Lipase derived from cladosporium, and coding gene sequence and application thereof

A gene sequence, coding gene technology, applied in genetic engineering, plant genetic improvement, microorganism-based methods, etc., can solve problems such as low lipase

Active Publication Date: 2014-06-18
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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  • Lipase derived from cladosporium, and coding gene sequence and application thereof
  • Lipase derived from cladosporium, and coding gene sequence and application thereof
  • Lipase derived from cladosporium, and coding gene sequence and application thereof

Examples

Experimental program
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preparation example Construction

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

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

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

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

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

[0088] In other preferred examples, the lipase activity prepared by the culture is strongly inhibited by 0-2% SDS or CTAB or a mixture of the...

Embodiment 1

[0184] Example 1. Cloning of lipase-encoding gene

[0185] Clone the lipase coding gene from bacterial strain Cladosporium strain WBRD3.10062425 by molecular biology method, concrete 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 WBRD3.10062425 slant 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. Cultivate at 28°C and 200rpm for 24h.

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

[0189] 1.2 Isolation and purification of wild lipase and acquisition of amino acid N-terminal sequence of lipase

[0190] The fermented liquid was collected ...

Embodiment 2

[0210] Example 2. Construction of recombinant expression vector

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

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

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

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Abstract

The invention relates to a lipase derived from cladosporium, and a coding gene sequence and an application thereof. Specifically, the invention relates to the gene sequence. The coding of the gene sequence has (i) an amino acid sequence as shown in SEQ ID NO:2, or an amino acid sequence as shown in 22nd-464th sequence of the (i), or a derived protein sequence of the sequence as shown in the (i), or a derived protein sequence of the 22nd-464th sequence of the (i). The invention also relates to a protein containing the sequence as shown in the SEQ ID NO:2 or a derived protein of the sequence as shown in the SEQ ID NO:2. The lipase has the characteristics of organic solvent resistance, wide pH stability, better temperature stability and the like, and has wide application values in multiple industrial fields, such as biodiesel, medical industry, food processing, oleochemical industry, environmental protection, detergents, textile manufacturing and papermaking.

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 application. Background technique [0002] Lipase (lipase, EC3.1.1.3), that is, triacylglycerol acyl hydrolase, is a special class of ester bond hydrolase, which widely exists in animals, plants and microorganisms. The basic unit of lipase is amino acid, mostly a polypeptide chain, and its catalytic activity is determined by its protein structure. Lipase can catalyze hydrophobic natural substrate oil into water-soluble fatty acid, glycerol or monoglyceride or diglyceride, maintain high catalytic activity and strong stability in inorganic solvents, and lipase is more abundant in organic solvents It will be denatured or its activity will decrease; but in a suitable enzymatic environment, lipase has the characteristics of broad spectrum or special specificity for the substrate, which ma...

Claims

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Application Information

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Patent Type & Authority Applications(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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