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Improved Rhizomucor miehei lipase gene and use thereof in yeast display

A technology of lipase gene and Rhizomucor miehei, applied in the field of yeast, can solve the problems of low yield and restrict the development of Rhizomucor mihei lipase enzyme preparation, etc., and achieve the effect of high catalytic performance and strong operation stability.

Active Publication Date: 2009-07-15
DONGGUAN HUAQI BIOLOGICAL SCI & TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Specific to the display of Rhizomucor miehei lipase, the inventors have displayed the unmodified original RML gene on the surface of Saccharomyces cerevisiae, the enzyme activity is 182U / g dry cells, and the yield is low, which greatly limits its application as a whole cell catalyst and rice Development of Rhizomucor miehei lipase enzyme preparation (Wei-GuoZhang, Shuang-YanHan, et al, Functional display of Rhizomucor miehei lipase on surface of Saccharomyces cerevisiae with higher activity and its practical properties; Journal of Chemical Technology and Biotechnology2008, 83: 329-3 )

Method used

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  • Improved Rhizomucor miehei lipase gene and use thereof in yeast display
  • Improved Rhizomucor miehei lipase gene and use thereof in yeast display
  • Improved Rhizomucor miehei lipase gene and use thereof in yeast display

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Embodiment 1

[0028] Embodiment 1: the synthesis of improved Rhizomucor miehei lipase gene

[0029] The existing Rhizomucor miehei lipase gene is shown in SEQ.ID.NO4. The source of the gene is Rhizomucor miehei, which is different from yeast. This may be the main reason why the expression level of this gene in Pichia pastoris is not high. reason.

[0030] The present invention is based on the amino acid sequence (SEQ.ID.NO1) of Rhizomucor miehei lipase (RML), adopts the preferred codon of Pichia pastoris to replace the codon with low frequency of use of RML in yeast, and designs The gene sequence with high frequency of use in Pichia pastoris was obtained to form the following specific gene sequence, such as SEQ.ID.NO2, so as to increase the expression level of Rhizomucor miehei lipase in yeast.

[0031] The gene designed in the present invention can be artificially synthesized by PCR, and the steps are as follows:

[0032] Firstly, 46 primers are synthesized, and the primers are shown as ...

Embodiment 2

[0034] Construction of PMD18-T-RML plasmid

[0035] The full-length PCR product obtained in the previous step was subjected to 0.8% agarose gel electrophoresis, and the target band with a size of about 1092 bp was cut out, and the target product was purified according to the instructions of QIAGEN’s PCR gel recovery kit. Take 25.5 μL of the recovered product, and add 25 mM 1 μL of dATP, 3 μL of 10XPCR buffer, 0.5 μL of Taq enzyme, and incubated at 72°C for 30 minutes.

[0036] The product after adding A (A is adenine) was cloned by TA using the pMD18-T simple vector kit and operated according to the instructions. The 10 μL volume reaction system is as follows: T vector 1 μL (50ng), add A-added PCR product 3uL, ATP-containing 10×Buffer 1 μL, T4 DNA ligase 1 μL, make up to 10 μL with ddH2O. Slightly centrifuge, and connect overnight in a 16°C water bath. The ligation product was transformed into E.coli DH5α, and then spread on the indicator plate containing 0.5mM IPTG, 40μg / ml...

Embodiment 3

[0038] Construction of recombinant plasmid pKFS-RML

[0039] The plasmid PMD18-T-RML was used as template and RMLp1 and RMLp2 were used as primers for PCR amplification. The system is 1 μL of template; 5 μL of 10×Taq DNA polymerase buffer (containing Mg 2+ ); 2.5mmol / L dNTP 4μL; 20μM mol / L upstream and downstream primers 1μL each; Taq DNA polymerase 0.75μL, add sterile water to a total volume of 50μL. The reaction conditions are: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 45 s, annealing at 45°C for 45 s, and extension at 72°C for 2 min, a total of 30 cycles; the 30th cycle was extended at 72°C for 10 min, and the PCR product was detected by 0.8% agarose gel electrophoresis And cut the gel to recover and purify.

[0040] Both the PCR product and the pKFS plasmid were digested with EcoRI and AvrII, and the recombinant plasmid pKFS-RML was constructed with CaCl 2 Transformation method into E.coli Top10F, in Amp + Plate on LB (50mg / mL) plate and culture ove...

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Abstract

The invention relates to an improved rhizomucor miehei lipase gene and an application to yeast display. The sequence of the improved rhizomucor miehei lipase gene is SEQ.ID.No2, with respect to a recombinant vector pMD18-T-RML containing the gene, RML means lipase gene; and the collection number of a bacterial strain Escherichia coli TOP10 / pMD18-T-RML carrying the plasmid is CCTCC M 208136. In the invention, the gene is transferred into pichia stipitis host strain, so that the rhizomucor miehei lipase is displayed and expressed in the pichia stipitis. The provided pichia stipitis can effectively display the rhizomucor miehei lipase. The lipase can be widely applicable for producing fatty acid methyl ester, ethyl caproate, triglycerides which have different melting points but does not contain various types of fatty acid and a few 'reconstructed esters'.

Description

technical field [0001] The present invention relates to a lipase gene sequence capable of highly expressing Rhizomucor miehei lipase in Pichia pastoris, a yeast displaying high-activity lipase, and a genetically engineered lipase obtained from the recombinant yeast . Background technique [0002] Lipase (Lipase EC 3.1.1.3) is triacylglycerol acyl hydrolase, which catalyzes the hydrolysis of natural substrate oil to generate fatty acid, glycerol and monoglyceride or diester, and is widely used in oil processing, food, medicine, daily chemical and other industries , is one of the important industrial enzyme preparations, its catalytic activity only depends on its protein structure, so lipases from different sources have different catalytic properties and catalytic activities. [0003] Rhizomucor miehei lipase (RML) is widely used in industry. Brady et al. have clarified the high-level structure of the enzyme, indicating that it has excellent Sn-1 stereospecificity, is more s...

Claims

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Application Information

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IPC IPC(8): C12N15/55C12N15/63C12N15/81C12N1/21C12N1/19C12R1/19C12R1/84
Inventor 林影韩双艳郑穗平韩振林黄登峰王小宁
Owner DONGGUAN HUAQI BIOLOGICAL SCI & TECH
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