Purification and characterization of CHpro1, a thermotolerant, alkali-stable and oxidation-resisting protease of Chumathang hotspring

Metagenomic approaches are recently used for searching novel open reading frames(ORFs) coding enzymes employed in pharmaceutical, food industries, etc.In this study, a metagenomic library was constructed from Chumathang hotspring sediment DNA. The library consisted of approximately 9,000 clones and was screened for protease activity. A clone exhibiting protease activity was identified and named CHpro1. Sequencing of CHpro1 revealed that the ORF encoded a functional protein of 363 amino acids belonging to peptidase S8–S53 superfamily.CHpro1 shared 41 % sequence similarity with a reported protease(subtilase family) and 35 % structural similarity with the crystal structure of Pro-Tk sps. of Thermococcus kodarkaenasis. In silico modeling the 3D structure of CHpro1 showed that it has two beta sheets, 10 alpha helices and 11 strands. Catalytic triad prediction implied CHpro1 to be a serine protease. The optimum temperature and p H of the purified protease were found to be 80 °C and 11.0,respectively. The enzyme was active at 5 % concentration of hydrogen peroxide and retained 60 % of activity at 10 %concentration. The thermotolerant, alkalophilic and oxidantstable properties of the protease make it a potential candidate for biotechnological applications.

Metagenomic approaches are recently used for searching novel open reading frames （ORFs） coding enzymes employed in pharmaceutical, rood industries, etc. In this study, a metagenornic library was constructed from Chumathang hotspring sediment DNA. The library consisted of approximately 9,000 clones and was screened for protease activity. A clone exhibiting protease activity was identified and named CHprol. Sequencing of CHprol revealed that the ORF encoded a functional protein of 363 amino acids belonging to peptidase S8-S53 superfamily. CHprol shared 41% sequence similarity with a reported protease （subtilase family） and 35 % structural similarity with the crystal structure of Pro-Tk sps. of Thermococcus kodarkaenasis. In silico modeling the 3D structure of CHprol showed that it has two beta sheets, 10 alpha helices and 11 strands. Catalytic triad prediction implied CHprol to be a serine protease. The optimum temperature and pH of the purified protease were found to be 80 ℃ and 11.0, respectively. The enzyme was active at 5 % concentration of hydrogen peroxide and retained 60 % of activity at 10 % concentration. The thermotolerant, alkalophilic and oxidant stable properties of the protease make it a potential can- didate for biotechnological applications.