Pichia yeast engineering strain for expressing Thermomyces lanuginosus gene 1n

A technology of Thermomyces and Pichia pastoris, applied in the field of bioengineering, can solve the problems of increased product cost and low enzyme production efficiency

Inactive Publication Date: 2008-11-19
SHANDONG AGRICULTURAL UNIVERSITY
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Problems solved by technology

It can produce thermostable lipase with important industrial value, but wild bacteria are used for large-scale industrial production of lipase, and there are still some unresolved problems
For example, the culture conditions of thermophilic bacteria are relatively harsh, and the large-scale fermentation production of thermophilic enzymes requires special equipment, and the enzyme production efficiency is low, resulting in increased product costs, thus limiting its application

Method used

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  • Pichia yeast engineering strain for expressing Thermomyces lanuginosus gene 1n
  • Pichia yeast engineering strain for expressing Thermomyces lanuginosus gene 1n
  • Pichia yeast engineering strain for expressing Thermomyces lanuginosus gene 1n

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Experimental program
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Embodiment Construction

[0022] 1. Isolation and identification of Thermomyces lanuginosus

[0023] (1) Specimen collection: collected from compost in Beijing.

[0024] (2) Separation and culture: take 0.5 g of the collected specimen and place it on a PDA plate for 3 days at 50°C.

[0025] Carry out separation and purification. The operating steps refer to Cooney and Emerson (1964) literature.

[0026] (3) Identification: refer to Cooney and Emerson (1964) literature.

[0027] 2. Cloning of lipase gene ln from Thermomyces lanuginosus

[0028] (1) Extraction of total RNA from Thermomyces lanuginosus: refer to the instructions of the Trizol kit.

[0029] (2) Synthesis of the first strand of cDNA: the first strand of cDNA was synthesized using Oligo(dT)20 as a primer according to the instructions of the Reverse Transcription Reaction kit from Promega. The reaction conditions are: 42°C for 1 hour; 85°C for 10 minutes.

[0030] (3) PCR reaction: upstream primer 5'-ATGAGGAGCTCCCTTGTGCTG-3', downstream...

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Abstract

The invention relates to a Pichia pastoris GS-LN-6 for expressing a Thermomyces lanuginosus thermo-stabilization lipase gene ln. The lipase gene ln is obtained from the Thermomyces lanuginosus through a method of RT-PCR, and is cloned and inserted into a Pichia pastoris integral expression vector pPIC9K. Then the expression vector pPIC9K / ln of the obtained lipase gene ln is inducted into a Pichia pastoris GS115 from which the Pichia pastoris GS-LN-6 for expressing the lipase is screened out. The engineering bacteria can have a lipase expression amount of 1.93mg / mL after undergoing six days of induction culture. The enzyme still has 82 percent of activity when the temperature is kept at 60 DEG C for 60 minutes, has 50 percent of activity when the temperature is kept at 70 DEG C for 60 minutes, and has 45 percent of activity when the temperature is kept at 80 DEG C for 60 minutes. The Pichia pastoris GS-LN-6 can be used as a production strain of the thermo-stabilization lipase with thermal stability and higher economic and social value.

Description

(1) Technical field: [0001] The invention relates to biological engineering, in particular to a Pichia pastoris engineering strain Pichiapastoris GS-LN-6 expressing the thermostable lipase gene ln of Thermomyces lanuginosus. (two) background technology: [0002] Lipase (lipase, EC 3.1.1.3), the full name of triacylglycerol acyl hydrolase, widely exists in organisms. It can catalyze the hydrolysis of the glyceride bond on the oil, and also catalyze the reactions of transesterification, esterification, alcoholysis and acidolysis. Lipases derived from microorganisms are widely used in industrial production due to their diverse functions, easy cultivation, and abundant yields. Among them, the research and development of thermostable lipases has important commercial value. [0003] Thermomyces lanuginosus is a fungus with a wide distribution and a high growth limit temperature. It can produce thermostable lipase with important industrial value, but there are still some unresolv...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/55C12N15/81C12N9/20C12R1/84
Inventor 李多川郑燕李安娜李华赵春青
Owner SHANDONG AGRICULTURAL UNIVERSITY
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