Characterization and improved properties of Glutamine synthetase from Providencia vermicola by site-directed mutagenesis.

In this study, a novel gene for Glutamine synthetase was cloned and characterized for its activities and stabilities from a marine bacterium Providencia vermicola (PveGS). A mutant S54A was generated by site directed mutagenesis, which showed significant increase in the activity and stabilities at a wide range of temperatures. The Km values of PveGS against hydroxylamine, ADP-Na2 and L-Glutamine were 15.7 ± 1.1, (25.2 ± 1.5) × 10-5 and 32.6 ± 1.7 mM, and the kcat were 17.0 ± 0.6, 9.14 ± 0.12 and 30.5 ± 1.0 s-1 respectively. In-silico-analysis revealed that the replacement of Ser at 54th position with Ala increased the catalytic activity of PveGS. Therefore, catalytic efficiency of mutant S54A had increased by 3.1, 0.89 and 2.9-folds towards hydroxylamine, ADP-Na2 and L-Glutamine respectively as compared to wild type. The structure prediction data indicated that the negatively charged pocket becomes enlarged and hydrogen bonding in Ser54 steadily promotes the product release. Interestingly, the residual activity of S54A mutant was increased by 10.7, 3.8 and 3.8 folds at 0, 10 and 50 °C as compared to WT. Structural analysis showed that S54A located on the loop near to the active site improved its flexibility due to the breaking of hydrogen bonds between product and enzyme. This also facilitated the enzyme to increase its cold adaptability as indicated by higher residual activity shown at 0 °C. Thus, replacement of Ala to Ser54 played a pivotal role to enhance the activities and stabilities at a wide range of temperatures.

Enhanced thermostability of halo-tolerant glutaminase from Bacillus licheniformis ATCC 14580 by immobilization onto nano magnetic cellulose sheet and its application in production of glutamic acid.

A halo-tolerant glutaminase gene (BlglsA) was isolated from Bacillus licheniformis. Heterologous expression of BlglsA revealed that it encodes for a 36 kDa protein containing 327 amino acid residues. The purified enzyme showed optimal activity at a pH of 9.5 while 35 °C was found to be the optimum temperature. The enzyme retained about 92 and 97% stability at pH 12 and temperature (40 °C) respectively. Subsequent immobilization of BlglsA on nano magnetic cellulose sheet (NMCS) led to an enhanced tolerance to higher temperature. NMCS-BlglsA showed optimum activity at 45 °C, although it was stable even at 60 °C. NaCl tolerance (≥90% in 0.3 M) was almost similar to BlglsA and NMCS-BlglsA. The metal ions Fe2+ (5 mM) and Mn2+ (2.5 mM) improved the BlglsA relative activity by 61 and 48%, respectively. In contrast, 5 mM Mn2+ was found suitable to enhance the activity of NMCS-BlglsA up to 72%. The production of glutamic acid by NMCS-BlglsA was 1.61 g/l in 48 h. Reusability test of NMCS-BlglsA showed 76 and 35% retention of the actual activity after 4th and 7th cycle, respectively. Such remarkable biochemical properties of NMCS-BlglsA make it an attractive enzyme for food industries.

Bio-affinity mediated immobilization of lipase onto magnetic cellulose nanospheres for high yield biodiesel in one time addition of methanol.

To synthesis biodiesel from palm oil in one-time addition of methanol and solvent-free medium using CBD fused with C-terminal of lipase from G. stearothermophilus (GSlip-CBD) was immobilized onto magnetic cellulose nanosphere (MCNS). The immobilized matrix traits were preconceived by FT-IR, TEM and XRD. Perceptible biodiesel yield 98 and 73% was synthesized by GSlip-CBD-MCNS in 4 h and GSlip-MCNS in 6 h under the optimized conditions of oil:methanol ratio (1:3.5), temperature (55 and 50 °C) and enzyme loading (15 U). Intriguingly, the operational stability of GSlip-CBD-MCNS was an easily attainable owing to the magnetic properties and could be reused up to 8th and19th cycles with 94 and 45% of biodiesel yield respectively, compared to GSlip-MCNS. Thus GSlip-CBD-MCNS could be a potential biocatalyst for higher yield of biodiesel and reusability in one step addition of methanol.