Effects of substrate binding site residue substitutions of xynA from Bacillus amyloliquefaciens on substrate specificity.

BACKGROUND: The aromatic residues of xylanase enzyme, W187, Y124, W144, Y128 and W63 of substrate binding pocket from Bacillus amyloliquefaciens were investigated for their role in substrate binding by homology modelling and sequence analysis. These residues are highly conserved and play an important role in substrate binding through steric hindrance. The substitution of these residues with alanine allows the enzyme to accommodate nonspecific substrates. RESULTS: Wild type and mutated genes were cloned and overexpressed in BL21. Optimum pH and temperature of rBAxn exhibited pH 9.0 and 50 °C respectively and it was stable up to 215 h. Along with the physical properties of rBAxn, kinetic parameters (Km 19.34 ± 0.72 mg/ml; kcat 6449.12 ± 155.37 min- 1 and kcat/Km 333.83 ± 6.78 ml min- 1 mg- 1) were also compared with engineered enzymes. Out of five mutations, W63A, Y128A and W144A lost almost 90% activity and Y124A and W187A retained almost 40-45% xylanase activity. CONCLUSIONS: The site-specific single mutation, led to alteration in substrate specificity from xylan to CMC while in case of double mutant the substrate specificity was altered from xylan to CMC, FP and avicel, indicating the role of aromatic residues on substrate binding, catalytic process and overall catalytic efficiency.

Enhanced catalysis of L-asparaginase from Bacillus licheniformis by a rational redesign.

L-Asparaginase (3.5.1.1) being antineoplastic in nature are used in the treatment of acute lymphoblastic leukemia (ALL). However glutaminase activity is the cause of various side effects when used as a drug against acute lymphoblastic leukemia (ALL). Therefore, there is a need of a novel L-asparaginase (L-ASNase) with low or no glutaminase activity. Such a property has been observed with L-ASNase from B. licheniformis (BliA). The enzyme being glutaminase free in nature paved the way for its improvement to achieve properties similar to or near to the commercially available L-ASNases. Rational enzyme engineering approach resulted in four mutants: G238N, E232A, D103V and Q112H. Among these the mutant enzyme, D103V, had a specific activity of 597.7IU/mg, which is higher than native (rBliA) (407.65IU/mg). Moreover, when the optimum temperature and in vitro half life were studied and compared with native BliA, D103V mutant BliA was better, showing tolerance to higher temperatures and a 3 fold higher half life. Kinetic studies revealed that the mutant D103V L-ASNase has increased substrate affinity, with Km value of 0.42mM and Vmax of 2778.9µmolmin(-1).

Purification and properties of an endoglucanase from Thermoascus aurantiacus.

An Endo-cellulase was purified to homogeneity using ammonium sulfate precipitation, ion exchange and size exclusion chromatography from newly isolated strain of Thermoascus aurantiacus RBB-1. The recovery and purification fold were 13.3% and 6.6, respectively, after size exclusion chromatography. The purified cellulase has a molecular mass (M) of 35 kDa. Optimum temperature for the enzyme was found to be 70 °C and stability was upto 80 °C for 1 h. Along with higher stability at 80 °C, enzyme showed half lives of 192 h and 144 h at 50 and 70 °C respectively. The purified cellulase was optimally active at pH 4.0 and was stable over a broad pH range of 3.0-7.0. The enzyme purified showed apparent Km and Vmax values of 37 mg/ml and 82.6 U/min/mg protein respectively with higher salt tolerance of 10% for 1 h.

Characterization of a recombinant glutaminase-free L-asparaginase (ansA3) enzyme with high catalytic activity from Bacillus licheniformis.

L-Asparaginase (3.5.1.1) is an enzyme widely used to treat the acute lymphoblastic leukemia. Two genes coding for L-asparaginase (ansA1 and ansA3) from Bacillus licheniformis MTCC 429 were cloned and overexpressed in Escherichia coli BL21 (DE3) cells. The recombinant proteins were purified to homogeneity by one-step purification process and further characterized for various biochemical parameters. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that both the enzymes are monomers of ∼37 kDa. Recombinant ansA1 was found to be highly unstable, and recombinant ansA3 was catalytically active and stable, which showed an optimum activity of 407.65 IU/mg at 37 °C and pH 8. Recombinant ansA3 showed higher substrate specificity for L-asparagine with negligible glutaminase activity. Kinetic parameters like K m , V max, k cat, and k cat/K m were calculated for recombinant ansA3.

Investigation on fixed bed column performance of fluoride adsorption by sugarcane charcoal.

The present study explores the potentiality of sugarcane charcoal for fluoride removal from synthetic fluoride solution. Column adsorption experiments with respect to variation of flow rate, pH, initial concentration, and column depths were carried out. Sugarcane charcoal exhibited almost consistent scavenging capacity at various bed depths with a flow rate 4.34 ml min(-1). Maximum adsorption capacity of sugarcane charcoal was recorded 7.33 mg g(-1). The adsorption studies were simulated using Thomas and Bed depth service time model. Both the models consistently predict its characteristic parameters and describe the breakthrough profiles in the whole range of sorption process.

Assessment of indoor pollutants generated from bio and synthetic fuels in selected villages of Burdwan, West Bengal.

The objective of the present study was to access the pollutant generated from bio-fuels like bamboo sticks, cow dung, paddy straw, carbon dust cake, gobar gas, jute stick, and mustard stick and synthetic fuel like LPG during cooking in rural villages of Burdwan, West Bengal, India and its fluctuation in living room. The average SO2 released from the fuels was found in the following order: bamboo stick > cow dung > paddy straw > carbon cake > gobar gas > jute stick > LPG > mustard stick; NO2 emission was in the following order : mustard stick > carbon dust cake > paddy straw > cow dung cake > LPG, jute stick > gobar gas > bamboo stick > and SPM was obtained in the following sequence: cow dung cake > bamboo stick > carbon dust cake > gobar gas > LPG > mustard stick > paddy straw > jute stick, respectively. The highest living room to kitchen room (L/K) ratio of SO2, NO, and SPM was found in LPG, gobar gas, jute stick respectively in 2009 and followed by bamboo stick > paddy straw > jute stick > cow dung cake, respectively in 2010. Results of this study suggest that different fuels released different amount of air pollutants, but more extensive study is needed to confirm the relationship between fuels and released air pollutants.

Utilization of Jatropha deoiled seed cake for production of cellulases under solid-state fermentation.

Toxic waste generated by Jatropha seed cake after utilization of biodiesel on one hand has stimulated the need to develop new technologies to treat the waste and on the other, forced us to reevaluate the efficient utilization of its nutritive potential for production of various high-value compounds and its conversion to non-toxic forms which could be used as animal feed stock. In this study, Jatropha seed cake was used for production of cellulases by new isolate of Thermoascus aurantiacus under solid-state fermentation. The interaction of nitrogen source concentration, moisture ratio, initial pH of the medium and inoculum size was investigated and modelled using response surface methodology (RSM) using Box-Behnken Design (BBD). Under optimized conditions endo-ß-1,4-glucanase, ß-glucosidase and filter paper activities were found to be 124.44, 28.86, 4.87 U/g of substrate, respectively. Characterization of endo-ß-1,4-glucanase, ß-glucosidase was done after partial purification by ammonium sulfate fractionation followed by desalting. The endo-ß-1,4-glucanase and ß-glucosidase showed maximum activity at 70 °C and pH 4. Saccharification studies performed with different lignocellulosic substrates showed that sugar cane bagasse was most susceptible to enzymatic hydrolysis. The study suggests that Jatropha seed cake can be used as a viable nutrient source for cellulase production without any pretreatment under solid-state fermentation by T. aurantiacus.

Low-dose intraocular triamcinolone injection for intractable macular oedema and inflammation in patients with uveitis.

AIMS: To evaluate the efficacy and safety of low-dose (2 mg in 0.05 ml) intraocular triamcinolone injection for patients with uveitis-related cystoid macular oedema and/or intractable intraocular inflammation. PATIENTS AND METHODS: Retrospective clinical case series. RESULTS: Cystoid macular oedema was eliminated in 24/30 eyes (80%). Intractable intraocular inflammation was eliminated in 4/8 eyes (50%). Snellen visual acuity was improved by two lines or more after 14/36 injections (38.9%). Intraocular pressure rose to above 21 mmHg after 8/36 injections (22%). There were no major complications. CONCLUSIONS: Low-dose (2 mg in 0.05 ml) intraocular triamcinolone acetonide injection is safe and effective for the management of refractory uveitic macular oedema. Its usefulness in controlling inflammation alone is questionable.