Factors influencing activity of enzymes and their kinetics

The conventional chemically based method of dehairing and fiber-opening discharges an enormous amount of pollutants in the processing of skins. Hence, bioprocessing of skin through a two-step process, dehairing using protease and fiber opening using α-amylase, has been developed. However, because this process involves two steps, we characterized commercial protease and α-amylase for their optimum activity and determine the influence of one enzyme on the activity of the other, in order to develop an integrated enzymatic dehairing and fiber-opening process. The influence of various factors, substrate concentration, time, pH, and temperature, on the activity of both protease and α-amylase was determined. Furthermore, the activity of protease on mixing with α-amylase and vice versa was investigated. It was found that there was no significant change in the activity of one enzyme in the presence of the other. Lineweaver-Burk plots showed K _m and V _max values of 31.6 mg/mL and 0.0106 mg/(mL@min) for protease and 8.79 mg/mL and 0.0912 mg/(mL@min) for α-amylase. This study provides substantial evidence for integrating the enzyme-based dehairing and fiber-opening processes using both the selected protease and α-amylase in one step.     

A multi-dimensional high performance liquid chromatographic method for fingerprinting polycyclic aromatic hydrocarbons and their alkyl-homologs in the heavy gas oil fraction of Alaskan North Slope crude

We report an offline multi-dimensional high performance liquid chromatography (HPLC) technique for the group separation and analysis of PAHs in a heavy gas oil fraction (boiling range 287-481 degrees C). Waxes present in the heavy gas oil fraction were precipitated using cold acetone at -20 degrees C. Recovery studies showed that the extract contained 93% (+/-1%; n=3) of the PAHs that were originally present while the wax residue contained only 6% (+/-0.5%; n=3). PAHs present in the extract were fractionated, based on number of rings, into five fractions using a semi-preparative silica column (normal-phase HPLC). These fractions were analyzed using reverse-phase HPLC (RP-HPLC) coupled to a diode array detector (DAD). The method separated alkyl and un-substituted PAHs on two reverse-phase columns in series using an acetonitrile/water mobile phase. UV spectra of the chromatographic peaks were used to differentiate among PAH groups. Further characterization of PAHs within a given group to determine the substituent alkyl carbon number used retention time matching with a suite of alkyl-PAH standards. Naphthalene, dibenzothiophene, phenanthrene and fluorene and their C1-C4 alkyl isomers were quantified. The concentrations of these compounds obtained using the current method were compared with that of a GC-MS analysis obtained from an independent oil chemistry laboratory.     

A high performance liquid chromatography-electrochemical array method for the measurement of oxidative/nitrative changes in human urine

Oxidative stress has been implicated in various pathologies as well as in environmental pollutant-induced negative health outcomes. In this work we have developed an analytical method to measure oxidative stress markers namely m-, o-tyrosine, 3-chlorotyrosine, 3-nitrotyrosine, and the DNA damage marker 8-hydroxy deoxyguanosine in human urine. The method involves the base hydrolysis of the urine sample followed by solid phase extraction of target analytes using a reverse phase polymeric sorbent column prior to the HPLC-EC analysis. The recovery studies indicated that the overall method recovery for all analytes were >60%. The limit of quantification of all target analytes was <10 nM with a linear range from 2 nM to 150 μM. The applicability of this method is demonstrated by analyzing the above markers in healthy human urine (n = 10) samples.     

Synthesis,spectroscopic investigations,antioxidants and DNA binding studies of a charge transfer of benzimidazole with 4-methylbenzenesulfonic acid

A new charge transfer (CT) complex benzimidazolium-4-methylbenzenesulfonate formed between benzimidazole and 4-methylbenzenesulfonic acid has been studied spectrophotometrically in methanol at room temperature. The binding of the novel CT complex with calf thymus DNA has been investigated by adsorption spectrum, the establish the ability of the CT complex for its interaction with the chosen DNA. The elemental stiochiometry and the X-ray crystallographic structure of the complex were studied, showing the significance of sulfonate anion in holding stability. Powder XRD confirms the crystalline nature of the compound. NMR signals explicitly confirmed the molecular structure crystal. Antioxidant activity of the CT complex was found to be potent. TG-DTA studies also proves the stability of the synthesized complex.     

Crystal Structure and Third Order Nonlinear Optical Studies on 2-Phenylbenzimidazolium-<Emphasis Type="Italic">p</Emphasis>-Toulenesulphonate

A new third order nonlinear optical single crystal of 2-phenylbenzimidazolium-p-toulenesulphonate is grown by the solvent evaporation technique using methanol as a solvent. The single crystal X-ray diffraction analysis reveals that crystals belong to the triclinic system. A nuclear magnetic resonance and Fourier transform infrared spectroscopic study confirms the formation and vibrational analysis for the compound. UV-visible absorption studies are also carried out for the crystal. Nonlinear refractive index, absorption coefficient, and third order nonlinear optical susceptibility of the crystals are evaluated by Z-Scan studies.