Purification, Cloning And Expression Of Thermostable Protease From Chaetomium Thermophilum

Protease was an important group of enzymes both in physiology and commerce fields. Microbial proteases dominate commercial applications. There has been an increasing interest in proteases from thermophiles, which were expected to produce thermostable proteases. Two thermostable extracellular proteases from culture supernatant of the thermophilic fungus Chaetomium thermophilum were purified to homogeneity by fractional ammonium sulphate precipitation, ion exchange chromatography on DEAE-Sepharose and Phenyl-Sepharose hydrophobic interaction chromatography. By SDS-PAGE, the molecular weight of the two purified enzymes was estimated to be 33 kDa and 63 kDa respectively. The two proteases were found to be inhibited by PMFS, while not by iodoacetamide and EDTA. The 33 kDa protease (PRO33) exhibited maximal activity at pH 10.0 and the 63 kDa protease (PRO63) at pH 5.0. The optimum temperature for the two proteases was 65oC. The PRO33 had a Km value of 6.6 mM and a Vmax value of 10.31μmol/L/min. The PRO63 was 17.6 mM and 9.08μmol/L/min with casein as substrate. They were thermostable at 60oC. The protease activity of PRO33 and PRO63 remained 67.2% and 17.31% respectively after incubation at 70oC for 1h. The thermal stability of the two enzymes was significantly enhanced by Ca2+. The residual activity of PRO33 and PRO63 at 70oC after 60 min was approximately 88.59% and 39.2% respectively when kept in the buffer containing Ca2+. These properties make them applicable for many biotechnological purposes.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 was used to generate full-length cDNA clones. The full length of pro cDNA gene is 2007bp, which contained an ORF of 1596bp encoding 532 amino acids. The cDNA and DNA sequence of gene pro has been registered in Genbank with accession number DQ839520 and EF100880 respectively.The alignment results of putative amino acids sequence showed the catalytic domain of pro was high homology with the catalytic domains of the subtilisin serine proteases. The pro gene and expression vector pET-22b(+) were digested with EcoRⅠand NotⅠ, and ligation was carried out in vitro. The recombinant plasmid pET/pro was generated, and transformed into E. coli BL21. The recombinant protein was produced in large amount after IPTG induction, approximately 41kDa protein was determined by SDS-PAGE, and this size was coincident with the protein molecular weight from the putative amino acid sequence; no interest protein was produced by inducing with IPTG after the pET-22b(+) was transformed into BL21. The solubility analysis showed the recombinant protein was presented in a fusion form. The high induce temperature results in E. coli BL21 rapid growth and over expression of recombinant protein in host cell maybe the reason.The pro gene and expression vector pPIC9K were digested with EcoRⅠand NotⅠ, and ligation was carried out in vitro. The recombinant expression plasmid pPIC9K/pro was constructed and sequenced to confirm the correct reading frame. The constructed plasmid pPIC9K/pro was linearized with a restriction enzyme SacI (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-PRO-16. The genetic and protease expression stability of recombinant P. pastoris GS-PRO-16 was tested and characterized.After streak culture for single colony of His+ transformant GS-PRO-16 on YPD plate for 10 generations, PCR analysis showed the interest gene was integrated in P. pastoris genome, and the expression level was also kept stable. The C. thermophilum protease was secreted into the culture medium by the yeast P. pastoris in a functionally activity form, and some specific properties of expressed protease were similar to those of the native strain C. thermophilum. It implied the expressed protease could be widely applied in the industry and biological fields. Therefore, we hope to reconstruct the pro gene and optimize the expression condition to obtain the yeast engineering strains suitable for industrial applications.