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Lipase mutant with increased optimum temperature and application of lipase mutant with increased optimum temperature

An optimal temperature and lipase technology, applied in the field of lipase mutants, can solve problems such as limiting the application range and reducing catalytic efficiency

Active Publication Date: 2014-12-03
宁夏夏盛实业集团有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, oil processing usually needs to be carried out at a higher temperature, and Rhizopus lipase belongs to mesophilic lipase, and the optimum temperature is 40°C, which limits its application range and reduces the catalytic efficiency

Method used

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  • Lipase mutant with increased optimum temperature and application of lipase mutant with increased optimum temperature
  • Lipase mutant with increased optimum temperature and application of lipase mutant with increased optimum temperature
  • Lipase mutant with increased optimum temperature and application of lipase mutant with increased optimum temperature

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Experimental program
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Effect test

Embodiment 1

[0030] The lipase mutant that embodiment 1 optimum temperature improves

[0031] A lipase mutant with improved optimum temperature, the 310th amino acid of the lipase lipase gene sequence published in GenBank EF405962 is mutated from Asp of the parent lipase to Val.

Embodiment 2

[0032] Example 2. Yeast expressing lipase Asp310Val point mutant.

[0033] The lipase mutant gene of the present invention can be obtained by chemical total synthesis or PCR method, and the PCR method is taken as an example below to introduce:

[0034] Nucleotide mutations were introduced into the lipase gene proRCL of Rhizopus sinensis in vitro by overlap extension PCR. The reaction conditions are as follows:

[0035] Reaction condition 1:

[0036]

[0037]

[0038] Among them, the sequences of the upstream primer 50bpFNEW and the downstream primer proRCLD190VR are:

[0039] 50bpFNEW:5'-AAAGAAGAAGGGGTATCTCTC-3';

[0040] proRCLD190VR:5'-TTCCGGTGCTAACAACGTAGTAAG-3'.

[0041] PCR amplification conditions: 98°C 10s; 98°C 10s, 56°C 15s, 72°C 1min, 30 cycles; 72°C 10min. The amplified product was purified by a DNA purification kit to obtain Fragment A.

[0042] Reaction condition 2:

[0043]

[0044] Among them, the sequences of the upstream primer 50bpRNEW and the...

Embodiment 3

[0051] The optimal temperature determination of embodiment 3 lipase mutants

[0052] In order to determine the optimum temperature of lipase, it is necessary to separate and purify the enzyme.

[0053] Shake flask fermentation: inoculum size 10% (V / V), in 25mL BMGY medium, shaking culture at 30°C for 16-20h to OD 600 2 to 6, the cells were collected by centrifugation and diluted to OD with BMMY medium 600 1, add 0.5% methanol every 24h to induce expression, after 3-4d of culture, collect the fermentation supernatant.

[0054] Separation and purification: the fermentation supernatant of the mutant strain is concentrated through a 10KD ultrafiltration membrane, followed by SP-Sepharose FF strong cation exchange chromatography and Phenyl-Sepharose 6FF hydrophobic chromatography column chromatography to obtain the purified active component of mutant lipase. Specific operation reference Yu Xiao-Wei et al.J Mol Catal B:Enzym,2009,57:304-311.

[0055] Determination of optimum temp...

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Abstract

The invention discloses a lipase mutant with increased optimum temperature, which belongs to the technical field of enzymatic genetic engineering. The lipase mutant with increased optimum temperature disclosed by the invention is obtained through taking rhizopus chinensis China center for type culture collection (CCTCC) M201021 lipase as a parent and utilizing a molecular biological technique, and the mutant amino acid of the mutant is Asp310Val. The optimum temperature of the lipase mutant is increased compare with the optimum temperature of the parent of rhizopus chinensis lipase and has an important industrial application value.

Description

technical field [0001] The invention relates to a lipase mutant, in particular to a lipase mutant with increased optimum temperature and application thereof. Background technique [0002] Lipase (EC 3.1.1.3) can not only catalyze the hydrolysis of oil, but also catalyze ester synthesis, transesterification, acid hydrolysis and other reactions in the non-aqueous phase. It is widely used in chemistry, food, pharmaceutical and detergent or bioenergy in industry. Microorganisms are an important source of industrial lipase, and Rhizopus is an important producer of microbial lipase. Today, more than 30 Rhizopus lipases have been commercially produced. Rhizopus lipase is often used in oil processing. However, oil processing usually needs to be carried out at a higher temperature, and Rhizopus lipase belongs to mesophilic lipase, and its optimum temperature is 40°C, which limits its application range and reduces its catalytic efficiency. [0003] Rational protein design is a use...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/20C12N15/55C12N15/10C12N1/19C12N5/10C12N15/81C12P7/64A21D8/04C12R1/84
CPCY02E50/13Y02E50/10
Inventor 喻晓蔚徐岩吴厚军
Owner 宁夏夏盛实业集团有限公司
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