Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Low-temperature organic solvent tolerant lipase derived from bacillus mojavensis

A lipase and amino acid technology, applied in the field of low-temperature organic solvent-resistant lipase, can solve the problem of no bacillus lipase, etc., and achieve the effect of broad organic solvent tolerance, good tolerance, and good heat resistance

Active Publication Date: 2014-01-22
WILMAR SHANGHAI BIOTECH RES & DEV CENT
View PDF8 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] It is reported that more than 70 kinds of Bacillus lipases have been successfully isolated and purified (M Guncheva, D Zhiryakova. J Mol catal B: Enzym, 2011, 68: 1-21), among which they have good stability at low and medium temperatures There are also many documents and patents on the lipase derived from bacillus resistant to organic solvents, but there is almost no report on the lipase from bacillus that has these three characteristics simultaneously

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Low-temperature organic solvent tolerant lipase derived from bacillus mojavensis
  • Low-temperature organic solvent tolerant lipase derived from bacillus mojavensis
  • Low-temperature organic solvent tolerant lipase derived from bacillus mojavensis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1: Cloning of lipase gene

[0056] 1. Acquisition of conserved sequences

[0057] According to the conserved Oxyanion hole of the lipase gene and the conserved sequences of the Activity center P(V / I)V(M / L)(V / L)HG and AHS(M / Q)GG, the designed degenerate primers are shown in Table 1:

[0058] Table 1 Primer Information

[0059]

[0060]

[0061] The genome of Bacillus mojave was extracted by SDS method (Fang Weiguo, Chinese Journal of Applied and Environmental Biology, 2002, 8(3): 305-307). Upstream primers and downstream primers were randomly combined, and there were 12 primer pairs in total. Using the genome as a template, the following degenerate PCR reactions were performed using the above primer pairs. PCR system: rTaq 0.5uL, 10× PCR buffer 5uL, dNTP mix 8uL, primer 11uL, primer 21uL, genome 0.3uL, water 34uL. Reaction conditions: 94℃ for 1s, 94℃ for 1min, 37℃ for 1s, 37℃ for 30s, 72℃ for 2min20s, 72℃ for 1min, 35 cycles (95℃ for 1min, 52℃ for 1min, ...

Embodiment 2

[0076] Example 2: Construction of lip2-2 recombinant Escherichia coli

[0077] 1. Cloning of lip2-2 Expressed Gene

[0078] Using the Bacillus mojave genome as a template, PCR was performed using the cloning primers lip-U / lip-D in the table. The PCR reaction system was shown in Example 1. 30s, 1min at 72°C), 10min at 72°C, and hold at 4°C.

[0079] 2. Enzymatic cleavage of genes and vectors and refinement of enzymatic cleavage products

[0080] The above PCR product was recovered by gel, and the PCR product and plasmid pET24a were double digested with EcoRI and Hind III. The digestion system: 20.0uL gel recovery product (or vector), 21.0uL ddH 2 O, 5.0uL 10×M Buffer, 2.0uL EcoRI and 2.0uL HindIII, digested overnight at 37°C. (2 tubes of 50uL restriction enzyme digestion system and 2 tubes of vector were used for the recovery product of gel injection), and the restriction enzyme digestion product was purified with Omega PCR product purification kit.

[0081] 3. ligation ...

Embodiment 3

[0087] Example 3: Induction and expression of recombinant Escherichia coli and isolation and purification of target protein

[0088] 1. Inducible expression of recombinant bacteria

[0089] After inoculating a loop of BL21 (pET24a-lip2-2) into LB liquid medium containing kanamycin for about 8 hours, inoculate BL21 (pET24a-lip) in 50mL LB liquid medium ( containing 50 μg / mL kanamycin), 37 ° C, 200 rpm shaking culture, while the empty host BL21 (DE3) as a control. Waiting for OD 600 Increase to about 0.4-0.6, add IPTG to a final concentration of 1.0 mM, and culture at 37° C. and 200 rpm for 5 h to induce the expression of exogenous proteins.

[0090] 2. Lipase activity determination method

[0091] Low temperature lipase activity was determined by colorimetric method. Taking p-nitrophenyl palmitate (p-NPP) as the substrate, the enzyme activity was calculated by the amount of p-nitrophenol (p-NP) produced by the enzymatic hydrolysis of unit volume of enzyme solution in unit...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention provides a lipase derived from Bacillus mojavensis WBRD1.10062302, CGMCC4961. The lipase provided by the invention has better tolerance on multiple organic solvents, such as methanol, ethanol, acetone, acetonitrile, dimethyl sulfoxide, normal hexane, and the like, thereby having application value in multiple industrial fields of biological diesel oil, medical industry, food, oil chemical industry, environmental protection, washing agents, spinning, papermaking, and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a low-temperature organic solvent-resistant lipase derived from Bacillus mojave. Background technique [0002] Lipase (EC3.1.1.3) is a special class of ester bond hydrolase, widely present in animal tissues, plants and microorganisms. Lipase has the biological function of catalyzing hydrophobic oils into water-soluble fatty acids and glycerol, maintaining high catalytic activity and strong stability in inorganic solvents, and lipase's broad-spectrum / special specificity for substrates, It endows lipase with great application value in food and oil processing, detergent, biodiesel, ester bond compound synthesis and chiral drug synthesis and other industries (CN201010599106). [0003] In the food and oil processing and oleochemical industries, lipase can be used to prepare fatty acids and glycerol, modify oil, prepare special oils, improve the yield of neutral oil, and prepa...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N9/20C12N15/55C12N15/63C12N1/21C12N15/70C11D3/386C02F3/00C02F3/34C12P7/64C10L1/02C12R1/19C12R1/07
CPCY02E50/13C02F3/342C11D3/38627C12N9/20C12Y301/01003Y02E50/10
Inventor 陈苗苗徐正军周美凤常桂芳许骏
Owner WILMAR SHANGHAI BIOTECH RES & DEV CENT
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com