Organic solvent resisting lipase, application thereof and bacteria for producing same

A technology resistant to organic solvents and lipases, applied in applications, bacteria, microorganisms, etc., can solve the problems of low tolerance to organic solvents, low enzyme production, and difficult purification, etc., to achieve strong tolerance, wide pH range, and easy The effect of purification

Inactive Publication Date: 2010-06-16
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there have been articles disclosing the organic solvent-resistant lipase produced by some extremophiles, the organic solvent tolerance is low, the enzyme yield is low, and it is not easy to purify, which affects its application in industrial catalysis

Method used

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  • Organic solvent resisting lipase, application thereof and bacteria for producing same
  • Organic solvent resisting lipase, application thereof and bacteria for producing same
  • Organic solvent resisting lipase, application thereof and bacteria for producing same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] This experiment illustrates the screening procedure for natural strains producing organic solvent-resistant lipase.

[0025] The following method was used for the initial screening: use different concentrations of cyclohexane, toluene, DMSO and other organic solvents as the screening pressure to obtain organic solvent-resistant extremophiles from oily soil samples, and then use olive oil rhodamine B plates to screen out 6 strains of fat Enzyme-producing strains. The specific formula of olive oil rhodamine B plate is: yeast extract 1g / L, corn steep liquor 5mL / L, K 2 HPO 4 1g / L, MgSO 4 ·7H 2 O 0.5g / L, olive oil 60mL / L, Rhodamin B 0.024g / L.

[0026] In order to obtain excellent organic-solvent-resistant lipase-producing bacteria, the above-mentioned 6 strains were re-screened by testing the enzyme-producing ability of shake flasks and the stability of lipase in organic solvents. Inoculate 6 strains into the enzyme-producing fermentation medium, the specific formula i...

Embodiment 2

[0030] This experiment illustrates the biological properties, identification and enzyme production conditions of the organic solvent-resistant lipase-producing strain LX1.

[0031] Biological properties of the strain LX1: Gram staining showed that the strain was a Gram-negative strain without spores. After growing in broth medium for 24 hours, the colony size and diameter are 1.5mm-2mm, the growth temperature range is 24°C-37°C, the optimum growth temperature is 27°C, the growth pH range is 6.0-11.0, and the optimum growth pH is 8.0, its physiological and biochemical characteristics are manifested in that the results of catalase reaction, oxidase reaction, nitric acid reduction reaction and gelatin reaction are positive, and it grows under aerobic conditions.

[0032] Species identification of strain LX1: BIOLOG automatic bacterial identification instrument and 16S rDNA sequence analysis showed that the strain was Pseudomonas aeruginosa, and named it Pseudomonas aeruginosa LX1...

Embodiment 3

[0035] This experiment illustrates the purification procedure for organic solvent-resistant LX1 lipase.

[0036] After culturing Pseudomonas aeruginosa LX1 in the enzyme-producing medium for 30 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; Ammonium sulfate was precipitated, and the supernatant was taken after centrifugation, and then precipitated with 50% saturated ammonium sulfate, and the precipitate was dissolved with 0.01M Tris-HCl buffer solution of pH 7.10, and dialyzed to remove salt. Purify the enzyme solution obtained from the above treatment with a DEAE-Sepharose FF ion-exchange chromatography column, elute with 0.01M Tris-HCl (pH 7.10, NaCl content is 1mol / L) buffer, and collect the lipase activity peak . By SDS-PAGE electrophoresis ( figure 1 ), found that the two-step purified organic solvent-resistant lipase (named as organic solvent-resistant LX1 lipase) had reach...

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Abstract

The invention aims at providing bacteria for producing organic solvent resisting lipase, the organic solvent resisting lipase produced from the bacteria and the application of the organic solvent resisting lipase to the enzymatic reaction for the synthesis of the biodiesel in the organic phase, in particular to the ester exchange reaction for the synthesis of the biodiesel. The bacteria of pseudomonas aeruginosa LX1 is obtained through screening, the organic solvent resisting LX1 lipase produced from the pseudomonas aeruginosa LX1 has the amino acid sequence showed in the sequence identifier (SEQ ID) No. 2, and the organic solvent resisting LX1 lipase encoding gene has the nucleotide sequence showed in the SEQ ID No. 2. The organic solvent resisting LX1 lipase is used for catalyzing the substrate of soybean oil and exchanging the soybean oil and the ester methanol in the organic solvent tert-butanol system or the solvent-free system to synthesize the clean energy of biodiesel which has the conversion rate of 80-90 percent.

Description

technical field [0001] The invention relates to an organic solvent-resistant lipase. The application of the organic solvent-resistant lipase in organic phase to catalyze the synthesis of biodiesel belongs to the field of microbiology and enzymology. technical background [0002] Lipase (Lipase, EC 3.1.1.3) is a class of hydrolytic enzymes that can catalyze the hydrolysis of natural oil substrates to produce diglycerides, monoglycerides, fatty acids and glycerol, and can catalyze the synthesis, transesterification, ammonia Therefore, it is widely used in fine chemical industry, washing, medicine, food, paper making, leather processing, textile and feed industry and other fields. [0003] Many substrates of lipase are insoluble, so the reaction in the non-aqueous phase is beneficial to increase the solubility of the substrate, thereby increasing the yield and reducing the cost. Non-aqueous catalysis also has the following advantages: it has a high degree of stereoselectivity ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/20C12N15/55C12P7/64C12R1/385
CPCY02E50/13Y02E50/10
Inventor 何冰芳刘晓宁季青春肖素静曹艳任伟吴斌
Owner NANJING UNIV OF TECH
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