Conserved cysteine variants of metagenomic derived polygalacturonase concurrently shift its optima at acidic pH and enhanced thermostability: structural and functional analysis.

To study the effect of conserved cysteins on biochemical properties of a previously cloned metagenomic polygalacturonase (PecJKR01), single point variants A42C, M283C, and double variants M283C + F24C, M283C + A42C were constructed. Mutations resulted in shifting the pH toward lower range and enhanced thermostability. The mutants were optimally active at pH 5.0 as compared to pH 7.0 for wild type. Point variants demonstrated slightly higher enzyme activity at 60o C than that of the wild type. In addition, the A42C/M283C + A42C variants displayed nearly 28-40% enhanced thermostability, while M283C + 24C was least thermostable among all variants/ wild type. Cys (pKa 8.18) possibly interfered in the ionization state resulting in change in pH optima of variants. Structure function analysis suggested that the increased activity in A42C could be due to van der Waals interactions in S···Ar with Phe29 and formation of an additional hydrogen bond between Cys42-S....HN-Ala31. Higher thermostability and decreased enzymatic activity of M283C might be attributed to the incorporation of additional disulfide linkage between Cys283 S=S Cys255 and decreased cavity size. Overall cysteine at position 42 was most promising in shifting the optimum pH toward lower range as well as for thermostability of enzyme.

Point mutation Arg153-His at surface of Bacillus lipase contributing towards increased thermostability and ester synthesis: insight into molecular network.

In order to design proteins with improved properties i.e. thermostability, catalytic efficiency and to understand the mechanisms underlying, a thermostable variant of Bacillus lipase was generated by site-directed mutagenesis with enhanced thermal (∆Tm = + 12 °C) and chemical (∆Cm denaturation for Gdmcl = + 1.75 M) stability as compared to WT. Arg153-His variant showed 72-fold increase in thermostability (t 1/2 = 6 h) at 60 °C as compared to WT (t 1/2 = 5 min). Increase in thermostability might be contributed by the formation of additional hydrogen bonds between His153/AO-Arg106/ANH2 as well as His153-Arg106/ANE. The variant demonstrated broad substrate specificity. A maximum conversion of 59 and 62% was obtained for methyl oleate and methyl butyrate, respectively, using immobilized variant lipase, whereas immobilized WT enzyme synthesizes 35% methyl oleate. WT enzyme was unable to synthesize methyl butyrate as it showed negligible activity with pNP-butyrate.

Structural and functional insights into thermostable and organic solvent stable variant Pro247-Ser of Bacillus lipase.

Thermostability of enzymes is an important issue in protein engineering and has been studied in detail. Still there is no hard and fast rule to define the conditions which will provide thermal stability. Understanding the various factors and mechanism responsible for thermal stability will add on new insights into our present knowledge in this area. Pro247-Ser variant was constructed based on homology modelling and rational design. It exhibited 60 fold increase in thermal stability at 60°C and+0.7M shift in C1/2 value for urea denaturation as compared to WT. Variant displayed noticeable tolerance to organic solvents. With decrease in Km, catalytic efficiency of Pro247-Ser variant was increased by 12 fold. The activity and stability assay including circular dichroism and fluorescence spectroscopy favoured increased thermal performance of variant. Hydrolytic activity of variant was found to be high in comparison to control for all p-nitrophenol esters investigated. The immobilized variant enzyme demonstrated nearly two fold enhanced conversion of methyl oleate than WT enzyme. The additional molecular interactions of variant residue might contribute to increased thermostability of lipase. The homology modeling predicted formation of additional hydrogen bonds between Ser247/O-Thr251/OG1 as well as Ser247/O-Glu250/N.