Bioprospecting of microbial hydrolytic enzymes in extreme environments

Acidic streamers and geothermal sites used serve as a part of natural reservoirs of extremophiles, including unculturable Bacteria and Archaea. They provide a large marine and terrestrial biodiversities especially for hydrolases. This study was followed the previous steps of a functional metagenomics research at Bangor University, where the lipolytic and cellulolytic activities have been ready screened using agar-based methods. To this research my supervisors provided me the DNA sequencing data of three fosmid pools A, B and C from V12 fosmid library, which was generated from a site sampled in the shallow hydrothermal vent of the Levante bay of Vulcano Island (Italy), and PCR products of various genes cloned from the chromosomal DNA of an Archaea Cuniculumplasma species. From the fosmid sequence data, I have annotated about 809 ORFs, but just only 30 of them were found to be related to hydrolytic enzymes of interest including lipases, esterases and glycosyl hydrolases. The 30 ORFs from V12 library were affiliated within a broad spectrum of bacterial phyla including Acidobacteria, Proteobacteria, Bacteroidetes, within others. Whereas, the CIB sequences were confirmed to belong at 100% to Cuniculiplasma divulgatum archaeal organism as expected. From the sequence analysis of the positive clones of V12 library, six were amplified and cloned into E.coli NovaBlue using pET/lic method and expressed in E. coli BL-21 (DE3) but only four of them were expressed in soluble forms. Eight genes from CIB genome sequences were also cloned into E.coli, but just three of them were successfully expressed. From CIB library we selected two beta-galactosidase, CIB_12 and CIB-13, and one alpha-glucosidase, CIB_14. Likewise, we selected three lipases/esterases and one glycosyl hydrolase from the V12 library. This study confirmed some habitat specificity both for V12 and CIB samples assessing the specific activity through the enzyme characterization. For the first, esterases from V12 library were found to be thermophilic and exhibited elevated stability under temperatures up to 90°C. On the other hand, putative glycosyl hydrolases showed high specific activity at pH 5.5 and even at pH 4.0. Active enzymes from both genomic and metagenomic libraries showed considerable activities with high concentration of organic solvents, especially with 1,2 propandiol and methanol for CIB GHs, with methanol and acetonitrile for V12 LIP enzyme. Detailed characterisation for the enzymes purified using Ni-NTA affinity chromatography have been reported in this work.