High temperature lipase, preparation for mutants thereof and application thereof

A lipase and carrier technology, applied in the fields of genetic engineering and enzyme engineering, can solve the problems of substrate water insolubility and instability

Active Publication Date: 2011-06-22
FUJIAN FUDA BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Different sources of lipase lead to problems such as structure and property diversity, instability, and substra

Method used

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  • High temperature lipase, preparation for mutants thereof and application thereof
  • High temperature lipase, preparation for mutants thereof and application thereof
  • High temperature lipase, preparation for mutants thereof and application thereof

Examples

Experimental program
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Embodiment 1

[0134] The cultivation of embodiment 1 Thermotoga petrophila and the extraction of genomic DNA

[0135] The anaerobic, heterotrophic, thermophilic and oleophilic bacteria Thermotoga petrophila (ATCC BAA-488) was cultured on YM medium at 85°C for 4-7 days, and the bacterial liquid was collected for use. The specific operation and medium preparation are as follows:

[0136] The composition of the YM medium is: ASW (artificial seawater) containing 0.2% (w / v) yeast extract, adjusted to pH 7.0 with 6N hydrochloric acid, divided into 30ml Hungate tubes (Hungate tubes), each tube 9ml, and then sterilized in a sterilizing pot at 121°C for 20min. Pass oxygen-free nitrogen and hydrogen into the Hungate tube (nitrogen and hydrogen need to be filtered with a 0.2um sterile filter membrane), and heat to reduce the copper wire in the tube. The tubes are then tightly sealed with sterile butyl rubber stoppers. Prepare the appropriate concentration of Na 2 S, use a 0.2um sterile filter memb...

Embodiment 2

[0157] Cloning and obtaining of embodiment 2 Thermotoga petrophila lipase coding gene

[0158] Design and synthesize degenerate primers based on the conserved sequences of anaerobic, thermophilic and oleophilic lipases:

[0159] Forward primer: 5'-RTGGCCTTYTTCGATWTRCCMCTYGARGAACTGA-3' (SEQ ID NO: 6)

[0160] Reverse primer: 5'-CTAGCCTTYCTCAAATAGTCTCTTCA-3' (SEQ ID NO: 7).

[0161] Wherein, W is A or T, Y is C or T or U, M is A or C, and R is A or G.

[0162] Degenerative PCR amplification was performed using the genomic DNA of Thermotoga petrophila (prepared in Example 1) as a template. The PCR reaction parameters were as follows: denaturation at 94°C for 3 minutes and cooling to 4°C; then denaturation at 94°C for 30 sec, annealing at 50°C for 30 sec, extension at 72°C for 1 min, and after 32 cycles, incubation at 72°C for 10 min. Obtain the whole gene fragment of lipase, perform agarose electrophoresis and recover the fragment with a gel recovery kit (E.Z.N.A. Gel Extracti...

Embodiment 3

[0167] Obtaining of embodiment 3 mutant lipase coding gene

[0168] Primers 5'-CCATGGTTCGCTTTCTTTGACTTACCGCT-3'(SEQ ID NO: 10), 5'-GCGGCCGCTTCCTGAAACGCCTGTT-3'(SEQ ID NO: 11) and 5'-CCATGGTTCGCTTTCTTTGACATGCCGCT-3'(SEQ ID NO: 12) were designed to introduce mutations Points M1F, L6M and K344E (enzyme cutting sites containing NcoI and NotI in the primers). Using the recombinant pET28a (+) obtained in Example 2 as a template, PCR was carried out with primer sequences SEQ ID NO: 10 and SEQ ID NO: 11, and the resulting product was subjected to agarose electrophoresis, and DNA fragments (E.Z.N.A. gel extraction kit). Using the recovered DNA fragment as a template, PCR was performed with primer sequences SEQ ID NO: 12 and SEQ ID NO: 11, and agarose electrophoresis was performed to recover the DNA fragment (E.Z.N.A. Gel Extraction Kit from OMEGA Company). Take 10ul of the recovered DNA fragment, digest it with restriction enzymes NcoI and NotI, then connect it with the pET28a(+) pla...

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Abstract

The invention discloses high temperature lipase, preparation for mutants thereof, and application thereof, and in particular discloses a separated lipase with an amino acid sequence as SEQ ID NO:4, a coding sequence thereof, a carrier, a cell and a composition containing the separated lipase, a preparation method for the separated lipase, and application of the separated lipase in catalyzing ester synthesis, performing chiral separation or transforming plant oil into biodiesel.

Description

technical field [0001] The present invention relates to the field of genetic engineering and enzyme engineering, in particular, relates to a kind of isolated lipase derived from the bacterium Thermotoga petrophila of the genus Thermospora, its encoding gene, and recombinant plasmids containing the encoding gene and used for expressing A recombinant strain of the target lipase protein; the present invention also relates to the use of the expressed thermostable lipase. Background technique [0002] Lipase (lipase, EC3.1.1.3, glyceride hydrolase) is an enzyme that decomposes fat. It is ubiquitous in animal and plant tissues and a variety of microorganisms. Its natural substrate is the natural oil produced by biology, and it is the first to be studied. One of the enzymes. Since the activity of rabbit pancreatic lipase was reported in 1834, the research on lipase has a history of hundreds of years. At the beginning of the 20th century, foreign researchers first discovered micro...

Claims

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Application Information

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IPC IPC(8): C12N9/20C12N15/55C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10C12P7/62C12P7/64C12P41/00
CPCY02E50/13Y02E50/10
Inventor 叶秀云靳伟刚张洋罗鋆琳陈萍
Owner FUJIAN FUDA BIOTECH
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