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Highly Expression Of Recombinant Proteinase K In Pichia Pastoris

Posted on:2014-10-16Degree:MasterType:Thesis
Country:ChinaCandidate:T WuFull Text:PDF
GTID:2250330425452209Subject:Fermentation engineering
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Serine protease is a kind of serine as an important proteolytic enzyme activecenter, and plays an important and extensive pHysiological function in organisms,whose activation or inhibition of protease and plays a role of regulating factor. In thispaper,a serine protein enzymes--proteinase K, is obtained from Tritirachium albumLimber. Due to microbial synthesis of this kind of protease in keratin (Kerantin) as thesole source of carbon and nitrogen environment to grow, so called it proteinase K.Proteinase K high enzyme activity and broad substrate specificity, can firstdecomposition and hydropHobic amino acids, sulfur-containing amino acids, aromaticamino acid C terminal adjacency ester linkage and peptide bond, are often used todegrade the protein production short peptide. It has typical serine protein enzymeshave catalytic triplet Asp39His69-Ser224characteristics and activity centers aroundtwo Ca2+binding sites in order to increase its stability, and keep it in a wider range ofconditions of high enzyme activity. Proteinase K can make the protease inactivationor degradation, using this feature, proteinase K would have important applications inareas such as the nucleic acid purification, silk, medicine, food and brewing.This experiment is to the original proteinase K gene codon optimization, then thegene synthesis. Connect the synthetic protease K gene carrier pPIC9K, recombinantplasmid pPIC9K-proteinase K, then the linearization of the recombinant plasmidpPIC9K-proteinase K into GS115cells, screening strains of multi-copy gene, asexperimental strains, known as GS11-pPIC9K-proteinase K. This paper alsostudied the temperature, initial PH, induction time and the induction time of straingrowth and protein expression of recombinant proteinase K, the optimal cultureconditions of recombinant proteinase K is: after BMGY medium cultivation ofrecombinant protease K to arrive OD6002.0centrifugal, bacteria to precipitate heavyhanging in BMMY medium cultivation,72h, each24h add0.5%(v/v) methanol. Article also column separation and purification of recombinant proteinase K Ni-NTAaffinity chromatograpHy purification. The final purified proteinase k product had aspecific bioactivity of12.94U/mg with93.3%purity, and the total recovery rate was45.4%.The purity of proteinase k was increased from69.4to91.6%after purification.Through these purification processes,105mg proteinase k with1100U activity wasrecovered from1L fermentation culture. The overall recovery of proteinase k purifiedfrom the culture supernatant was68.9%.And studies the enzymatic properties ofrecombinant proteinase K, experimental results show that the purified proteinase khad similar enzymatic characteristics as the native enzyme and was good tolerant tomany denaturant. It also has good temperature resistance and acid and alkaliresistance, all these conclusions that recombinant proteinase K is a widely usedprotease.
Keywords/Search Tags:proteinase k, recombinant expression, Pichia pastoris, enzymatic property
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