Debarati Paul

Purification, biochemical characterization and self-assembled structure of a fengycin-like antifungal peptide from Bacillus thuringiensis strain SM1

An antifungal lipopeptide fengycin, producing strain SM1 was isolated from farm land soil sample and identified as Bacillus thuringiensis strain SM1 by using 16S rDNA analysis. Fengycin detected in the culture extract was further purified using HPLC and showed a molecular mass of 1492.8 Da by MALDI-TOF-MS analysis. Purified fengycin was allowed to construct their self-assembled structure onto a hydrophobic surface showing a clear improvement of antibacterial activity. In self-assembly, fengycin adapts a spherical micelle core shell like structure. Self-assembled fengycin may be a successful antimicrobial compound modifying its action from confined antifungal function. Besides it can open up a new area of research in supramolecular lipopeptide based compound making. This can revealed the mode of action of this unique self-assembled structure to fully evaluate its potential for use as an antimicrobial drug to control the emergence of bacterial infection.

Genome Sequence of the Oleaginous Yeast Rhodotorula glutinis ATCC 204091.

ABSTRACT Rhodotorula glutinis ATCC 204091 is an oleaginous oxidative red yeast that can accumulate lipids to >50% of its biomass when grown with appropriate carbon and nitrogen ratios. It produces a red pigment consisting of useful antioxidants, such as carotenoids, torulene, and torularhodin, when cultivated under carbon-deficient conditions.

Concomitant Production of Lipids and Carotenoids in Rhodosporidium toruloides under Osmotic Stress Using Response Surface Methodology.

As a replacement to existing fossil fuels, biofuels, have proven their worth; however, their widespread use is limited due to inconsistent yields, higher costs and poor productivity. An oleaginous yeast, Rhodosporidium toruloides has been reported to accumulate substantial amounts of lipids (that can be converted to biofuels) and therefore, it was selected for study and optimization. Apart from lipids, R. toruloides is also reported to produce carotene that can be used as a therapeutic agent. In this study, the culture medium was statistically modeled and optimized for concomitant production of lipids and carotenoids and for improving and maximizing the productivity of lipids as well as carotenes. The two metabolites were expressed differentially in the growth cycle of the organism. Culture medium components were simultaneously varied at five different levels using statistical modeling employing response surface methodology (RSM). Osmotic stress was introduced in order to simulate saline conditions and optimize the carotenoid as well as lipid production process, to be used in conditions with high salt contents. We observed a 10% (w/v) increase in carotenoid production in initial experiments under osmotic stress due to high salt concentration, while the increase in lipid synthesis was not pronounced. In this study, we demonstrate 36.2% (w/v) lipid production and 27.2% (w/v) carotenoid production, under osmotic stress with high salt concentrations, for the first time.

Genome comparison of Listeria monocytogenes serotype 4a strain HCC23 with selected lineage I and lineage II L. monocytogenes strains and other Listeria strains.

More than 98% of reported human listeriosis cases are caused by specific serotypes within genetic lineages I and II. The genome sequence of Listeria monocytogenes lineage III strain HCC23 (serotype 4a) enables whole genomic comparisons across all three L. monocytogenes lineages. Protein cluster analysis indicated that strain HCC23 has the most unique protein pairs with nonpathogenic species Listeria innocua. Orthology analysis of the genome sequences of representative strains from the three L. monocytogenes genetic lineages and L. innocua (CLIP11262) identified 319 proteins unique to nonpathogenic strains HCC23 and CLIP11262 and 58 proteins unique to pathogenic strains F2365 and EGD-e. BLAST comparison of these proteins with all the sequenced L. monocytogenes and L. innocua revealed 126 proteins unique to serotype 4a and/or L. innocua; 14 proteins were only found in pathogenic serotypes. Some of the 58 proteins unique to pathogenic strains F2365 and EGD-e were previously published and are already known to contribute to listerial virulence.

Modeling and optimization of a continuous bead milling process for bacterial cell lysis using response surface methodology

Efficient cell lysis for intracellular protein recovery is a major bottleneck in the economics and commercial feasibility of any biotechnological process. Grinding of cells with abrasive beads, also known as bead milling remains a method of choice, as it can handle a large volume of cells. Bead mills when operated in a continuous mode substantiate to be economical, and more productive as compared to a batch mode process. In this study, the recovery of recombinant cholesterol oxidase (COD) was investigated and optimized using response surface methodology (RSM) based on Central Composite Design (CCD) in a continuous bead milling process. Process parameters, viz. slurry feed rate (A), bead loading (B), cell loading (C) and process time (D) were found to be significant during the continuous bead milling process. A polynomial model was developed to correlate the participating factors for efficient cell disruption. Optimized conditions yielded 3.20 g L−1 (∼90%) of COD with A = 300.6 mL h−1, B = 77.5% (v/v), C = 69.9 (OD600 nm) and D = 29.7 (min), when compared to existing batch mode operations (3.56 g L−1). This is the very first study that attempts to optimize a continuous bead milling process using RSM to maximize the intracellular protein (COD in this case) recovery with minimum inputs to make the process economical and scalable to industrial levels. The developed model in this study can be scaled-up to large-scale for efficient recovery of intracellular proteins in similar expression systems.

Induction of nodD Gene in a Betarhizobium Isolate, Cupriavidus sp. of Mimosa pudica, by Root Nodule Phenolic Acids.

Abstract A range of phenolic acids, viz., p-coumaric acid, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid, protocatechuic acid, caffeic acid, ferulic acid, and cinnamic acid have been isolated and identified by LC–MS analysis in the roots and root nodules of Mimosa pudica. The effects of identified phenolic acids on the regulation of nodulation (nod) genes have been evaluated in a betarhizobium isolate of M. pudica root nodule. Protocatechuic acid and p-hydroxybenzoic acid were most effective in inducing nod gene, whereas caffeic acid had no significant effect. Phenylalanine ammonia lyase, peroxidase, and polyphenol oxidase activities were estimated, indicating regulation and metabolism of phenolic acids in root nodules. These results showed that nodD gene expression of betarhizobium is regulated by simple phenolic acids such as protocatechuic acid and p-hydroxybenzoic acid present in host root nodule and sustains nodule organogenesis.

Augmenting pentose utilization and ethanol production of native Saccharomyces cerevisiae LN using medium engineering and response surface methodology

Economics of ethanol production from lignocellulosic biomass depends on complete utilization of constituent carbohydrates and efficient fermentation of mixed sugars present in biomass hydrolysates. Saccharomyces cerevisiae, the commercial strain for ethanol production uses only glucose while pentoses remain unused. Recombinant strains capable of utilizing pentoses have been engineered but with limited success. Recently, presence of endogenous pentose assimilation pathway in S. cerevisiae was reported. On the contrary, evolutionary engineering of native xylose assimilating strains is promising approach. In this study, a native strain S. cerevisiae LN, isolated from fruit juice, was found to be capable of xylose assimilation and mixed sugar fermentation. Upon supplementation with yeast extract and peptone, glucose (10%) fermentation efficiency was 78% with ~90% sugar consumption. Medium engineering augmented mixed sugars (5% glucose + 5% xylose) fermentation efficiency to ~50 and 1.6% ethanol yield was obtained with concomitant sugar consumption ~60%. Statistical optimization of input variables Glucose (5.36%), Xylose (3.30%), YE (0.36%), and peptone (0.25%) with Response surface methodology led to improved sugar consumption (74.33%) and 2.36% ethanol within 84 h. Specific activities of Xylose Reductase and Xylitol Dehydrogenase exhibited by S. cerevisiae LN were relatively low. Their ratio indicated metabolism diverted toward ethanol than xylitol and other byproducts. Strain was tolerant to concentrations of HMF, furfural and acetic acid commonly encountered in biomass hydrolysates. Thus, genetic setup for xylose assimilation in S. cerevisiae LN is not merely artifact of xylose metabolizing pathway and can be augmented by adaptive evolution. This strain showed potential for commercial exploitation.

Comparative analysis of biodiesel produced by acidic transesterification of lipid extracted from oleaginous yeast Rhodosporidium toruloides

This study investigated the potential of oleaginous yeast Rhodosporidium toruloides strain (ATCC20409) for the sustainable production of microbial lipids as biodiesel feedstock and other economically important fatty acids in comparison to algal or plant-based biodiesel. The strain exhibited high lipid content (76% of dry cell weight biomass) through consolidated bioprocessing which was transesterified to produce biodiesel. Physico-chemical properties of the biodiesel produced showed that they were in accordance with ASTM standards, although few parameters such as acid value, calorific value and free fatty acid value differed to some extent, as also reported in plant-based/microalgal biodiesel. Fatty acid methyl esters analysis of biodiesel showed 50.18% unsaturated fatty acid and 49.81% saturated fatty acid. Total content of (monounsaturated fatty acid) MUFA was higher than (polyunsaturated fatty acid) PUFA, being 44.36% and 2.69%, respectively. Considering the yield and cost, lipid extracted from R. toruloides may become a promising alternative feed in biodiesel production.

Fungi Fights Fungi: Tip-off in Antifungal Chemotherapy

Fungal infections have taken a new spectrum due to the increased incidence of multi-drug resistant fungal pathogens. Freedom of choice for drugs to treat fungal infections is also narrow because of lesser probability of discovering drugs that would bypass affecting human cells and target fungal cells producing fewer side effects in patients. An approach has gained prominence in research is to look for bioactive antifungal compounds from natural sources and discover new classes of antifungals to control the recent emergence of fungal infections. Most of antifungal drugs are originated from fungi. A conservative estimate of total number of fungal species on this planet would exceed 106 if taken into account the ones yet to be discovered from diverse habitats ranging from forest land to marine ecosystem. While attempting to summarize the status of reported fungi-derived antifungal compounds discovered since ancient times, the subset of such compounds were found to be anticancer too. Antifungal compounds with the promise of inducing challenge to rediscover the new effective molecules from drug prototype are also discussed.