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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 restricting the development of Rhizomucor mihei lipase enzyme preparation, low yield and the like, and achieve the effect of strong operational stability and high catalytic performance

Inactive Publication Date: 2010-12-15
DONGGUAN HUAQI BIOLOGICAL SCI & TECH +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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, eta1, 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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Experimental program
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Effect test

Embodiment 1

[0030] The present invention is based on the basis of the amino acid sequence (SEQ.ID.NO1) of Mi Black -rooted Modelin (RML).In Bickebast yeast, a gene sequence with high frequency is used to form the following specific gene sequences, such as seq.id.no2, thereby increasing the expression of rice black root coat lipase in yeast.

[0031] The genes designed by the present invention can be artificially synthesized by PCR. The steps are as follows:

[0032] First synthesize 46 primers, as shown in seq.id.no3.like figure 1 It shows that the primers include 23 positive primers (odd numbers, positive arrows), 23 reverse primers (even order, reverse arrows).Add 46 oligonucleotides (primers) to the PCR reaction system at 200NMol / L's final concentration, add 10 × TAQ DNA polymerase Buffer 5μL, 2.5 mmol / L DNTPS 4 μl, TAQ DNA polymerase 0.5 μl, noThe bacterial double steaming water is supplemented to 50 μL, and the first round of PCR reaction is performed: 94 ° C degeneration 10s, 50 ° C for...

Embodiment 2

[0036] After adding A, the product (A is adenine), use the PMD18-T Simple Vector kit to be checked and operate according to the instructions.The volume reaction system of 10 μl is as follows: 1 μL (50ng) of the T carrier, 3 μL of PCR products after adding A, 10 × Buffer 1 μL of ATP, 1 μL of T4DNA connection enzymes, and ddh2O to 10 μL.Slightly centrifuged, 16 ° C water bath connecting night.Connect the product to transform the E.Coli DH5α, and then apply it to the 0.5mm IPTG, 40 μg / ml x-gal indicator tablet, cultivate overnight, select the white spots to extract plasmid enzymes to identify, and send the reorganized plasmid PMD18-T-RML to Shanghai ShengshengThe sequencing of the Industrial Engineering Co., Ltd., the sequencing result is like image 3 It shows that the cloning gene is consistent with us designing genes.

[0037] Example 3:

[0038] Construction of reorganized plasmid PKFS-RML

Embodiment 3

[0040] Both PCR products and PKFS plasmid are cut with ECORI and AVRII dual enzymes to build a reorganized plasmid PKFS-RML 2 Transformation method to e.coli top10F, in AMP + LB (50mg / ml) plate coating plate, cultivating overnight.Extract -positive transformer plasmids for ECORI and AVRII dual enzyme cutting identification.After the appraisal is correct, the Shanghai Biological Engineering Biological Engineering also has a limited company for sequencing.

[0041] Example 4

[0042] The cultivation of Bi Chi yeast engineering bacteria that expresses high vitality lipase

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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 involves a lipase gene sequence that can efficiently express rice -black -rooted lipase in Bichibasted yeast and the yeast with high vibrant lipase, and the genetic engineering lipase obtained from the reorganized yeastEssence Background technique [0002] Lipase EC3.1.1.3) is trigyl glyceride hydrolytic enzyme. It catalyzes natural substrate oil and hydrolysis of fatty acids, glycerin and glycerin monocles or dharma, which is widely used in oil processing, food, medicine, dailyization, etc.Industry is one of the important industrial enzyme preparations. Its catalytic activity only depends on its protein structure, so the lipases of different sources have different catalytic characteristics and catalytic vitality. [0003] Rhizomucor Miehei (RML) lipase (RML) industrial applications are more widely used.Brady and others have elaborated the advanced structure of the enzyme clearly, indicating that it has excellent SN-1 three-dimensional...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/55C12R1/84C12R1/19
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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