Comparative biophysical characterization: A screening tool for acetylcholinesterase inhibitors.

Among neurodegenerative diseases, Alzheimer's disease (AD) is one of the most grievous disease. The oldest cholinergic hypothesis is used to elevate the level of cognitive impairment and acetylcholinesterase (AChE) comprises the major targeted enzyme in AD. Thus, acetylcholinesterase inhibitors (AChEI) constitutes the essential remedy for the treatment of AD. The study aims to evaluate the interactions between natural molecules and AChE by Surface Plasmon Resonance (SPR). The molecules like alkaloids, polyphenols and substrates of AChE have been considered for the study with a major emphasis on affinity and kinetics. To better understand the activity of small molecules, the investigation is supported by both experimental and theoretical approach such as fluorescence, Circular Dichroism (CD) and molecular docking studies. Amongst the screened ones tannic acid showed promising results compared with others. The methodology followed here have highlighted many molecules with a higher affinity towards AChE and these findings may take lead molecules generated in preclinical studies to treat neurodegenerative diseases. Additionally, we suggest a unique signature for the heterogeneous analyte model using competitive experiments for analyzing simultanous interactions of both the analytes.

Statistical optimization of process parameters for inulinase production from Tithonia weed by Arthrobacter mysorens strain no.1.

Tithonia rotundifolia is an easily available and abundant inulin rich weed reported to be competitive and allelopathic. This weed inulin is hydrolyzed by inulinase into fructose. Response surface methodology was employed to optimize culture conditions for the inulinase production from Arthrobacter mysorens strain no.1 isolated from rhizospheric area of Tithonia weed. Initially, Plackett- Burman design was used for screening 11 nutritional parameters for inulinase production including inulin containing weeds as cost effective substrate. The experiment shows that amongst the 11 parameters studied, K2HPO4, Inulin, Agave sisalana extract and Tithonia rotundifolia were the most significant variables for inulinase production. Quantitative effects of these 4 factors were further investigated using Box Behnken design. The medium having 0.27% K2HPO4, 2.54% Inulin, 6.57% Agave sisalana extract and 7.27% Tithonia rotundifolia extract were found to be optimum for maximum inulinase production. The optimization strategies used showed 2.12 fold increase in inulinase yield (1669.45 EU/ml) compared to non-optimized medium (787 EU/ml). Fructose produced by the action of inulinase was further confirmed by spectrophotometer, osazone, HPTLC and FTIR methods. Thus Tithonia rotundifolia can be used as an eco-friendly, economically feasible and promising alternative substrate for commercial inulinase production yielding fructose from Arthrobacter mysorens strain no.1.

Interaction of small molecules with fungal laccase: A Surface Plasmon Resonance based study.

Laccases have a great potential for use in industrial and biotechnological applications. It has affinity towards phenolics and finds major applications in the field of bioremediation. Here, Surface Plasmon Resonance (SPR) as a biosensor with immobilized laccase on chip surface has been studied. Laccase was immobilized by thiol coupling method and compounds containing increasing number of hydroxyl groups were analyzed for their binding affinity at various concentrations in millimolar range. The small molecules like phloroglucinol (1.532×10(-8) M), crocin (3.204×10(-3) M), ascorbic acid (8.331×10(-8) M), kojic acid (6.411×10(-7) M) and saffron (3.466×10(-7) M) were studied and respective KD values are obtained. The results were also confirmed by inhibition assay and IC50 values were calculated. All these molecules showed different affinity towards laccase in terms of KD values. This method may be useful for preliminary screening and characterization of small molecules as laccase substrates, inhibitors or modulators of activity. This method will be useful for rapid screening of phenolics in waste water because of high sensitivity.

Evaluation of Various Factors Affecting Bioconversion of l-Tyrosine to l-DOPA by Yeast Yarrowia lipolytica-NCIM 3450 Using Response Surface Methodology.

ABSTRACT: 3,4-Dihydroxy l-phenylalanine (l-DOPA) is considered a potent drug for the treatment of Parkinson disease. Physical and nutritional parameters where optimized by using Yarrowia lipolytica-NCIM 3450 to accomplished the highest production of l-DOPA. Screenings of critical components were completed by using a Plackett-Burman design, while further optimization was carried out using the Box-Behnken design. The optimized factor levels predicted by the model were pH 6.1, 1.659 g L(-1) yeast extract, 1.491 g L(-1)l-tyrosine and 0.0290 g L(-1) CuSO4. The predicted yield of l-DOPA with these levels was 1.319 g L(-1), while actual yield obtained was 1.273 g L(-1). The statistical analysis revealed that model is significant with F value 19.55 and R(2) value 0.9514. This process resulted in a 3.594-fold increase in the yield of l-DOPA. l-DOPA was confirmed by HPTLC and HPLC analysis. Thus, Yarrowia lipolytica-NCIM 3450 has potential to be a new source for the production of l-DOPA.

Optimization of Biotransformation of l-Tyrosine to l-DOPA by Yarrowia lipolytica-NCIM 3472 Using Response Surface Methodology.

l-DOPA (3,4-dihydroxyphenyl-l-alanine) is the most widely used drug for treatment of Parkinson's disease. In this study Yarrowia lipolytica-NCIM 3472 biomass was used for transformation of l-tyrosine to l-DOPA. The process parameters were optimized using response surface methodology (RSM). The optimum values of the tested variables for the production of l-DOPA were: pH 7.31, temperature 42.9 °C, 2.31 g l(-1) cell mass and 1.488 g l(-1)l-tyrosine. The highest yield obtained with these optimum parameters along with recycling of the cells was 4.091 g l(-1). This optimization of process parameters using RSM resulted in 4.609-fold increase in the l-DOPA production. The statistical analysis showed that the model was significant. Also coefficient of determination (R(2)) was 0.9758, indicating a good agreement between the experimental and predicted values of l-DOPA production. The highest tyrosinase activity observed was 7,028 U mg(-1) tyrosine. l-DOPA production was confirmed by HPTLC and HPLC analysis. Thus, RSM approach effectively enhanced the potential of Y. lipolytica-NCIM 3472 as an alternative source to produce l-DOPA.

Optimization of L-DOPA production by Brevundimonas sp. SGJ using response surface methodology.

L-DOPA (3,4-dihydroxyphenyl-L-alanine) is an extensively used drug for the treatment of Parkinson's disease. In the present study, optimization of nutritional parameters influencing L-DOPA production was attempted using the response surface methodology (RSM) from Brevundimonas sp. SGJ. A Plackett-Burman design was used for screening of critical components, while further optimization was carried out using the Box-Behnken design. The optimized levels of factors predicted by the model were pH 5.02, 1.549 g l(-1) tryptone, 4.207 g l(-1) L-tyrosine and 0.0369 g l(-1) CuSO(4) , which resulted in highest L-DOPA yield of 3.359 g l(-1). The optimization of medium using RSM resulted in a 8.355-fold increase in the yield of L-DOPA. The anova showed a significant R(2) value (0.9667), model F-value (29.068) and probability (0.001), with insignificant lack of fit. The highest tyrosinase activity observed was 2471 U mg(-1) at the 18th hour of the incubation period with dry cell weight of 0.711 g l(-1). L-DOPA production was confirmed by HPTLC, HPLC and GC-MS analysis. Thus, Brevundimonas sp. SGJ has the potential to be a new source for the production of L-DOPA.

Efficient microbial conversion of L-tyrosine to L-DOPA by Brevundimonas sp. SGJ.

L-DOPA (3,4-dihydroxyphenyl-L-alanine), the most widely used drug for the treatment of Parkinson's disease, was produced in buffer using biomass of Brevundimonas sp. SGJ. The effects of enhancers, such as carrageenan, diatomaceous earth, and activated charcoal, on the L-DOPA production were evaluated to obtain the maximum yield. The optimal process conditions found were pH 8, 2 g l⁻¹ cell mass, 2 g l⁻¹ L-tyrosine, 0.04 g l⁻¹ CuSO4, 0.02 g l⁻¹ L-ascorbic acid, 0.5 g l⁻¹ carrageenan, and 40 °C temperature. In addition, repeated use of cells resulted in the highest yield of 3.81 g l⁻¹ (95.2%) of L-DOPA with utilization of 4 g l⁻¹ L-tyrosine, and the highest tyrosinase activity (9,201 U mg⁻¹) was observed at 18 h of incubation. Furthermore, the produced L-DOPA was confirmed by high-performance thin-layer chromatography, high-performance liquid chromatography, and gas chromatography-mass spectroscopy. Kinetic studies showed significant values of Y (p/s), Q (s), and q (s) after optimization of the process. Thus, Brevundimonas sp. SGJ could be an eventual new source for large-scale production of L-DOPA.