Nature versus nurture in two highly enantioselective esterases from Bacillus cereus and Thermoanaerobacter tengcongensis.
Bottom Line: The principal findings of this study are: (i) the complete sequenced genomes of Bacillus cereus ATCC 14579 and Thermoanaerobacter tengcongensis MB4 contain a hitherto undescribed enantioselective and alkaliphilic esterase (BcEST and TtEST respectively) that is specific for the production of (R)-2-benzyloxy-propionic acid ethyl ester, a key intermediate in the synthesis of levofloxacin, a potent antibiotic; and (ii) directed evolution targeted for increased thermostability of BcEST produced two improved variants, but in either case the 3-5 °C increase in the apparent melting temperature (T(m)) of the mutants over the native BcEST that has a T(m) of 50 °C was outperformed by TtEST, a naturally occurring homologue with a T(m) of 65 °C.Protein modelling of BcEST mapped the S148C and K272R mutations at protein surface and the I88T and Q110L mutations at more buried locations.This work expands the repertoire of characterized members of the α/β-fold hydrolase superfamily.
Affiliation: Biotechnology Research Institute, National Research Council Canada, Montreal, Quebec H4P 2R2, Canada.Show MeSH
Mentions: The four specified esterases were purified to electrophoretic homogeneity (Fig. 3) using a three‐step chromatographic procedure as described in Experimental procedures. By SDS‐PAGE (12%) gel, the proteins appear as single bands with estimated Mr between 35 and 38 kDa, in good agreement with the calculated Mr(s). The 2IC12 and TtEST proteins showed a slightly higher Mr than expected (34 336 and 34 388 respectively). The purified esterases eluted at 36–40 kDa by gel filtration on Superose‐12 indicating monomeric enzymes. Analysis by UV‐Vis showed no other absorbance peak besides that at 280 nm.
Affiliation: Biotechnology Research Institute, National Research Council Canada, Montreal, Quebec H4P 2R2, Canada.