The soluble, catalytically self-sufficient cytochrome P450 BM3 from Bacillus megaterium is a good candidate as biocatalyst for the synthesis of drug metabolites. To this end, error-prone polymerase chain reaction (PCR) was used to generate a library of P450 BM3 mutants with novel activities toward drugs. The double mutant Asp251Gly/Gln307His (A2) with activities towards diclofenac, ibuprofen and tolbutamide was identified by screening with the alkali method. This is based on the detection of NADPH oxidation during enzymatic turnover on whole Escherichia coli cells heterologously expressing the P450 BM3 mutants in the presence of the target substrates. The three drugs screened are marker substrates of human liver cytochromes P450 belonging to the 2C subfamily. Interestingly the mutations Asp251Gly/Gln307His are located on the protein surface and they are not directly involved in substrate binding and turnover. Dissociation constants and K(M) values of mutant A2 for diclofenac, ibuprofen and tolbutamide are in the micromolar range. Catalysis leads to hydroxylations in specific positions, producing 4'-hydroxydiclofenac, 2-hydroxyibuprofen and 4-hydroxytolbutamide, respectively.

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Bioethanol is recognized as a valuable substitute for renewable energy sources to meet the fuel and energy demand of the nation, considered an environmentally friendly resource obtained from agricultural residues such as sugarcane bagasse, rice straw, husk, wheat straw and corn stover. The energy demand is sustained using lignocellulosic biomass to produce bioethanol. Lignocellulosic biomass (LCBs) is the point of attention in replacing the dependence on fossil fuels. The recalcitrant structure of the lignocellulosic biomass is disrupted using effective pretreatment techniques that separate complex interlinked structures among cellulose, hemicellulose, and lignin. Pretreatment of biomass involves various physical, chemical, biological, and physiochemical protocols which are of importance, dependent upon their individual or combined dissolution effect. Physical pretreatment involves a reduction in the size of the biomass using mechanical, extrusion, irradiation, and sonification methods while chemical pretreatment involves the breaking of various bonds present in the LCB structure. This can be obtained by using an acidic, alkaline, ionic liquid, and organosolvent methods. Biological pretreatment is considered an environment-friendly and safe process involving various bacterial and fungal microorganisms. Distinct pretreatment methods, when combined and utilized in synchronization lead to more effective disruption of LCB, making biomass more accessible for further processing. These could be utilized in terms of their effectiveness for a particular type of cellulosic fiber and are namely steam explosion, liquid hot water, ammonia fibre explosion, CO2 explosion, and wet air oxidation methods. The present review encircles various distinct and integrated pretreatment processes developed till now and their advancement according to the current trend and future aspects to make lignocellulosic biomass available for further hydrolysis and fermentation.

A Gram-negative bacterial strain designated LS2 isolated from Lahaul-Spiti valley of North India was shown to produce pink pigment while utilizing methanol as sole source of carbon and energy. Interestingly, pigment production was inducible in nature since the organism did not produce any pigment when grown on other carbon sources. Based on phenotypic and phylogenetic characterization the non-pigmented methylotroph was identified as a novel strain of Acinetobacter lwoffii MTCC 8288 (DQ144736). By means of spectral and mass analyses the pigment was characterized as bacterioruberin-like carotenoid molecule. Here, the carotenoid pigment may form an important part of the antioxidant defense mechanism against oxidative stress imparted by methanol. The methanol utilization pathway in strain LS2 was deciphered by showing the presence of functional methanol dehydrogenase and formaldehyde dehydrogenase genes. In addition, to investigate methanol induced physiological changes, comparative fatty acid profile was analysed and distinctive qualitative as well as quantitative differences in fatty acid content were observed. Therefore, we suggest that strain LS2 exhibiting such unique phenotypic property should be assigned a taxonomic position other than the pigmented and non-pigmented methylotrophs.