Lipase gene, recombinant expression vector, recombinant expression strain, lipase and preparation method thereof and preparation method of biodiesel

A technology of lipase gene and expression vector, applied in the field of genetic engineering, can solve the problem that heterologous genes are difficult to obtain high-efficiency expression, and achieve the effects of high yield, simple preparation process and increased expression amount

Active Publication Date: 2018-03-20
金堂佰信维科技有限公司
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Problems solved by technology

However, due to the codon usage preference of Pichia pastoris for heterologous genes, the complexity of the secondary structure of mRNA, the abundance of A / T or G / C segments in genes, protease cleavage sites, and the abundance of genes in the host Influenced by factors such as the degree of concentration, it is difficult to obtain high-efficiency expression of heterologous genes in Pichia pastoris

Method used

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  • Lipase gene, recombinant expression vector, recombinant expression strain, lipase and preparation method thereof and preparation method of biodiesel
  • Lipase gene, recombinant expression vector, recombinant expression strain, lipase and preparation method thereof and preparation method of biodiesel
  • Lipase gene, recombinant expression vector, recombinant expression strain, lipase and preparation method thereof and preparation method of biodiesel

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preparation example Construction

[0055] The present invention also proposes a method for preparing the above-mentioned recombinant expression vector, comprising the following steps:

[0056] Step S11, linking the synthesized lipase gene TLLsyn to the intermediate vector pUC57 through the restriction site EcoR Ⅰ to obtain the pUC-TLLsyn vector;

[0057] First, add restriction sites EcoR Ⅰ to both ends of the synthetic lipase gene TLLsyn, and after EcoR Ⅰ digestion, connect to the intermediate vector pUC57 that has also been digested by EcoR Ⅰ to obtain the pUC-TLLsyn vector.

[0058] Step S12, digesting the pUC-TLLsyn vector and the pAO815 vector with EcoR I respectively, electrophoresis and gel recovery of lipase gene TLLsyn fragment and pAO815 fragment;

[0059] Optionally, in steps S11 and S12, the enzyme digestion system is: 30 μL of vector, 2 μL of EcoRI, 10 μL of 10×Buffer, ddH 2 Make up to 200 μL with O, and digest for about 2 hours at 37°C.

[0060] Step S13, linking the lipase gene TLLsyn fragment a...

Embodiment 1

[0086] The construction of lipase gene TLLsyn single-copy recombinant expression strain is as follows:

[0087] (1) Design the lipase gene TLLsyn sequence with the aid of DNA2.0 software, and obtain the lipase gene TLLsyn fragment by artificial synthesis.

[0088] (2) Add restriction site EcoR I to both ends of the synthesized TLLsyn gene, and after EcoR I digestion, connect to the intermediate carrier pUC57 carrier (Suzhou Jinweizhi Biotechnology Co., Ltd.) after EcoR I restriction, The pUC-TLLsyn vector was obtained; the enzyme digestion system was: 30 μL of vector, 2 μL of EcoR I, 10 μL of 10×Buffer, ddH 2 Make up to 200 μL with O, and digest for 2 hours at 37°C.

[0089] (3) The pUC-TLLsyn vector and the pAO815 vector (purchased from Invitrogen, USA, the map is as follows figure 1 (shown) were digested with EcoRI, electrophoresis, gel recovery of the lipase gene TLLsyn fragment and pAO815 fragment; wherein, the enzyme digestion system was: 30 μL of vector, 2 μL of EcoRI,...

Embodiment 2

[0094] The construction of lipase gene TLLsyn multi-copy recombinant expression strain is as follows:

[0095] (1) After the pAO815-TLLsyn recombinant expression vector single copy recombinant expression vector obtained in Example 1 was digested with Bgl II and BamH I, the large fragment was recovered from the gel to obtain the pAO815-TLLsyn recombinant expression vector fragment, wherein the enzyme digestion system For: 30 μL pAO815-TLLsyn recombinant expression vector, 1.5 μL BamH I, 1.5 μL Bgl II, 20 μL 10×Buffer K, 20 μL BSA, 20 μL Triton X-100, ddH 2 Make up to 200 μL with O, and digest for 4 hours at 37°C.

[0096] (2) Gel recovery after the new pAO815-TLLsyn recombinant expression vector recombinant expression vector is digested with BamH I, and it is connected with the pAO815-TLLsyn recombinant expression vector fragment obtained in step (1) with T4DNA ligase to obtain lipase-containing The pAO815-TLLsyn recombinant expression vector of the gene TLLsyn two copies of t...

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Abstract

The invention discloses a lipase gene, a recombinant expression vector, a recombinant expression strain, lipase and a preparation method thereof and a preparation method of biodiesel. The lipase geneTLLsyn is used for coding lipase, and a nucleotide sequence of the lipase gene TLLsyn is shown as SEQ ID NO: 1. According to the lipase gene TLLsyn provided by the invention, the original low-frequency codon is replaced with a high-frequency codon in pichia pastoris, and the expression quantity of the synthetic lipase gene TLLsyn in pichia pastoris cells is obviously improved. In addition, in thepreparation process of the lipase, the synthetic lipase gene TLLsyn is transferred into the pichia pastoris, and multi-copy construction of target genes is combined, so that the expression quantity ofthe synthetic lipase gene TLLsyn in the lipase is improved, and the enzyme activity of the lipase is improved. The preparation process is simple, the yield is high, and the production cost of the lipase is reduced.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a lipase gene, a recombinant expression vector, a recombinant expression strain, a lipase and a preparation method thereof, and a preparation method of biodiesel. Background technique [0002] Biodiesel refers to oil crops such as soybeans, rapeseed, cotton, palm, etc., wild oil plants and engineering microalgae and other aquatic vegetable oils, animal fats, and catering waste oils, etc. Renewable diesel fuel, which replaces petrochemical diesel, has attracted widespread attention because of its excellent environmental protection properties. The preparation method of biodiesel includes chemical method and biological enzyme synthesis method, wherein the biological enzyme synthesis method uses animal fat and low-carbon alcohol as raw materials, and carries out transesterification reaction through the catalysis of lipase to prepare corresponding fatty acid methyl ester a...

Claims

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Application Information

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IPC IPC(8): C12N15/55C12N9/20C12N15/81C12N1/19C12N15/66C12P7/64C12R1/84
CPCC12N9/20C12N15/66C12N2800/22C12P7/649C12Y301/01003Y02E50/10
Inventor 杨江科尤逊
Owner 金堂佰信维科技有限公司
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