, strong solvent tolerance, wide acting pH range and high temperature resistant. The protease is provided with organic phase catalysis peptides synthesis etc. industry applications value.

Description

technical field [0001] The invention relates to an organic solvent-resistant protease high-yielding bacterium, its organic solvent-resistant protease gene, and the application of the organic solvent-resistant protease in organic phases to catalyze peptide synthesis, belonging to the fields of microbiology and enzymology. technical background [0002] Protease refers to a class of enzymes that can catalyze the hydrolysis of peptide bonds. Since it was tested as an additive ingredient in detergents in 1913, it has attracted widespread attention due to its important commercial value. Due to the huge market demand, protease has gradually become one of the three major industrial enzymes. Today, the production of protease has occupied more than 40% of the enzyme market, and it is widely used in detergent, food, medicine, leather, organic synthesis, waste treatment and other fields. Although proteases widely exist in almost all organisms, since the proteases produced by microorgan...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N9/52C12N15/57C12P21/00C12R1/385
Inventor 何冰芳欧阳平凯唐啸宇何小丹潘瑶姚忠柏中中
Owner NANJING TECH UNIV
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