Prokaryotic Expression And Functional Verification Of Soybean Lipoxygenase LOX-3

Soybean Lipoxygenase,an oxidoreductase,in plant vegetative growth,defense responses and stress resistance.Especially in the production of flavor food and other food processing and other aspects have a significant impact.However,due to different varieties,different years and different planting sites,it will have a greater impact on the activity of soybean lipoxygenase,which seriously restricts its development and utilization.Therefore,it is necessary to use genetic methods to study the Lipoxygenase gene and establish a prokaryotic expression system to obtain a stable soybean Lipoxygenase enzyme protein.The purpose is to provide raw materials for soybean Lipoxygenase in food addition,and Lay a theoretical foundation for the industrial development and utilization of soybean Lipoxygenase."Heinong 66" was a soybean cultivar which has high activity to LOX-3 enzyme,obtained through different soybean cultivars.In this experiment,the LOX-3 gene in "Heinong 66" was homologously cloned through genetic engineering techniques to construct the prokaryotic expression vector pET32b(+)-LOX-3.The constructed prokaryotic expression vector pET32b(+)-LOX-3 was transferred into the protein-compETent E.coli Rosetta strains and cultured.IPTG was used to induce the expression of E.coli Rosetta strains,and the target protein was purified using the purification kit.The concentration of obtained purified Lipoxygenase protein was determined by BCA method.Spectrophotometry was used to measure the changes of the enzyme activity of soybean Lipoxygenase and the change of the absorbance of soybean fat oxidase to ?-carotene,the bleaching effect was analyzed by the change in absorbance and color of ?-carotene.The main results are as follows:(1)LOX-3 gene was obtained by homologous cloning technique.The similarity of the LOX-3 gene sequencing sequence with the NCBI sequence was 99.95%,and the similarity of the encoded amino acid sequence alignment was 100%.The resulting gene was identified as the Lipoxygenase LOX-3 gene.(2)Successfully constructed the LOX-3 gene into the prokaryotic expression vector pET32b(+).Expression of Rosetta bacteria containing the pET32b(+)-LOX-3 prokaryotic expression vector was induced by IPTG.The purified soybean lipoxygenase LOX-3 protein had a concentration of 0.612354 ?g/?L and an enzyme activity of 4267.4 U.Prokaryotic Expression proves that the soybean protein has lipoxygenase activity.(3)LOX-3 was shown to be bleachable by the determination of the change in the bleached absorbance of the ?-carotene by LOX-3 and the change in the color of ?-carotene.