Molecular Characterization of the Iron-Containing Alcohol Dehydrogenase from the Extremely Thermophilic Bacterium Pseudothermotoga hypogea.

Pseudothermotoga hypogea is an extremely thermophilic bacterium capable of growing at 90 °C and producing ethanol, which is catalyzed by an alcohol dehydrogenase (ADH). The gene encoding P. hypogea ADH (PhADH) was cloned, sequenced and over-expressed. The gene sequence (1164 bp) was obtained by sequencing all fragments of the gene, which were amplified from the genomic DNA. The deduced amino acid sequence showed high identity to iron-containing ADHs from other Thermotoga species and harbored typical iron- and NADP-binding motifs, Asp195His199His268His282 and Gly39Gly40Gly41Ser42, respectively. Structural modeling showed that the N-terminal domain of PhADH contains an α/ß-dinucleotide-binding motif and that its C-terminal domain is an α-helix-rich region containing the iron-binding motif. The recombinant PhADH was soluble, active, and thermostable, with a subunit size of 43 ± 1 kDa revealed by SDS-PAGE analyses. The recombinant PhADH (69 ± 2 U/mg) was shown to have similar properties to the native enzyme. The optimal pH values for alcohol oxidation and aldehyde reduction were 11.0 and 8.0, respectively. It was also thermostable, with a half-life of 5 h at 70 °C. The successful expression of the recombinant PhADH in E. coli significantly enhanced the yield of enzyme production and thus will facilitate further investigation of the catalytic mechanisms of iron-containing ADHs.

Comparative modelling, molecular docking and immobilization studies on Rhizopus oryzae lipase: evaluation of potentials for fatty acid methyl esters synthesis.

Elucidation of lipase-substrate interactions will guide the proper industrial use and applicability of the enzyme. The aim of this study was to predict the 3 D structure of Rhizopus oryzae ZAC3 (RoZAC3) lipase, study its interactions with some natural substrates and evaluate the feasibility of fatty acid methyl esters (FAME) production by the immobilized lipase. Protein identification of RoZAC3 lipase was carried out using LC-MS/MS. The 3 D structure of the lipase was built using homology modelling and natural substrates such as tributyrin, tripalmitin and triolein were docked to the optimized 3 D model for investigation of enzyme-ligand interactions. RoZAC3 lipase, immobilized by adsorption on Lewatit VP OC 1600 was applied in the synthesis of fatty acid methyl esters (FAME). From the phylogenetic analysis, it was observed that RoZAC3 lipase was closely related (48%) to Rhizopus javanicus lipase (Q7M4U7). The predicted 3 D model was validated using the SWISS model validation server. Ramachandran and ERRAT plots were used to assess the amino acid environment and overall quality of the model. From the docking studies, the values of the binding energies obtained for tributyrin, tripalmitin and triolein were - 5.37, -5.27 and -5.77 respectively. At an enzyme:immobilization support ratio of 50 mg/g, transesterification reaction duration of 18 h and a temperature of 40 oC, the conversion reached above 80%. The molecular docking studies provided information on the interaction/modifications between the RoZAC3 lipase and triacylglycerols that can be exploited for numerous applications. The immobilized lipase could serve in hydro-esterification reactions adaptable for biodiesel production.Communicated by Ramaswamy H. Sarma.

Strain improvement and statistical optimization as a combined strategy for improving fructosyltransferase production by Aureobasidium pullulans NAC8.

Strain improvement of a low fructosyltransferase-producing Aureobasidium pullulans NAC8 (Accession No. KX023301) was carried out using chemical mutagens such as ethidium bromide and ethyl methane sulfonate. The wild-type and mutant strain were distinguished using Random amplified polymorphic DNA PCR and DNA fingerprinting analysis. Plackett-Burman and Box Behnken design were statistical tools used to determine important media parameters and optimization, respectively. Phenotypically and genetically, the new improved strain was different from the wild-type. The most important media parameters from PDB influencing fructosyltransferase production were ammonium chloride, sucrose and yeast extract at p < 0.05. Some significant parameters obtained with the BBD exhibited quadratic effects on FTase. The F values (35.37 and 32.11), correlation coefficient (0.98 and 0.97) and the percent coefficient of variation (2.53% and 2.40%) were obtained for extracellular and intracellular FTase respectively. The validation of the model in the improved strain resulted in an overall 6.0 and 2.0-fold increase in extracellular and intracellular FTase respectively compared to the wild-type. A relatively low FTase-producing strain of Aureobasidium pullulans NAC8 was enhanced for optimum production using a two-pronged approach involving mutagenesis and statistical optimization. The improved mutant strain also had remarkable biotechnological properties that make it a suitable alternative than the wild-type.