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A low-temperature organic solvent-resistant lipase derived from Bacillus mojave

An organic solvent and lipase technology, applied in the biological field, can solve the problem of no Bacillus lipase, etc., and achieve the effects of broad organic solvent tolerance, good heat resistance, and broad pH stability

Active Publication Date: 2018-07-31
WILMAR SHANGHAI BIOTECH RES & DEV CENT
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  • 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, DZhiryakova. J Mol catal B: Enzym, 2011, 68: 1-21), among which have good stability at low or medium temperature or There are also many literatures and patents on Bacillus-derived lipases resistant to organic solvents, but there are almost no reports on Bacillus-derived lipases with these three characteristics at the same time

Method used

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  • A low-temperature organic solvent-resistant lipase derived from Bacillus mojave
  • A low-temperature organic solvent-resistant lipase derived from Bacillus mojave
  • A low-temperature organic solvent-resistant lipase derived from Bacillus mojave

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

[0058] Table 1 Primer Information

[0059]

[0060]

[0061] The genome of Mojave bacillus was extracted by SDS method (Fang Weiguo, Acta 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, respectively. PCR system: rTaq 0.5uL, 10×PCR buffer 5uL, dNTP mixture 8uL, primer 11uL, primer 21uL, genome 0.3uL, water 34uL. Reaction conditions: 94°C for 1s, 94°C for 1min, 37°C for 1s, 37°C for 30s, 72°C for 2min20s, 72°C for 1min, 35 cycles (95°C for 1min...

Embodiment 2

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

[0077] 1. Cloning of lip2-2 expression gene

[0078] Use the Bacillus mojave genome as a template and use the cloning primers lip-U / lip-D in the table to perform PCR. See Example 1 for the PCR reaction system. Reaction conditions: 94°C for 5min, 35 cycles (95°C for 30s, 47°C 30s, 1min at 72°C), 10min at 72°C, keep warm at 4°C.

[0079] 2. Digestion of genes and vectors and purification of digested products

[0080] Recover the above PCR product from the gel, and simultaneously digest the PCR product and plasmid pET24a with EcoRI and Hind III, enzyme 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, digestion at 37°C overnight. (Make 2 tubes of 50uL enzyme digestion system for the recovered product from gel injection, and 2 tubes for the carrier), and purify the enzyme digestion product with Omega PCR Product Purification Kit. ...

Embodiment 3

[0087] Example 3: Induced expression of recombinant Escherichia coli and separation and purification of target protein

[0088] 1. Induced expression of recombinant bacteria

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

[0090] 2. Method for Determination of Lipase Activity

[0091] Low-temperature lipase activity was determined by colorimetry. With p-nitrophenyl palmitate (p-NPP) as the substrate, the enzymatic activity is calculated by the amount of p-nitrophenol (p-NP) produced by enzymatic hydrolysis per unit volume of enzyme solution per unit time. ...

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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 Mojave bacillus. Background technique [0002] Lipase (lipase, EC3.1.1.3) is a special class of ester bond hydrolase, which widely exists in animal tissues, plants and microorganisms. Lipase has the biological function of catalyzing hydrophobic oils into water-soluble fatty acids and glycerol, maintains high catalytic activity and strong stability in inorganic solvents, and lipase has broad-spectrum / special specificity for substrates, It endows lipase with great application value in industries such as food oil processing, detergent, biodiesel, synthesis of ester bond compounds and chiral drug synthesis (CN201010599106). [0003] In food oil processing and oleochemical industries, lipase can be used to produce fatty acids and glycerol, modify oils, prepare special oils, increase the yield of neutral oils, and...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/20C12N15/55C12N15/63C12N1/21C12N15/70C11D3/386C02F3/00C02F3/34C12P7/64C10L1/02C12R1/19C12R1/07
CPCC02F3/342C11D3/38627C12N9/20C12Y301/01003Y02E50/10
Inventor 陈苗苗徐正军周美凤常桂芳许骏
Owner WILMAR SHANGHAI BIOTECH RES & DEV CENT
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