Uma Ghosh

ISOLATION AND CHARACTERIZATION OF CYANOBACTERIA POSSESSING ANTIMICROBIAL ACTIVITY FROM THE SUNDARBANS, THE WORLD’S LARGEST TIDAL MANGROVE FOREST1

Eight obligately halophilic, euryhaline cyanobacteria from intertidal soil were isolated in artificial seawater nutrients III (ASN‐III) medium. Antimicrobial activity, 16S rRNA gene sequences, phenotypic characters as well as growth and antibiosis in response to variable salinity, temperature, phosphate concentration, and pH were studied. Minimum inhibitory concentrations (MIC) of the extracts against Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, and multiple drug‐resistant clinical isolates ranged between 0.25 and 0.5 mg · mL−1. Cytotoxicity tests showed 73%–84% human colon adenocarcinoma (HT‐29/C1) cell survival at MIC values, indicating that the extracts were nontoxic. Morphologically, six cyanobacteria were assigned to the Lyngbya‐Phormidium‐Plectonema (LPP) group B, and one each was assigned to Oscillatoria and Synechocystis genera. Glycerol, mannitol, and starch supported better photoheterotrophic growth than simpler mono‐ and disaccharides. No heterocyst formation was observed when grown under nitrogen‐starved conditions. All isolates survived 7‰ salinity, grew at minimum 32‰ salinity, and showed sustained growth throughout 32‰–82‰ salinity but matured poorly in freshwater medium supplemented with 30.0 g · L−1 NaCl. Antimicrobial production occurred only at 32‰ salinity. While four of the eight isolates demonstrated sustained growth at 37°C, maximum antimicrobial activity was obtained at 25°C. All strains showed maximum growth and antimicrobial elaboration at 0.04 g · L−1 phosphate. All isolates thrived at pH 9.5; six grew at pH 4.5, though antimicrobial production occurred only at pH 7.5. Molecular phylogenetic analysis based on 16S rRNA gene sequences of the filamentous isolates validated the previous taxonomic affiliations established on morphological characteristics. This is the first study of antimicrobial‐producing halophilic cyanobacteria from the mangroves.

Study on Thermodynamics and Adsorption kinetics of Purified endoglucanase (CMCase) from Penicillium notatum NCIM NO-923 produced under mixed solid-state fermentation of waste cabbage and Bagasse

In the current study, one thermostable endoglucanase was purified from Penicillium notatum NCIM NO-923 through mixed solid state fermentation of waste cabbage and bagasse. The molecular weight of the purified enzyme was 55kDa as determined by SDS polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme had low activation energy (Ea) of 36.39KJ mol(-1) for carboxymethyl cellulose hydrolysis and the enthalpy and entropy for irreversible inactivation was 87 kJ mol (-1) and 59.3 J mol (-1) K(-1) respectively. The enzyme was quite thermostable with a Tm value of 62.2°C. The pKa1 and pKa2 of ionizable groups of the active sites were 2.5 and 5.3 respectively. Apparent Km, Vmax and Kcat of the enzyme were found to be 5.2 mg mL(-1), 80 U/gds and 322.4 sec(-1) respectively. The enzyme showed about 1.4 fold increased activity in presence of 10mM MgSO4. Adsorption of endoglucanase on Avicel at wide pH range was studied at different temperatures. Langmuir type adsorption isotherm at 10°C showed maximum adsorption strength of enzyme at pH 3.0, which was in a range of optimum pH of the enzyme.

Pectinmethylesterase Production from mixed agro- wastes by Penicillium notatum NCIM. 923 in Solid-State fermentation

The enzyme pectinmethylesterase (PME; EC 3.1.1.11) catalyzes the hydrolysis of the methyl ester groups from pectin and has been found in plants as well as in pathogenic fungi and bacteria. PME is of significance to the citrus industry because it has been established as the causative agent for juice clarification and gelation of frozen concentrates. The fruit processing industries produce a large amount of waste material, which poses considerable disposal problems and ultimately leads to pollution. Dried citrus peel is rich in carbohydrates, proteins and pectin; pectin acts as the inducer for production of pectinolytic enzymes by microbial systems. Thus, in the present study, dried citrus peel and wheat bran was used as substrate for the production of pectin methyl esterase (PME; EC 3.1.1.11) by fungus Penicillium notatum . Maximum enzyme activity was obtained with 1:1(w/w) substrate ratio, which gives a solid mass of initial pH 5.5, when incubated at 30°C for 120 h at 1:1 (w/v) initial moisture content ratio under static condition.

Purification and Characterization of Tannin Acyl Hydrolase Produced by Mixed Solid State Fermentation of Wheat Bran and Marigold Flower by Penicillium notatum NCIM 923

Tannin acyl hydrolase produced extracellularly by the fungal strain Penicillium notatum NCIM 923 in mixed solid state fermentation of wheat bran and marigold flower in the ratio 4 : 1 was purified from the cell-free extract broth by ammonium sulphate fractionation followed by diethylaminoethyl-cellulose column chromatography. Tannase was purified by 19.89-fold with yield of 11.77%. The specific activity of crude tannase was found to be 1.31 U/mg protein while that of purified tannase was 22.48 U/mg protein. SDS-PAGE analysis indicated that the enzyme is dimeric with one major band of molecular mass 97 kDa and a very light band of molecular mass 43 kDa. Temperature of 35 to 40°C and pH 5 were optimum for tannase activity. The enzyme retained more than 60% of its stability at 60°C and 40% stability at pH 3 and 8, respectively. K m was found to be 0.33 × 10−2 M and V max = 40 U/mg. Since the enzyme is active over a wide range of pH and temperature, it could find potential use in the food processing industry.

Value Addition to Horticultural Solid Waste by Applying It in Biosynthesis of Industrially Important Enzyme: Cellulase

Banana (Musa sp.) which is a popular fruit consumed worldwide has a yearly production of over 145 million tons approximately. The peel is generally discarded after consumption of the fruit, which eventually leads to the generation of a significant amount of organic waste causing environmental pollution. Meaningful utilization of this nutrient-rich waste has not drawn much attention. The peel can be biotechnologically converted to value-added products which can reduce environmental pollution to a great extent. Cellulases represent a major group of the industrially important enzyme. Its production has been studied widely by the technique of solid-state fermentation (SSF). The present study deals with the cost-effective biosynthesis of endoglucanase (CMCase) utilizing solid waste, banana peel. Optimization of nutritional parameters was done by the conventional one variable at a time (OVAT) method followed by chemical characterization of banana peel by energy-dispersive X-ray spectroscopy (EDX). By applying OVAT methodology for optimization of nutritional parameters for cellulase production, the specific activity of endoglucanase (CMCase) was found to be 54 U/mg. EDX analysis showed that, as the solid waste was found to be rich in carbon source, no additional carbon supplement was essential for the growth of cellulase-producing microorganism used for the production of secondary metabolite by SSF.

Isolation and Identification of Potent Alkaline Protease Producing Microorganism and Optimization of Biosynthesis of the Enzyme Using RSM

Abstract In this study, 6 isolates out of 30, forming comparatively zone as a result of casein and skim milk hydrolysis were selected and further studied for selection of the isolate producing maximum alkaline protease activity. Isolate No. F19 was the most potential alkaline protease producing fungal strain. Upon 18S rRNA analysis, it displayed maximum similarity with Alternaria alternata. Agricultural residue, cauliflower leaves were used as main substrate for production of alkaline protease under solid state fermentation. Supplementation of different nutritional components parameters such as carbon, nitrogen and metals were statistically optimized for maximum enzyme production. The enzyme secretion improved 1.55 fold, when the variables were optimized by Response surface methodology. Under the optimized conditions, the protease experimental yield (930 U/gds) closely matched the yield predicted by the statistical model (931 U/gds) with R2 value of 0.9965.

Statistical Optimization of Fermentation Parameters for Cellulase Production Utilizing Banana Peel

Aim: To optimize the physical parameters of solid state fermentation (SSF) by implementing statistical tool for production of cellulase, one of the potential biocatalyst in industries. Study Design: Fermentation was carried out by the employment of response surface methodology (RSM) based on the Box-Behnken design (BBD) available in software Design-Expert (Version 7) Stat-Ease, Inc. Place and Duration of Study: Department of Food Technology and Biochemical Engineering, Jadavpur University, Kolkata, India, between June 2014 and December 2014. Methodology: The optimization of environmental factors for production of cellulase (FPase) was carried out by response surface methodology (RSM) based on the Box-Behnken design (BBD). The design included a total of 29 experimental trials that comprised time and temperature of fermentation, amount of substrate and hydration ratio as model factors for three levels. Results: The mutual interaction between the independent variables under optimized conditions yielded FPase at the level of 8.05 U/ gds and total dissolved protein at the level of 1.4 mg/ml which Original Research Article Mandal and Ghosh; JABB, 7(3): 1-9, 2016; Article no.JABB.26982 2 were close to the predicted values from the model (7.95 U/gds and 1.39 mg/ml respectively for FPase and protein). The ideal parameters for cellulase production by Aspergillus sp. were 25% of hydration, 33oC, 3.1 g of substrate and 7 days of fermentation. Conclusion: A good correspondence between the predicted values from the model and the experimental values were seen. Consequently, we can use this methodology to adequately describe the interaction effect between the important independent variables influencing the fermentation process and the responses.