Screening, Identification And Characterization Of Novel Proteases And Esterases From The Sediments Of The Arctic And The Atlantic Based On Functional Metagenomic Analysis

As one of the three enzyme industry, but also one of the most widely used enzyme preparation, proteases accounted for60%of total world sales of enzymes have a good prospect in the food, pharmaceutical, detergents, cosmetics, textiles, leather, health chemicals and waste treatment and other industries. A novel fibrinolytic metalloprotease, two serine proteases and a metalloproteinase with a novel domain were isolated by screening of mategenomic libraries, and these mategenomic libraries were constructed using total genomic DNA extracted from the mud in the west coast of korea, surface sand from Gobi and Death deserts, the soil from mining shaft and compost, respectively. These proteases enrich the types of proteases and provide resources for industrial applications, but these current proteases screened by metagenomic techniques are only for basic research in the theory and are not applied to industrial production.Lipolytic enzymes (including esterase and lipase) are stable in the organic solvent, and reactions catalyzed by lipolytic enzymes proceed with high enantioselectivity, high regioselectivity, and did not be require for cofactors, Which make the lipolytic enzymes widely use in industry, pharmaceuticals, food production and other fields. Up to date, Lipolytic enzymes have been screened from soil, fresh water, hydrothermal vents, surface and deep marine sediments and other environments using the method of metagenomics, but most of the Lipolytic enzymes are not appropriate for industrial applications. Therefore, we need more researches by metagenomic to obtain proteases and lipolytic enzymes with greater industrial applicability.I. Screen of proteases and lipolytic enzymes from metagenomic libraries constructed from the marine sediment of the Arctic and the Atlantic.A metagenomic library was constructed using genomic DNA extracted from the marine sediment sample stn.3of Arctic. The metagenomic library included2700Escherichia coli clones which covered94.5Mbp DNA. Two positive clones were identified by screening active proteases in the ST3fosmid library. Based on the construction of subcloning libraries and subsequent sequencing, the gene sequences coding for proteases on2fosmids (12-3C,21-5G) were determined. The BLAST results show that protease3C subordinates M4_uncharacterized family, protease5G subordinate M10A/M12A family. And the proteins similar to3C and5G are mostly predicted proteins and hypothetical proteins in the NCBI nr database.The DNA extracted from deep-sea sediment samples26II-NAR-S009-TVG05/07of the Atlantic was used to construct fosmid metagenomic library which included about17,000Escherichia coli clones contained595Mbp DNA. Functional assays were carried out to screen active lipolytic enzymes and proteases in the TVG5/7fosmid library, and a total of3lipolytic enzymes positive clones and5proteases positive clones were identified.Ⅱ. Amino acid sequence analysis, heterogeneous expression and biochemical characterization of protease3CBased on multiple sequence alignment analysis, the homologous sequence most similar to3C was a hypothetical protein of Janthinobacterium lividum, which belonged to M4_uncharacterized family, and the similarity between the hypothetical protein with3C was51%. The protease3C was screened from ST3fosmid metagenomic library based on functional metagenomic analysis. Protease3C not only enriched the diversity of protease sequences in M4family, but also clarified the characteristics of this family.3C was heterogeneously expressed in E. coli BL21(DE3) and recombinant3C was purified and biochemically characterized. The purified intracellular3C was prothermolysin. The thermolysin is synthesized as a pre-proenzyme and the prosequence assists the refolding of the proenzyme in vitro and inhibits enzyme activity. The prosequence cleavage from the mature enzyme is autocatalytic. Recombinant3C was purified by Superdex-75gel filtration chromatography, and make specific activity increased by2times. Protease activity analysis of3C purified by gel filtration chromatography shows that3C has a variety of active mature form.Protease3C was completely inhibited by EDTA, EGTA and o-phenanthroline, which confirmed the BLAST results.3C was a mesophilic enzyme, displaying optimal activity at40℃and its optimal pH was7.3C was remarkably decreased by NaCl. NaCl concentrations similar to seawater had a little effect on the activity of3C, but the activity was inhibited with increasing NaCl concentrations increasing. The activity of3C was decreased by most of divalent metal ions, but the effect of univalent metal ions was weak, and its activity was only improved by Mn2+.