Cloning, Expression And Characterization Of Thermostable Protease From Thermoascus Aurantiacus Var. Levisporus

Protease is one of the most important groups of industrial enzymes that form a major portion of worldwide enzyme sales. They have been widely used in industrial production for a long time, especially in the food, chemical, weaving, washing detergent and leather industries. The alkaline proteases from bacteria have been intensively studied. Recently, the application of protease in organic synthesis has increased rapidly. There has been an increasing interest in proteases from thermophiles fungus, which were expected to produce thermostable proteases.Thermophilic fungus is the group of extremophiles which grow at high temperature and alkaline pH. Studies on thermostable enzymes from thermophilic fungi have been paid more attention with the expectation to produce thermostable enzyme. In thermophilic fungi, studies on proteases are very limited, only few protease genes have been cloned. Thermoascus aurantiacus var. levisporus is a widely distributed soilinhibiting fungus with the potential of thermostable enzymes production. Thermoascus aurantiacus var. levisporus also produced thermostable protease, however, no further information has been reported and the protease gene has not been cloned yet.Degenerate primers designed on the conserved domain of other reported serine proteases, and a cDNA fragment encoding the protease gene was obtained through RT-PCR. The RACE and TAIL were used to generate full-length cDNA clones. The full length of Tapro cDNA gene is 2704bp, which contained an ORF of 1482bp encoding 494 amino acids. The cDNA and DNA sequence of gene Tapro has been registered in GenBank with accession number EU364816. The alignment results of putative amino acids sequence showed the catalytic domain of Tapro appear to have independently and convergently evolved an Asp183/Ser384/His215catalytic triad, like that found in the trypsin serine proteasesThe Tapro gene and expression vector pPIC9K were digested with EcoRâ… and Notâ… , and ligation was carried out in vitro. The recombinant expression plasmid pPIC9K/Tapro was constructed and sequenced to confirm the correct reading frame. The constructed plasmid pPIC9K/Tapro was linearized with a restriction enzyme Xbaâ… (insertion at 5'AOX1), and transformed into Pichia pastoris GS115 competent cell by electroporation methods, and Screened for His+Mut+ transformants on MD and MM plates. The parent vector was linearized with the same restriction enzyme and transformed GS115 as a control. PCR analysis of P. pastoris integrants and G418 screening determined the multicopy integrants to induce by methanol. These integrants were used to analyze expression levels of interest protein every 24 hours. The engineering strain with highest expression level was called GS-TAPRO-18. The genetic and protease expression stability of recombinant P. pastoris GS-TAPRO-18 was tested and characterized.By SDS-PAGE, the molecular weight of the expressed protease was estimated to be 59 kDa, respectively. The protease was found to be inhibited by PMFS. The protease exhibited maximal activity at pH 8.0. The optimum temperature for the proteases was 50oC. It was thermostable at 60oC. The protease activity of protease remained 60% after incubation at 70oC for 1h. The thermal stability of the enzyme was significantly enhanced by Ca2+. It implied the expressed protease could be widely applied in the industry and biological fields. Therefore, we hope to reconstruct the Tapro gene and optimize the expression condition to obtain the yeast engineering strains suitable for industrial applications.