Abstract
Screening of 20,000 clones of a fosmid gene bank, constructed from DNA extracted from North West Himalaya (NWH) glacier soil sample, using functional approach identified 10 esterase/lipase-producing clones. Of these, a clone designated pFG43 with an insert size of 45 kb which produced the highest concentration of enzyme (467.43 U/mg) was sequenced. Clone pFG43 contained 61 open reading frames (ORF) and of these an ORF of 1155 bp designated ME-003, was found to be closely related to a hydrolase from Acidobacteria sps (77% sequence identity and E value = 1e-164) and subsequently identified as a putative cocaine esterase. ORF ME-003 was amplified and sub-cloned using a TA vector system into E. coli (DH5α). The purified recombinant enzyme with a molecular weight of 43 kDa had optimal activity at 40 °C, pH 6 and the highest activity with shorter chain fatty acids than with higher chain length fatty acids. There is insignificant effect of inhibitors on the enzyme activity of ME-003, except PMSF which completely inhibited its activity. ME-003 activity was also inhibited in the presence of copper oxide but remained stable in presence of other metal ions. The enzyme activity was also inhibited in the presence of organic solvents; however, in the presence of 10% isopropanol, 12% of enzymatic activity was retained. Among various detergents, SDS completely inhibited enzymatic activity. The recombinant enzyme also shows enantio-specific activity against the racemic drug intermediates/precursors and exhibited 90% ee against racemic 1-phenyl ethanol and fluoxetine.