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Lipase L-1 and coding gene and application thereof

A lipase gene and lipase technology are applied to lipase L-1 and its encoding gene and application fields, and can solve the problems of production technology restriction, high lipase price and the like

Inactive Publication Date: 2016-02-03
SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a new lipase L-1 and its coding gene and application for the deficiencies in the prior art that the price of lipase is expensive and the production technology is restricted.

Method used

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  • Lipase L-1 and coding gene and application thereof
  • Lipase L-1 and coding gene and application thereof
  • Lipase L-1 and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Lipase gene 1-1 primer design and open reading frame boundary determination

[0027] Genomic DNA of actinomycetes (Streptomycessp.) SCSIO13580 was extracted, and after being verified by 16SrRNA, it was submitted to Shanghai Meiji Biotechnology Co., Ltd. for sequencing. Use bioinformatics means to annotate the genome, analyze the lipase gene wherein, and determine the open reading frame of lipase gene 1-1 wherein, its nucleotide sequence is as shown in SEQIDNO.1, full length is 1005bp (from start codon to stop codon), the amino acid sequence of lipase L-1 encoded by it is as shown in SEQ ID NO.2, a total of 334 amino acids; The similarity is 51%. According to the lipase gene 1-1 sequence obtained by analysis, design full-length amplification primers as follows: Forward primer: 5'-CAC GGATCC ATGAGGATCGATCCGCCCG-3′, the underlined part is the BamHI restriction site; reverse primer: 5′-CAT CTCGAG TTAGGCGGGCTGTGGGGTC-3', the underlined part is the XhoI restri...

Embodiment 2

[0028] Embodiment 2: Cloning and vector construction of lipase gene 1-1

[0029] 2.1PCR amplification

[0030] The primer designed in Example 1 (forward primer 5'-CAC GGATCC ATGAGGATCGATCCGCCCG-3′, reverse primer 5′-CAT CTCGAG TTAGGCGGGCTGTGGGGTC-3') was sent to Shanghai Bioengineering Co., Ltd. to synthesize primers, and the synthesized primers were diluted to 10 μM with TE, and the total DNA of actinomycetes (Streptomycesssp.) SCSIO13580 was extracted as a DNA template, and the reaction system shown in Table 1 was established:

[0031] Table 1 PCR reaction system

[0032]

[0033] Amplify lipase gene l-1 using the following PCR amplification program: a. Denaturation at 95°C for 5 min; b. Denaturation at 95°C for 1 min, annealing at 55-65°C for 0.5 min, extension at 72°C for 1 min and 20 s, for 30 cycles; c.72 Extend at ℃ for 10min, then cool to 10℃.

[0034] The PCR product was electrophoresed in 1% agarose gel for 20 min at 120V, and observed in a gel imaging syste...

Embodiment 3

[0045] Example 3: High expression of lipase L-1 in Escherichia coli BL21 (DE3)

[0046] 3.1 Preparation of Escherichia coli BL21(DE3) Competent Cells

[0047] a. Inoculate Escherichia coli BL21(DE3) into 5mL LB liquid medium, shake overnight at 37°C, 250rpm;

[0048] b. Inoculate the Escherichia coli BL21 (DE3) bacterium solution after overnight shaking culture into LB shake flasks at an inoculation rate of 1% volume ratio, and shake at 37° C. for 3 hours (≥ 300 rpm) to obtain the original culture;

[0049] c. Rapidly cool the shake flask to 0°C in ice water, divide the original culture into ice-precooled centrifuge tubes (50 mL), and place on ice for several minutes;

[0050] d. Centrifuge at 4000rpm for 10 minutes at 4°C to recover the cells, and dry the residual liquid in air (rapidly);

[0051] e. 10mL of 0.1M CaCl pre-cooled on ice 2 Resuspend the cells and centrifuge at 4000rpm for 10min at 4°C to recover the cells;

[0052] f. 10mL of 0.1M CaCl 2 Resuspend the cell...

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Abstract

The invention discloses lipase L-1 and a coding gene and application thereof. The lipase gene l-1 is obtained from Streptomyces sp. SCSIO 13580 in a cloned mode, the nucleotide sequence of the lipase gene l-1 is shown as SEQ ID NO.1, the overall length of the lipase gene l-1 is 1005 bp, and the amino acid sequence of the coded lipase L-1 is shown as SEQ ID NO.2 and contains 334 amino acids in all. The lipase gene l-1 is cloned and connected with an expression vector pET-28a(+), then conversion is performed to obtain escherichia coli BL21 (DE3), and after culture and induced expression, the recombinant expression lipase L-1 is obtained. The lipase L-1 serving as a catalyst catalyzes a reaction between cinnamyl alcohol and an acyl donor, and cinnamyl acetate is prepared, wherein the yield of the obtained cinnamyl acetate can reach 48.3%. The lipase L-1 has the advantages of being high in stability and catalysis efficiency and can be applied to the fields of biological medicine, cosmetics, fine chemical engineering and the like.

Description

technical field [0001] The invention belongs to the fields of biochemical industry and biotechnology, and in particular relates to a lipase L-1 and its coding gene and application. Background technique [0002] Cinnamyl acetate is one of the spice ingredients extracted from cinnamon, and it is a spice that can be used in my country. Most cinnamyl acetate is chemically synthesized, and only a small amount is extracted from natural plants. The chemical method has high requirements on conditions and needs to be completed under high temperature and high pressure. The consumption of equipment and energy consumption are high, and various by-products will be produced during the synthesis process. The subsequent extraction process is complicated and pollutes the environment. Enzyme as a biocatalyst has the advantages of mild reaction conditions, high specificity, less by-products, and less environmental pollution. It can overcome the above-mentioned shortcomings of chemical synthes...

Claims

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Application Information

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IPC IPC(8): C12N9/20C12N15/55C12P7/62
CPCC12N9/20C12P7/62C12Y301/01003
Inventor 胡云峰王召贺张云孙爱君
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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