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Organic solvent tolerant protease and producing strain thereof

A technology resistant to organic solvents and organic solvents, applied in the direction of bacteria, microorganisms, hydrolytic enzymes, etc., can solve the problems that have not been seen, and achieve the effect of good organic solvent tolerance

Inactive Publication Date: 2009-11-25
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, there is no report of Serratia marcescens producing protease resistant to organic solvents

Method used

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  • Organic solvent tolerant protease and producing strain thereof
  • Organic solvent tolerant protease and producing strain thereof
  • Organic solvent tolerant protease and producing strain thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] This experiment illustrates the screening procedure for natural strains producing organic solvent-resistant proteases.

[0027] Preliminary screening: Obtain strains that can tolerate organic solvents and produce protease. Using different concentrations of cyclohexane, DMF, DMSO and other organic solvents as the screening pressure, extremophiles resistant to organic solvents were screened from oily soil samples. Then, the milk agar plate medium was used to screen the protease-producing strains from the above-mentioned extremophiles resistant to organic solvents. According to the ratio of the colonies formed by each strain on the milk agar plate medium to the diameter of the transparent circle, 6 protease high-producing bacteria were preliminarily screened ( figure 1 ). Because these 6 strains can tolerate organic solvents, the protease produced by them is also very likely to have tolerance to organic solvents.

[0028]Re-screening: By testing the protease-producing a...

Embodiment 2

[0033] This experiment illustrates the physiological and biochemical properties and identification of organic solvent-resistant protease-producing bacteria MH6

[0034] The strain Serratia marcescens MH6 is a Gram-negative short bacilli strain, without spores, with perinatal flagella, and the size is 1 μm×1.5~0.7×1.0 μm. After growing in LB medium for 24 hours, the colony is red and the size of the colony is 1.5-2.5 mm. The growth temperature range of the strain is 20-45 DEG C, the optimum growth temperature is 35 DEG C, the growth pH is 5-9, the optimum pH is 8.0, and it grows under aerobic conditions. See its physiological and biochemical properties (Table 1).

[0035] LB medium formula: tryptone 10g / L, yeast powder 5g / L, NaCl 1.0g / L.

[0036] Table 1 Some physiological and biochemical characteristics of strain MH6

[0037] feature

[0038] oxidase reaction

[0039] Note: + (positive): can be used; - (negative): can not be used.

[0040] After BIOLOG ...

Embodiment 3

[0047] This experiment illustrates the purification procedure of the organosolvent-resistant protease produced by the strain Serratia marcescens MH6.

[0048] The strain Serratia marcescens MH6 was cultured in the enzyme-producing medium for 28 hours, the obtained fermentation liquid was centrifuged (9000 rpm, 4° C.) for 20 minutes, and the supernatant was taken as the crude enzyme liquid.

[0049] Preliminary purification of organic solvent-resistant protease by ammonium sulfate precipitation. Place the crude enzyme solution in an ice bath, and slowly add (NH 4 ) 2 SO 4 to 50% saturation, then centrifuge (9000rmp, 4°C) for 20min, take the supernatant and continue to add (NH 4 ) 2 SO 4 To 60% saturation, centrifuge again (9000rmp, 4°C) for 20min, and dissolve the obtained precipitate with Tris-HCl buffer (50mmol / L, pH8.0). After testing, the recovery rate of protease activity in the precipitate reached 69.1%.

[0050] The organic solvent-resistant protease was further p...

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Abstract

The invention provides a serratia strain with organic solvent tolerance, which is named Serratiamarcescens MH6, and has a preservation registration number of CCTCC M 208205. The invention also provides organic solvent tolerant protease produced by the strain, the organic solvent tolerant protease has an amino acid sequence shown in SEQ ID NO: 2, and an encoding gene of the organic solvent tolerant protease has a nucleotide sequence shown in SEQ ID NO: 1. The strain Serratiamarcescens MH6 is an extreme microorganism capable of tolerating organic solvents, can produce the organic solvent tolerant protease, and enrich the variety of extreme microorganisms and the sources of the organic solvent tolerant protease. The protease has good organic solvent tolerance compared with other natural proteases, and is expected to be applied to the synthesis of polypeptides in nonaqueous phases and the theoretical research related to a mechanism of organic solvent tolerance of the protease.

Description

technical field [0001] The invention relates to an organic solvent-resistant protease producing strain and the organic solvent-resistant protease, belonging to the fields of microbiology and enzymology. Background technique [0002] Proteases are widely used in detergents, food, pharmaceuticals, tanning, diagnostic reagents, sewage treatment and other fields. In 1984, Klibanov et al. found that protease was still active in some organic solvents for a long time, and could catalyze the reaction of synthesizing peptide bonds. This discovery promoted the rise of non-aqueous enzymology. A large number of studies have shown that enzymatic reactions in organic solvents have many advantages, such as greatly enhanced solubility of organic substrates, improved enzyme stability, prevention of microbial contamination, and easy purification of products. More importantly, the use of protease to catalyze the synthesis of peptides in organic solvents can avoid the racemization of amino ac...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N9/52C12N15/57C12R1/425
Inventor 何冰芳吴斌万茂华柏中中姚忠
Owner NANJING UNIV OF TECH
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