Peptidoglycan in Mycobacteria: chemistry, biology and intervention.

Peptidoglycan, a major glycoconjugate in the mycobacterial cell envelope provides strength to resist osmotic stress and plays a pivotal role in maintaining the cellular morphology. Several unique growth stage specific structural alterations occur in its constituent monosaccharides and peptides that allow Mycobacterium to survive nutrient starvation and environmental stress. Here, we discuss the enzymes involved in its intricate biosynthesis that are novel targets for therapeutic intervention and provide an opportunity for potential antibiotic adjuvants. We also revisit the enzymatic steps which are critical for maintaining the equilibrium between peptidoglycan synthesis and hydrolysis during cellular growth and division specifically focused on the importance of cell wall remodelling during "exit from dormancy" in Mycobacterium, a phenomenon with tremendous physiological and therapeutic importance for intervention in mycobacterial infections.

Biocatalytic synthesis of flavor ester "pentyl valerate" using Candida rugosa lipase immobilized in microemulsion based organogels: effect of parameters and reusability.

Pentyl valerate was synthesized biocatalytically using Candida rugosa lipase (CRL) immobilized in microemulsion based organogels (MBGs). The optimum conditions were found to be pH 7.0, temperature of 37 °C, ratio of concentration of water to surfactant (Wo) of 60, and the surfactant sodium bis-2-(ethylhexyl)sulfosuccinate (AOT) for MBG preparation. Although kinetic studies revealed that the enzyme in free form had high affinity towards substrates (K(m) = 23.2 mM for pentanol and 76.92 mM for valeric acid) whereas, after immobilization, the K(m) values increased considerably (74.07 mM for pentanol and 83.3 mM for valeric acid) resulting in a slower reaction rate, the maximum conversion was much higher in case of immobilized enzyme (~99%) as compared to free enzyme (~19%). Simultaneous effects of important parameters were studied using response surface methodology (RSM) conjugated with Box-Behnken design (BBD) with five variables (process parameters), namely, enzyme concentration, initial water content (Wo), solvent used for MBG preparation, substrate ratio and time, and response as the final product formation, that is, pentyl valerate (%). The MBGs were reused for 10 consecutive cycles for ester synthesis. Efficacy of AOT/isooctane as dehydrating agent for extracting excess water from MBGs was found to exert a positive effect on the esterification reaction.

Enhanced conjugation of Candida rugosa lipase onto multiwalled carbon nanotubes using reverse micelles as attachment medium and application in nonaqueous biocatalysis.

Three liquid phases (viz. aqueous, nonaqueous, and reverse micelles) were scrutinized as medium for attachment of the enzyme Candida rugosa lipase (CRL) onto multiwalled carbon nanotubes (CNTs). The nanotubes were functionalized to attain carboxyl and amino groups on their surfaces before enzyme conjugation. Transmission electron microscopy and Fourier transformation infrared spectroscopic studies were used for characterization of the nanotubes during the course of functionalization. High enzyme loadings associated with the functionalized CNTs were observed when reverse micelles were used as the attachment medium. In addition, high activity in terms of ester synthesis in organic solvents was also observed while using those preparations. The nanobioconjugates prepared using reverse micelles were found to be highly sturdy and exhibited appreciable operational stability of around 95 ± 3% at 20th cycle (in case of carboxylated nanotubes) and 90 ± 5% at 10th cycle (in case of aminated nanotubes) for esterification. This shows the potential application of reverse micelles as the attachment medium for surface active enzymes such as CRL onto CNTs.

Robust nanobioconjugates of Candida antarctica lipase B--multiwalled carbon nanotubes: characterization and application for multiple usages in non-aqueous biocatalysis.

Present work elucidates two approaches for covalent attachment of the enzyme Candida antarctica lipase B (CALB) on multiwalled carbon nanotubes (MWCNTs). One method of enzyme immobilization involved carbodiimide chemistry while in the other approach, the cross linker (3-Aminopropyl) triethoxysilane (APTES) followed by succinic acid anhydride (SAA) were employed prior to carbodiimide activation. Modified MWCNTs were characterized by transmission electron microscopy (TEM), Fourier transformation infrared spectroscopic (FTIR), Raman spectroscopy and thermal gravitometric analysis (TGA). The lipase-MWCNTs conjugates were applied for synthesis of the flavor ester 'pentyl valerate' in cyclohexane and effects of solvent, temperature and agitation on ester synthesis were studied. Upon subject to reusability studies for 50 cycles, the bionanoconjugates were found to be highly sturdy and exhibited ≈ 79% activity (immobilization using carbodiimide) whereas the nanoconjugate prepared using APTES and SAA retained only up to ≈ 30% activity.

An alkaline lipase from organic solvent tolerant Acinetobacter sp. EH28: Application for ethyl caprylate synthesis.

A mesophilic bacterium producing a thermostable alkaline lipase was isolated from oil rich soil sample and identified as Acinetobacter sp. EH28. The lipase was partially purified by ammonium sulphate precipitation followed by hydrophobic interaction chromatography with 24.2-fold purification and 57.1U/ml specific activity. The partially purified enzyme exhibited maximum activity at pH 10.0 and at 50 degrees C and was highly stable at 50 degrees C retaining 100% of its activity up to 90min. It was highly stable and retained more than 80% of its initial activity upon exposure to various organic solvents. The EH28 lipase was used for synthesis of the flavor ester ethyl caprylate in organic solvents, thus providing a concept of application of Acinetobacter sp. lipase in non-aqueous catalysis. Reaction parameters best suited for this esterification reaction were 40 degrees C reaction temperature, 1.3:1 ratio of caprylic acid to ethanol and cyclohexane as the medium.

A thermostable alkaline lipase from a local isolate Bacillus subtilis EH 37: characterization, partial purification, and application in organic synthesis.

A mesophilic bacterial culture producing a novel thermostable alkaline lipase was isolated from oil rich soil sample and identified as Bacillus subtilis EH 37. The lipase was partially purified by ammonium sulfate precipitation and hydrophobic interaction chromatography with 17.8-fold purification and 41.9 U/ml specific activity. The partially purified enzyme exhibited maximum activity at pH 8.0 and at 60 degrees C. It retained 100% of activity at 50 degrees C and 60 degrees C for 60 min. The presence of Ca2+, Mg2+, and Zn2+ exhibited stimulatory effect on lipase activity, whereas Fe3+ and Co2+ reduced its activity. The enzyme retained more than 80% of its initial activity upon exposure to organic solvents, exhibited 107% and 115% activity in the presence of 15% isopropyl alcohol and 30% n-hexane, respectively. The EH 37 lipase also proved to be an efficient catalyst in synthesis of ethyl caprylate in organic solvent, thus providing a concept of application of B. subtilis lipase in non-aqueous catalysis.