Characterization of a novel thiol activated phospholipase TAPLB1 from Trichosporon asahii MSR 54.

Phospholipases are hydrolytic enzymes that play crucial roles in vivo and also possess immense biotechnological potential. In the present study, the phospholipase B of Trichosporon asahii MSR54 was overexpressed in E. coli and characterized. The 68-kDa enzyme was monomeric in solution and possessed phospholipase, lysophospholipase, esterase and acyltransferase activities. It was maximally active at pH 8.0 and 40 °C. The enzyme retained >50% activity between pH 3.0-8.0 and had a half-life of 30 min at 60 °C. Its activity was not metal dependent and was stable in the presence of most metal ions. Its catalytic efficiency on lysophosphatidyl choline was 1.0 × 103 mM-1 h-1. Site directed mutagenesis revealed R121 (present in the GYRAMV motif), S194 (present in the conserved GLSGG motif) and D420 (present in LVDXGE motif) to be the crucial amino acid residues for esterolytic activity. S194 and D420 were also the catalytic amino acids for lysophospholipase and phospholipase activities of the enzymes, while R121 was not involved in catalysis of phospholipid substrates. Further, it was found that cysteine residues in C61 and C354 were involved in disulphide linkages that imparted the properties of thiol activation and thermostability, respectively.

Functional characterization of hormone sensitive-like lipase from Bacillus halodurans: synthesis and recovery of pNP-laurate with high yields.

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 1/2 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 cat/K m with pNP-palmitate as a substrate was 2.52 × 105 mM-1 min-1 while that with glyceryl tripalmitin was 4.06 × 102 mM-1 min-1, 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.

Recombinant exochitinase of the thermophilic mould Myceliopthora thermophila BJA: Characteristics and utility in generating N-acetyl glucosamine and in biocontrol of phytopathogenic fungi.

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 × 103 U L-1 ). 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 t1/2 values of 113 and 48 min at 65 and 75°C, respectively. Kinetic parameters Km , Vmax , kcat , and kcat /Km of the enzyme are 4.655 mg mL-1 , 34.246 nmol mg-1  s-1 , 3.425 × 106 min-1 , and 1.36 × 10-6 mg mL-1  min-1 , 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.