Novel Cold-Adapted Esterase MHlip from an Antarctic Soil Metagenome

An Antarctic soil metagenomic library was screened for lipolytic enzymes and allowed for the isolation of a new cytosolic esterase from the a/b hydrolase family 6, named MHlip. This enzyme is related to hypothetical genes coding esterases, aryl-esterases and peroxydases, among others. MHlip was produced, purified and its activity was determined. The substrate profile of MHlip reveals a high specificity for short p-nitrophenyl-esters. The apparent optimal activity of MHlip was measured for p-nitrophenyl-acetate, at 33 °C, in the pH range of 6–9. The MHlip thermal unfolding was investigated by spectrophotometric methods, highlighting a transition (Tm) at 50 °C. The biochemical characterization of this enzyme showed its adaptation to cold temperatures, even when it did not present evident signatures associated with cold-adapted proteins. Thus, MHlip adaptation to cold probably results from many discrete structural modifications, allowing the protein to remain active at low temperatures. Functional metagenomics is a powerful approach to isolate new enzymes with tailored biophysical properties (e.g., cold adaptation). In addition, beside the ever growing amount of sequenced DNA, the functional characterization of new catalysts derived from environment is still required, especially for poorly characterized protein families like α/b hydrolases

Interaction of an odorant lactone with model phospholipid bilayers and its strong fluidizing action in yeast membrane.

Some odorant lactones are naturally present in fruits or in fermented products; they can also be used as food additives and can be produced by microorganisms at the industrial scale by biotechnological processes. Gamma-decalactone was previously shown to have antimicrobial properties. We determined by infrared spectroscopy measurements that this compound rapidly diffused into model phospholipid bilayers (within 2 min), modifying the general physical state of a dimyristoyl-L-alpha-phosphatidylcholine (DMPC) film. In vivo, the lactone strongly increased membrane fluidity in the model yeast Yarrowia lipolytica, as evaluated by fluorescence anisotropy measurements. This effect was more important than that of benzyl alcohol, which is known as a fluidizing agent in living cells, and may explain the toxic action of gamma-decalactone in microorganisms

Directed evolution of mammalian paraoxonases PON1 and PON3 for bacterial expression and catalytic specialization

Serum paraoxonases (PONs) are a group of enzymes that play a key role in organophosphate (OP) detoxification and in prevention of atherosclerosis. However, their structure and mechanism of action are poorly understood. PONs seem like jacks-of-all-trades, acting on a very wide range of substrates, most of which are of no physiological relevance. Family shuffling and screening lead to the first PON variants that express in a soluble and active form in Escherichia coli. We describe variants with kinetic parameters similar to those reported for PONs purified from sera and others that show dramatically increased activities. In particular, we have evolved PON1 variants with OP-hydrolyzing activities 40-fold higher than wild type and a specificity switch of >2,000-fold, producing PONs specialized for OP rather than ester hydrolysis. Analysis of the newly evolved variants provides insights into the evolutionary relationships between different family members