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A kind of lipase derived from Cladosporium, its coding gene sequence and use thereof

A gene sequence, lipase technology, applied in the fields of bioengineering and enzyme industry, can solve the problem of less lipase

Active Publication Date: 2018-02-16
WILMAR SHANGHAI BIOTECH RES & DEV CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there are many microorganisms that can produce lipase, there are still few lipases that can be used under actual conditions and can be produced by microbial fermentation, that is, commercialized lipases.

Method used

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  • A kind of lipase derived from Cladosporium, its coding gene sequence and use thereof
  • A kind of lipase derived from Cladosporium, its coding gene sequence and use thereof
  • A kind of lipase derived from Cladosporium, its coding gene sequence and use thereof

Examples

Experimental program
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preparation example Construction

[0083] In the fifth aspect of the present invention, there is provided a method for preparing a lipase, the method comprising: culturing the cell under conditions suitable for the cell of the present invention to produce the lipase of the present invention; The lipase produced by the cell.

[0084] In some preferred examples, the culture process is a fermentation production process.

[0085] In other preferred examples, the culture temperature is 20-70°C, 25-60°C, 30-55°C, 35-50°C, or 40-45°C.

[0086] In other preferred examples, the culture pH is 3.0-11.0, 4.0-10.0, 5.0-9.0 or 6.0-8.0.

[0087] In some other preferred examples, the lipase activity prepared by the culture is strongly inhibited by one or more of sodium dodecyl sulfate (SDS) and CTAB, or by Zn 2+ Or Co 2+ One or more of the ions are strongly suppressed.

[0088] In some other preferred examples, the lipase activity prepared by the culture is strongly inhibited by 0~2% SDS or CTAB or a mixture of both, or by 0~50mM Zn 2+...

Embodiment 1

[0184] Example 1. Cloning of lipase encoding gene

[0185] The lipase coding gene was cloned from the strain Cladosporium strain WBRD3.10062425 by molecular biology method, the specific steps are as follows:

[0186] 1.1 Production of wild lipase

[0187] Add about 10 mL of sterile normal saline to a freshly cultured Cladosporium WBRD 3.10062425 slope to prepare a spore suspension, and then take 1 mL of the suspension and inoculate it into a 250 mL seed medium with a volume of 30 mL. Incubate at 28°C and 200 rpm for 24 hours.

[0188] After the seeds are cultivated, 3 mL of the seed culture solution is inoculated into 250 mL of fermentation medium at 28°C, 200 rpm, and fermented for about 67 hours to detect the activity of extracellular lipase. The results showed that the lipase activity in the fermented supernatant reached 1.03 units / mL.

[0189] 1.2 Separation and purification of wild lipase and obtaining the amino acid N-terminal sequence of lipase

[0190] Collect the fermentation ...

Embodiment 2

[0210] Example 2. Construction of recombinant expression vector

[0211] 2.1 Cloning of Cladosporium target lipase gene (mlics)

[0212] The lipase derived from Cladosporium WBRD3.10062425 has the highest consistency of 39% with the lipase derived from Malassezia furfur (GI:73765555) at the amino acid level. And like other lipases, the conserved sequence of the active center is "GYSGG". Among them, the amino acids 1-21 in the amino acid sequence of SEQ ID NO: 2 are the signal peptide region, the amino acids 22-464 are the mature peptide region, and the molecular weight of the mature peptide is 46796 Da. The gene encoding the mature peptide is named mlipcs.

[0213] Extract the genome of Cladosporium WBRD3.10062425 with TakaRa Mini BEST Universal Genomic DNA Extraction kit Ver.4.0 (Bao Biological Engineering (Dalian) Co., Ltd.), and then use it as a template to use primer lipcs-U (SEQ ID NO: 9) and primer lipcs-D (SEQ ID NO: 10) are upstream and downstream primers for PCR. Among t...

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Abstract

The invention relates to a lipase derived from Cladosporium, its coding gene sequence and its application. Specifically, the present invention relates to a gene sequence, which encodes (i) the amino acid sequence shown in SEQ ID NO: 2; or the amino acid sequence shown in the 22-464th position of (i); or (i) A derivative protein sequence of the sequence shown, or a derivative protein sequence of the sequence at position 22-464 of (i). The present invention also relates to a protein comprising the sequence shown in SEQ ID NO:2 or a derivative protein of the sequence shown in SEQ ID NO:2. The lipase of the present invention has the characteristics of resistance to organic solvents, broad pH stability, good temperature stability, etc. It has a wide range of application value in the industrial field.

Description

Technical field [0001] The invention belongs to the fields of bioengineering and enzyme industry. More specifically, the present invention relates to a lipase, its coding gene sequence and its use. Background technique [0002] Lipase (EC3.1.1.3), or triacylglycerol acylhydrolase, is a special kind of ester bond hydrolase, which is widely present in animals, plants and microorganisms. The basic constituent unit of lipase is amino acid, mostly a polypeptide chain, and its catalytic activity is determined by its protein structure. Lipase can catalyze the hydrophobic natural substrate oil into water-soluble fatty acid, glycerol or monoglyceride or glycol diester, maintaining high catalytic activity and strong stability in inorganic solvents, and more lipase in organic solvents It may denature or decrease its vitality; but in a suitable environment for enzyme activity, lipase has broad-spectrum or special specificity to the substrate, which makes it widely used in fine chemicals, w...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/55C12N9/20C12N15/63C12N1/21C12N1/15C12N1/19C12N5/10C12R1/645
Inventor 徐正军陈苗苗周美凤许骏
Owner WILMAR SHANGHAI BIOTECH RES & DEV CENT
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