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Construction method of Pichia pastoris genetic engineering strain producing lipase

A technology of genetically engineered strains and construction methods, applied in the field of constructing genetically engineered strains of Pichia pastoris, can solve the problems of high price and high cost of biodiesel

Active Publication Date: 2019-10-29
BEIJING UNIV OF CHEM TECH
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  • Abstract
  • Description
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Problems solved by technology

[0005] The present invention aims to solve the problem that most of the lipases are imported, and the high price leads to the high cost of biodiesel, and provides a method for constructing lipase-producing genetically engineered strains, as well as fermentation of lipase and determination of its effect on short-, medium- and long-chain lipids. hydrolytic activity

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  • Construction method of Pichia pastoris genetic engineering strain producing lipase
  • Construction method of Pichia pastoris genetic engineering strain producing lipase
  • Construction method of Pichia pastoris genetic engineering strain producing lipase

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Embodiment Construction

[0025]1. The construction method of the Pichia genetically engineered strain producing lipase is characterized in that it is carried out in the following steps: one, the lipase gene is derived from the genome of Candida sp.99-125; the Candida sp.99-125 genome is extracted by the extraction kit Candida sp.99-125 genome; 2. PCR amplification of Lip14 gene: PCR amplification was performed using the Candida sp.99-125 genome extracted in step 1 as a template, the product was detected by electrophoresis, and the target gene band Purify and recover the target gene Lip14 by gel recovery kit; the forward primer for PCR amplification is 5'-GGAATTCGTTCTATTCTACAGTCATTGGAGAATGGT-3', and the reverse primer is 5'-GCTCTAGAAAAAAGATGGTGGAACCGTTGG-3' 3. Recombinant expression vector pPICZα-Lip14 Construction: The Lip14 gene purified in step 2 was digested with EcoR I and Xba I, and then purified and recovered by a PCR recovery kit. The expression vector pPICZαA was double digested with EcoR I and...

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Abstract

The invention relates to a pichia pastoris gene engineering strain for producing lipase and a building method of the pichia pastoris gene engineering strain. During the enzymic catalytic reaction of the lipase, if the reaction promotes esterolysis in the oil-water interface, enzymatic synthesis and ester exchange can be achieved in the organic phase, and accordingly biodiesel can be synthesized by using waste edible oil, waste industrial oil and low-carbon alcohol as the raw materials. The lipase is massively fermented by transferring recombinant lipase Lip14 genes into pichia pastoris X33. P-nitrophenyl palmitate is used as the substrate to measure the long-chain lipid hydrolysis ability of the lipase, p-nitrophenyl laurate and p-nitrophenyl octoate are used to measure the medium-chain lipid hydrolysis ability of the lipase, p-nitrophenyl butyrate is used to measure the short-chain lipid hydrolysis ability of the lipase, and accordingly the long-chain, medium-chain and short-chain lipid hydrolysis activity of the lipase can be determined and a foundation is laid for the application of the lipase to the catalytic production of biodiesel.

Description

technical field [0001] The invention relates to a method for constructing a genetic engineering strain of Pichia pastoris producing lipase. Background technique [0002] In enzyme-catalyzed reactions, lipase can promote ester hydrolysis at the oil-water interface, and can enzymatically synthesize and transesterify in the organic phase. Therefore, waste oil or industrial waste oil and low-carbon alcohol can be used as raw materials to synthesize biodiesel. [0003] The basic raw material of biodiesel is gutter oil or industrial waste oil, which can be used repeatedly and continuously, which has completely changed the situation that one barrel of oil resources is less than one barrel. The development of biodiesel has far-reaching significance for achieving sustainable economic development, promoting energy substitution, reducing environmental pressure, and enhancing national strategic security. Moreover, biodiesel does not contain harmful substances such as sulfur dioxide, l...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/81C12N15/55C12N1/19C12R1/84
CPCC12N9/20C12N15/815C12N2800/102C12Y301/01003
Inventor 范立海王在宇张子剑谭天伟
Owner BEIJING UNIV OF CHEM TECH
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