Pichia pastoris engineering strain expressing thermoascus aurantiacus var. levisporus lgh gene

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

Inactive Publication Date: 2013-03-20
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, the culturing conditions of Thermoascomyces photospora are relatively harsh, high-temperature fermentation requires special equipment, and the efficiency of enzyme production is low, resulting in increased costs, thus limiting its application.

Method used

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  • Pichia pastoris engineering strain expressing thermoascus aurantiacus var. levisporus lgh gene
  • Pichia pastoris engineering strain expressing thermoascus aurantiacus var. levisporus lgh gene
  • Pichia pastoris engineering strain expressing thermoascus aurantiacus var. levisporus lgh gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Example 1: Isolation and identification of T.aurantiacus var.levisporus

[0016] (1) Specimen collection: collected from compost.

[0017] (2) Isolation and culture: Take 0.5 g of the collected specimens and place them on a PDA plate for 3 days at 50°C, then isolate and purify. The operating steps refer to Cooney and Emerson (1964) literature.

[0018] (3) Identification: Refer to the documents of Cooney and Emerson (1964) and LaTouche (1950).

Embodiment 2

[0019] Embodiment 2: Cloning of lipase gene lgh

[0020] (1) Extraction of total RNA from Thermoascus var. photospora: refer to the instructions of the Trizol kit.

[0021] (2) Synthesis of the first strand of cDNA: according to the instructions of the TaKaRa RNA PCR kit (AMV) Ver3.0 kit from Takara Company: take 1-2 μg of total RNA, add RNase Free ddH 2 0 to 9.5 μL, denature the RNA sample at 75°C for 5 min, immediately cool it in an ice bath for 5 min, then centrifuge slightly, and add the following components in sequence in the ice bath: 10 mmol / L dNTP Mixture 2 μL, 10×RTBuffer (Mg 2+ )2μL, 25mmol / L MgCl 2 4 μL, Oligo d(T)-Adaptor Primer 1 μL, RNase Inhibiter 0.5 μL, AMV Reverse Transcriptase 1 μL (Final Volume 20 μL), after mixing the reaction solution, put it at room temperature for 10 minutes, then incubate at 42°C for 60 minutes, and boil for 5 minutes to inactivate the reverse transcriptase . Add 180 μL DEPC-treated ddH 2 O, dilute to 200 μL, mix well, centrifuge s...

Embodiment 3

[0072] Embodiment 3: Construction of expression vector

[0073] (1) Design expression primers according to the nucleotide sequence of the isolated lgh gene, and introduce EcoRI and NotI restriction sites at the 5' ends of the primers:

[0074] Upstream primer: 5'-CG GCCCCGTATAAACCCGAAC-3'

[0075] Downstream primer: 5'-TTG CTCATTGGCATGCAGAAATAGGT-3'

[0076] (2) Extraction of total RNA of Thermoascomyces var. photospora: using Trizol reagent to extract.

[0077] (3) Synthesis of the first strand of cDNA by reverse transcription: according to the instructions of the TaKaRa RNA PCR kit (AMV) Ver3.0 kit from TaKaRa Company, the first strand of cDNA was synthesized with Oligo dT-Adaptor primer as a primer. The reaction conditions are: incubate at 42°C for 60 minutes, and then boil for 5 minutes to inactivate the reverse transcriptase. Add 180 μL DEPC-treated ddH 2 O, dilute to 200 μL, mix well, centrifuge slightly, and store at -20°C for later use.

[0078] (4) PCR reacti...

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Abstract

The invention relates to a Pichia pastoris engineering strain GS-TA-LGH expressing a thermoascus aurantiacus var. levisporus thermostable lipase gene lgh. According to the invention, the lipase gene lgh is obtained from thermoascus aurantiacus var. levisporus through RT-PCR (reverse transcription-polymerase chain reaction), RACE (rapid-amplification of cDNA (complementary deoxyribonucleic acid) ends) and other methods, an expression vector pPIC9K/lgh is constructed and introduced into Pichia pastoris GS115, and then the Pichia pastoris engineering strain GS-TA-LGH expressing the lipase is screened out from the Pichia pastoris GS115. The enzyme activity of the lipase of the engineering strain can achieve 19.92U/mg, when heat insulation is performed on the enzyme at the temperature of 50 DEG C for 60 minutes, the loss of the activity can be avoided; and when the heat insulation is performed at the temperature of 60 DEG C for 60 minutes, the enzyme activity can still achieve 66%. The Pichia pastoris engineering strain GS-TA-LGH has higher thermal stability and has economical value and social value as the strain for producing the thermostable lipase.

Description

(1) Technical field [0001] The invention relates to bioengineering, in particular to a Pichia pastoris engineering strain Pichiapastoris GS-TA-LGH expressing thermoascu aurantiacus var. levisporus thermostable lipase gene lgh. (2) Background technology [0002] The full name of lipase (lipase) is triacylglycerol acyl hydrolase, which belongs to the α / β-fold enzyme family. It can decompose various natural oils and fats produced by organisms, and participate in important life activities such as intracellular lipid metabolism in organisms. The application of lipase involves detergent, food, oil, leather, medicine and other industries. In recent years, it has been studied for the preparation of chiral compounds that are difficult to obtain by chemical methods, and for the production of biodiesel as a green renewable energy source, making it It has once again become a research hotspot in the biological and chemical industries. Because microbial lipase has many kinds, short cycl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/55C12N15/81C12R1/84
Inventor 李多川李萌郭晓红黄刚
Owner SHANDONG AGRICULTURAL UNIVERSITY
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