Chitinase from the thermophilic mould Myceliopthora thermophila BJA (MtChit) is an acid tolerant, thermostable and organic solvent stable biocatalyst which does not require any metal ions for its activity. To produce high enzyme titres, reduce fermentation time and overcome the need for induction, this enzyme has been heterologously expressed under GAP promoter in the GRAS yeast, Pichia pastoris. The production medium supplemented with the permeabilizing agent Tween-20 supported two-fold higher rMtChit production (5.5 × 10 U L ). The consensus sequences S(132)xG(133)G(134) and D(168)xxD(171)xD(173)xE(175) in the enzyme have been found to represent the substrate binding and catalytic sites, respectively. The rMtChit, purified to homogeneity by a two-step purification strategy, is a monomeric glycoprotein of ∼48 kDa, which is optimally active at 55°C and pH 5.0. The enzyme is thermostable with t values of 113 and 48 min at 65 and 75°C, respectively. Kinetic parameters K , V , k , and k /K of the enzyme are 4.655 mg mL , 34.246 nmol mg s , 3.425 × 10 min , and 1.36 × 10 mg mL min , respectively. rMtChit is an unique exochitinase, since its action on chitin liberates N-acetylglucosamine NAG. The enzyme inhibits the growth of phytopathogenic fungi like Fusarium oxysporum and Curvularia lunata, therefore, this finds application as biofungicide at high temperatures during summer in tropics. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:70-80, 2017.

The HSL-like lipase encoding gene (Blip) from the polyextremophile Bacillus halodurans C-125 has been heterologously expressed in E.coli BL21(DE3). The enzyme is a monomer of ~42 kDa. It has extremely high thermal stability with a t of 35 min at 100 °C. Thermal denaturation/renaturation studies by CD and fluorescence analysis revealed complete refolding of the protein back to its native conformation even after 30 min at 90 °C. Blip prefers substrates with mid to long chain fatty acids. It has a higher catalytic efficiency on para-nitrophenyl fatty acyl esters as opposed to triacylglycerides (k/K with pNP-palmitate as a substrate was 2.52 × 10 mM min while that with glyceryl tripalmitin was 4.06 × 10 mM min, respectively). The enzyme also has a unique selectivity for hydrolysis of unsaturated fatty acyl esters. The enzyme catalyses the synthesis of pNP-laurate with an optimized conversion of 95.94 ± 0.24%. A simple procedure for purification of the product has been developed that led to 89.91 ± 0.33% product recovery.

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