Mutagenesis Of Metallothionein Producing Strains And Purification Of Protein

This article is intended to mutagenesis high-yielding metallothionein strains of Saccharomyces cerevisiae,and its hydroxyl radical scavenging ability comparative studies, for the future of metallothionein in scavengingfree radicals and chelating heavy metals in the application provides a theoretical support, but also foundationfor the big scale production of metallothionein.In this paper, as the starting strain of Saccharomyces cerevisiae31206, After initial screen，complexscreen，and complex screen method selection，Uv-colorimetry， microwave and NTG compound mutationwere adopted to screen a high-yield metal sulfur protein from Saccharomyces Cerevisiae in the experiment.The results showed that the mutant strains had high genetic stability maintained over90%after15generation.The total protein content，metallothionein content simplified sulfhydryl reagents was determinedwith coomassie brilliant blue G-250，enzyme-linked immunosorbent assay（ELISA） and sulfhydryl active（DTNB method）respectively.As result，the total protein content of mutated Saccharomyces Cerevisiae wasincreased from44.6mg/g mycelium to170.2mg/g，the metallothionein content increased from38.3ng/L to163.4ng/L and the mercapto activity was increased from0.029μmol to0.146μmoL.Further optimization of fermentation conditions of the mutant strains N-8.CuCl2as the inducer with aconcentration of1.0mmol/L，activated for1days，induced pH6.5，added1mL activated culture into50mL，culture media in a250mL flask and induced in a rotate shaker （140r/min）at30℃for2days.response surfaceselect4factors：concentration，time，pH，temperature.The best proposal：induced concentration of0.7mmol/L，pH6.4，at30℃for62h.In selective condition，the metallothionein content is170.91ng/L.After Cu-MT was separated and purified using Sephadex G-100gel filter column and DEAE-celluloseanion exchange column，three subtypes were obtained，MT-Ⅰ，MT-Ⅱ，MT-Ⅲ，desalination using SephadexG-25gel filter column，obtain MT sterling by freeze dehydration with a molecular weight of7254Da，respectively，analyzed by SDS-PAGE gel electrophoresis. Comparing ascorbic acid’s ability of eliminatinghydroxy radical，Each subtype metallothionein-like protein shows a good effect.