Engineering and characterization of a novel low temperature active and thermo stable esterase from marine Enterobacter cloacae.

Esterases are one of the most important industrial enzymes. Here, a novel estA was cloned from Enterobacter sp. and characterized. The sequence alignment results showed that it was a novel esterase. The purified EstA had a molecular weight of 26 KDa with an optimum temperature and pH of 40 °C and 9.0. EstA retained >70% activity between 0 °C and 20 °C, indicating it was a low temperature active enzyme. EstA exhibited low activity after incubation at 45 °C for 120 min or 50 °C for 30 min. In the presence of organic solvents, detergents and different concentrations of NaCl, EstA retained high activity. In order to improve thermal stability, a mutant A92D with better thermal stability than EstA was obtained by random mutation. ESTA92D showed high activity at 45 °C for 120 min and maintained 85% of the original activity at 50 °C for 30 min, approximately a 3.4-fold increase over EstA. Homology modeling analysis showed that the improved thermostability of ESTA92D was attributed to hydrophilic Asp rather than hydrophobic Ala, leading to an increase of the interaction and solubility as well as the surrounding area. The improved thermostability of low-temperature-active EstA suggests its immense applications in industrial applications.

Cloning, purification and characterization of novel Cu-containing nitrite reductase from the Bacillus firmus GY-49.

Nitrite is generated from the nitrogen cycle and its accumulation is harmful to environment and it can be reduced to nitric oxid by nitrite reductase. A novel gene from Bacillus firmus GY-49 is identified as a nirK gene encoding Cu-containing nitrite reductase by genome sequence. The full-length protein included a putative signal peptide of 26 amino acids and shown 72.73% similarity with other Cu-containing nitrite reductase whose function was verified. The 993-bp fragment encoding the mature peptide of NirK was cloned into pET-28a (+) vector and overexpressed as an active protein of 36.41 kDa in the E.coli system. The purified enzyme was green in the oxidized state and displayed double gentle peaks at 456 and 608 nm. The specific activity of purified enzyme was 98.4 U/mg toward sodium nitrite around pH 6.5 and 35 °C. The K m and K cat of NirK on sodium nitrite were 0.27 mM and 0.36 × 103 s-1, respectively. Finally, homology model analysis of NirK indicated that the enzyme was a homotrimer structure and well conserved in Cu-binding sites for enzymatic functions. This is a first report for nitrite reductase from Bacillus firmus, which augment the acquaintance of nitrite reductase.