Surface displaying system for rhizopus oryzaelipase, and preparation method and application of surface displaying system

A technology of rhizopus oryzae lipase and surface display system, which is applied in the field of genetic engineering, can solve the problems of long catalysis time, low yield of methyl esterification products, and easy shedding of displayed proteins, so as to reduce toxic and side effects, easy to operate, and good The effect of economic benefits and social effects

Inactive Publication Date: 2013-01-16
NANJING FORESTRY UNIV
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

At present, the lipase immobilized by the cell surface display method has a long catalytic time and a low yield of methylated products, and the most widely used N-terminal anchor protein FLO1p (FS) is linked by a weak non-covalent bond On the cell wall, so that the displayed protein is easy to fall off

Method used

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  • Surface displaying system for rhizopus oryzaelipase, and preparation method and application of surface displaying system
  • Surface displaying system for rhizopus oryzaelipase, and preparation method and application of surface displaying system
  • Surface displaying system for rhizopus oryzaelipase, and preparation method and application of surface displaying system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 S. cerevisiae The extraction and PCR amplification of the EBY100 genome yielded the mature ankyrin gene mpir1;

[0032] 1) Saccharomyces cerevisiae EBY100 ( S. cerevisiaeEBY100) culture: activate the strain on the YPD plate, pick a single colony in the YPD shake flask, and culture overnight at 30°C. Centrifuge at 6000rmp for 10min at 4°C to collect the strains, add 10% glycerol, and store the strains in a -80°C refrigerator. S. cerevisiae EBY100 was purchased from Invitrogen.

[0033] 2) Extraction of Saccharomyces cerevisiae EBY100 genomic DNA, steps: Pick a single colony from the activated Saccharomyces cerevisiae EBY100 plate, inoculate it in YPD medium, and culture it overnight at 30 ℃; take the cells in the logarithmic growth phase, centrifuge at 6000rpm for 5min, and the bacteria Wash the body once with sterile distilled water, remove the supernatant; add 5mL DNA wall breaking buffer (100mmol / L Tris-HCl, pH8.0, 10mmol / L EDTA, 1%SDS), mix well, an...

Embodiment 2

[0039] Example 2 Construction of surface display recombinant plasmids;

[0040] (1) Construction of AOX-mPir1 and GAP-mPir1 recombinant vectors

[0041] mPir1 (prepared in Example 1) and pPICZαA (Invitrogen) and pGAPZαA (Invitrogen) were digested with EcoRI and KpnⅠ, purified and recovered with the kit, and ligated with T4 DNA ligase to obtain the recombinant Pichia pastoris surface display expression plasmid GAP -mPir1, AOX-mPir1. The resulting GAP-mPir1 and AOX-mPir1 plasmids were transformed into Escherichia coli host TOP10F (Novagen). Transformants were screened with 50 μg / ml zeocin LLB plates, zeocin-positive transformants were picked, plasmids were extracted, identified by EcoRI and KpnⅠ double enzyme digestion and sequenced, the results showed that the wall protein mPir1p gene sequence was inserted correctly.

[0042] (2) Construction of AOX-mPir1-ProROL and GAP-mPir1-ProROL vectors

[0043] Cloning of the Rhizopus oryzae lipase gene prorol with a flag tag protein, f...

Embodiment 3

[0048] Example 3 Expression and identification of recombinant plasmids in Pichia pastoris;

[0049] The AOX-mPir1-ProROL plasmid was linearized with restriction enzyme sac I, and the GAP-mPir1-ProROL plasmid was linearized with AvrII. Pichia pastoris KM71H (Invitrogen) was electrotransformed, grown on a YPD plate containing 100 μg / mL zeocin for 72 hours, and resistance-positive transformants were picked, that is, transformants integrated with fusion gene sequences (KM71H / AOX-mPir1- ProROL, KM71H / GAP-mPir1-ProROL), spot on the high-resistant YPD plate containing 500 μg / mL zeocin, select larger strains, that is, transformants containing more copies, and spot on the olive oil-MMH-RB plate to form The obvious fluorescent circle indicates that ProROL is expressed in Pichia pastoris and exhibits lipase hydrolysis activity.

[0050] Inoculate the positive transformants KM71H / AOX-mPir1-ProROL, KM71H / GAP-mPir1-ProROL in 50mL BMGY medium, culture at 30°C, 200rpm shaking for 16-20h to O...

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Abstract

The invention discloses a surface displaying system for rhizopus oryzaelipase, and a preparation method and application of the surface displaying system. According to the surface displaying system, a rhizopus oryzaelipase ProROL gene is connected to a C end of an ankyrin Pir1p gene; and by normal secretion and expression of an expression strain KM71H, a fusion protein is fixed on the surface of pichia pastoris KM71H strain by a covalent bond mode by using N-end repeated sequences of Pir1p as anchoring areas. By a gene fusion method, the rhizopus oryzaelipase gene and the C end of the mature ankyrin Pir1p are fused, the gene mpir1 is inserted into a vector containing different promoters pAOX1 and pGAP, recombinant plasmids AOX-mPir1 and GAP-mPir1 are obtained, the surface displaying of the pichia pastoris KM71H can be realized, and the high-efficiency expression can also be realized. Compared with the promoter pAOX1, the promoter pGAP has the advantages that the carbon source conversion is not required; the promoter pGAP is easy and convenient to prepare; methanol is not needed to be added in the induction process; the toxic and side effects of methanol on thallus growth are reduced; and the highest enzyme activity is 614.5U/L (180.74[dry cell weight]) and is greater than that of the yeast strain using the promoter pAOX1. The surface displaying system is high in practical applicability and has high economic and social benefit.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a surface display system of Rhizopus oryzae lipase and its preparation method and application. Background technique [0002] Biodiesel, also known as fatty acid methyl ester or ethyl ester, is made from oil crops, wild oil crops and aquatic oil plants of engineered microalgae, as well as animal fats and waste cooking oil, etc., through transesterification reaction with methanol or ethanol Methyl or ethyl ester fuels can effectively reduce the molecular weight and viscosity of natural oils through transesterification. [0003] The preparation methods of biodiesel include physical method and biological method. The physical method includes direct mixing method and microemulsion method, and the chemical method includes pyrolysis method and transesterification (or transesterification) method (Marchetti J M, Renewable and Sustainable Energy Reviews , 2007, 11:1300-1311). ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N11/16C12N15/81C12N9/20C12R1/84C12R1/845
Inventor 王飞王烨李迅
Owner NANJING FORESTRY UNIV
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