Thermo-alkali-stable lipase from a novel Aspergillus niger: statistical optimization, enzyme purification, immobilization and its application in biodiesel production.

The influences of nutritional components affecting lipase production from the new Aspergillus niger using wheat bran as substrate were studied by employing Plackett-Burman and central composite statistical designs. Out of the 11 medium components tested, sucrose, KH2PO4 and MgSO4 at final concentrations of 3.0, 1.0 and 0.5 g/L, respectively, were reported to contribute positively to enzyme production (20.09 ± 0.98 U/g ds). The enzyme was purified through ammonium sulfate precipitation followed by Sephadex G-100 gel filtration. Molecular mass of the purified lipase was 57 kDa as evident on SDS-PAGE. Different methods of immobilization were studied and the highest immobilization yield of 81.7 ± 2.18% was reported with agarose (2%) and the optimum temperature was raised from 45 to 50 °C. Immobilized lipase could retain 80% of its original activity at 60 °C after 1 hr of incubation, and was stable at pH values between neutral and alkaline pH. Lipase-catalyzed transesterification process of fungal oil resulted in a fatty acid methyl ester yield consisting of a high percentage of polyunsaturated fatty acids (83.6%), making it appropriate to be used as winter-grade biodiesel. The operational stability studies revealed that the immobilized lipase could keep 70% of its total activity after 5 cycles of the transesterification process.

Purification and partial characterization of NAD aminohydrolase from Aspergillus oryzae NRRL447.

Aspergillus oryzae aminohydrolase free acid phosphodiesterase catalyzes nicotinamide adenine dinucleotide to deamino-NAD and ammonia. The enzyme was purified to homogeneity by a combination of acetone precipitation, anion exchange chromatography and gel filtration chromatography. The enzyme was purified 230.5 fold. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band of MW 94 kDa. The enzyme displayed maximum activity at pH 5 and 40 °C with NAD as substrate. The enzyme activity appeared to be stable up to 40 °C. The enzyme activity was enhanced slightly by addition of Na⁺ and K⁺, whereas inhibited strongly by addition of Ag⁺, Mn²âº, Hg²âº and Cu²âº to the reaction mixtures. The enzyme hydrolyzes several substrates, suggesting a probable non-specific nature. The enzyme catalyzes the hydrolytic cleavage of amino group of NAD, adenosine, AMP, CMP, GMP, adenosine, cytidine and cytosine to the corresponding nucleotides, nucleosides or bases and ammonia. The substrate concentration-activity relationship is the hyperbolic type and the apparent Km and Kcat for the tested substrates were calculated.

NAD deamidation "a new reaction" by an enzyme from Aspergillus terreus DSM 826.

NAD deamidation is a non-previously recognized reaction. This reaction has been found to be catalyzed by extracts of Aspergillus terreus DSM 826. Conversion of NAD to the biosynthetic intermediate, deamido NAD, by these extracts, at the optimum pH and temperature did not exceed about 55 of the amount of the substrate added. Completion of the reaction was achieved when the extracts were pre-heated at 50 degrees C for 15 min in absence of the substrate. In a very similar manner, the extracts catalyzed hydrolytic cleavage of the amide linkages of different biomolecules such as nicotinamide, nicotinamide riboside, nicotinamide mononucleotide, L-glutamine, L-asparagine and acetamide. Polyacrylamide was also deamidated under the same conditions. In addition, complete dephosphorylation of the dinucleotide molecule was also effected by the same extracts. Separation of the NAD deamidating enzyme from the NAD dephosphorylating enzyme was achieved on using either DEAE - Sephadex A-25 or Sephadex G-200 column chromatography. The obtained phosphohydrolase-free-deamidase showed optimum activity at pH 8 of 0.1 M phosphate buffer and 50 degrees C. It exhibited broad substrate specificity and hyperbolic substrate saturation kinetics. It was isosterically inhibited by the product of its activity and this inhibition was prevented by heating the extracts at 50 degrees C for 15 min. Its activity was not affected in presence of sodium fluoride, partially inhibited in presence of magnesium chloride and was retained in the freezer for some months.