Extracellular beta-Glucosidase production by marine Aspergillus sydowii BTMFS 55

The beta-glucosidase enzyme purified from the marine fungus, Aspergillus sydowii BTMFS 55 showed a good yield of enzyme production under solid state fermentation. The statistical optimization of the media components revealed that moisture content, concentration of peptone and inoculum are the major parameters which supported the maximal enzyme production. The purified enzyme showed low pH activity and stability, glucose tolerance and activation by ethanol. It could produce ethanol from wheat bran and rice straw by simultaneous saccharification and fermentation with yeast.The glucosidase purified from Aspergillus sydowii BTMFS 55 shows great potential for several biotechnological applications such as the production of bio-ethanol from agricultural biomass and improvement in the aromatic character of wines and fruit juices through the hydrolysis of flavour glucosidic precursors. There is immense scope for the application of this marine fungus in the biofuel production besides in other industries provided further studies are pursued in exploiting this enzyme and the organism particularly scale up studies with respect to application. There is also ample scope for cloning of the gene encoding beta-glucosidase in domesticated hosts such as Pichia pastoris or S. cerevisiae that can produce ethanol directly from cellulosic biomass.

L-Glutaminase production by Marine Bacteria

L-glutaminases (L—glutamine amidohydrolase EC.3.5.l.2) is proposed as a prospective candidate for enzyme therapy cnf cancer and also as zui important additive during enzymatic digestion of shoyu koji since it could enhance glutamate content of soysauce. Commercial production of glutaminase could make possible its wide application in these areas, which would demand availability of potential sources and suitable fermentation techniques. The ‘present investigation highlighted marine environment as a potential source of efficient glutaminase producing bacteria mainly species of pseudomonas, aeromonas ,vibrio,alcaligenes, acinetobacter bacillus and planococci.Among them pseudomonas fluorescens ACMR 267 and v.cholerae ACMR 347 were chosen as the ideal strains for glutaminase production.Extracellular glutaminase fraction from all strains were in higher titres than intracellular enzymes during growth in mineral media, nutrient broth and nutrient broth added with glutamine.Glutaminase from all strains were purified employing (NH4)2SO4 fractionation followed tnr dialysis and ion exchange chromatography. The purified glutaminase from all strains were observed to be active and stable over a wide range of gfii and temperature.Optimization studies cflf environmental variables that normally influence time yiehi of glutaminase indicated that the optimal requirements of these bacteria for maximal glutaminase production remained stable irrespective of the medium, they are provided with for enzyme production. However, solid state fermentation technique was observed to be the most suitable process for the production of Glutaminase.

“Studies on extracellular enzyme production during growth of Pleurotus sp. on lignocellulosic agriwaste and the utilization of spent mushroom substrate”

Commercially, Pleurotus spp. of mushroom are cultivated in bags. After mushroom cultivation, spent substrate remains as residual material. Proper recycling of spent substrate is beneficial for our economy. Spent substrate can be utilized for various other value added purposes through the proper knowledge of its components. Composition of various components depends on the activity of extracellular enzymes in the spent substrate. The present study was conducted to know the enzyme profile of some major extracellular enzymes - cellulase, hemicellulase (xylanase), pectinase and ligninase (lignin peroxidase and laccase) and to estimate cellulose, hemicellulose, pectin and lignin in the substrate. The use of spent substrate as a source of fibre and ethanol, and in the biodegradation of phenol by Pleurotus spp. was also investigated

Distribution, Extracellular Virulence Factors and Drug Resistance of Motile Aeromonads in Fresh Water Ornamental Fishes and Associated Carriage Water

During last decades there has been a continuous growth of aquaculture industries all over the world and taking into consideration the spurt in freshwater ornamental fish aquaculture and trade in Kerala, the present study was aimed to assess the prevalence of various motile Aeromonas spp. in fresh water ornamental fishes and associated carriage water. The extracellular virulence factors and the antibiogram of the isolates were also elucidated. Various species of motile aeromonads such as Aeromonas caviae, A. hydrophila, A. jandaei, A. schubertii, A. sobria, A. trota and A. veronii were detected. Aeromonas sobria predominated both fish and water samples. Extracellular enzymes and toxins produced by motile aeromonds are important elements of bacterial virulence. The production of extracellular virulence factors - proteases, lipase, DNase and haemolysin by the isolates were studied. All the isolates from both fish and water samples produced gelatinase and nuclease but the ability to produce lipase, caseinase and haemolysins was found to vary among isolates from different sources. Among the 15 antibiotics to which the isolates were tested, all the isolates were found to be sensitive to chloramphenicol, ciprofloxacin and gentamicin and resistant to amoxycillin. Local aquarists maintain the fish in crowded stressful conditions, which could trigger infections by the obligate/ opportunistic pathogenic members among motile aeromonads

Extracellular production of L-glutaminase by alkalophilic Beauveria bassiana BTMF S10 isolated from marine sediment

Beauveria sp. BTMF S10 isolated from marine sediment produced extracellular L-glutaminase. Maximal L- glutaminase yield (46.9 U/ml) was obtained in a medium supplemented with 1% (w/v) yeast extract and sorbitol, 9% (w/v) sodium chloride and 0.2% (w/v) methionine, initial pH 9.0 and at 27 °C after 108 h. This enzyme was inducible and growth-associated.

Extracellular L-Glutaminase Production By Marine Bacteria

Four species of bacteria which included Pseudomonas fluorescens, Vibrio cho!erae and Vibrio costicola were observed to produce glutaminase both as extracellular and intracellular fractions. Comparatively both the fractions were higher in mineral media supplemented with 1% glutamine than in nutrient broth added with or without glutamine. Extracellular glutaminase production was about 2.6-6.8 times greater than the intracellular production by all the tested strains

Extracellular β-glucosidase Production by a Marine Aspergillus sydowii BTMFS 55 under Solid State Fermentation Using Statistical Experimental Design

A potential fungal strain producing extracellular β-glucosidase enzyme was isolated from sea water and identified as ^ëéÉêJ Öáääìë=ëóÇçïáá BTMFS 55 by a molecular approach based on 28S rDNA sequence homology which showed 93% identity with already reported sequences of ^ëéÉêÖáääìë=ëóÇçïáá in the GenBank. A sequential optimization strategy was used to enhance the production of β-glucosidase under solid state fermentation (SSF) with wheat bran (WB) as the growth medium. The two-level Plackett-Burman (PB) design was implemented to screen medium components that influence β-glucosidase production and among the 11 variables, moisture content, inoculums, and peptone were identified as the most significant factors for β-glucosidase production. The enzyme was purified by (NH4)2SO4 precipitation followed by ion exchange chromatography on DEAE sepharose. The enzyme was a monomeric protein with a molecular weight of ~95 kDa as determined by SDS-PAGE. It was optimally active at pH 5.0 and 50°C. It showed high affinity towards éNPG and enzyme has a hã and sã~ñ of 0.67 mM and 83.3 U/mL, respectively. The enzyme was tolerant to glucose inhibition with a há of 17 mM. Low concentration of alcohols (10%), especially ethanol, could activate the enzyme. A considerable level of ethanol could produce from wheat bran and rice straw after 48 and 24 h, respectively, with the help of p~ÅÅÜ~êçãóÅÉë=ÅÉêÉîáëá~É in presence of cellulase and the purified β-glucosidase of ^ëéÉêÖáääìë=ëóÇçïáá BTMFS 55.

Continuous Production of Extracellular L-Glutaminase by Ca-Alginate-Immobilized Marine Beauveria bassiana BTMF S-10 in Packed-Bed Reactor

L-Glutamine amidohydrolase (L-glutaminase, EC 3.5.1.2) is a therapeutically and industrially important enzyme. Because it is a potent antileukemic agent and a flavor-enhancing agent used in the food industry, many researchers have focused their attention on L-glutaminase. In this article, we report the continuous production of extracellular L-glutaminase by the marine fungus Beauveria bassiana BTMF S-10 in a packed-bed reactor. Parameters influencing bead production and performance under batch mode were optimized in the order-support (Na-alginate) concentration, concentration of CaCl2 for bead preparation, curing time of beads, spore inoculum concentration, activation time, initial pH of enzyme production medium, temperature of incubation, and retention time. Parameters optimized under batch mode for L-glutaminase production were incorporated into the continuous production studies. Beads with 12 × 108 spores/g of beads were activated in a solution of 1% glutamine in seawater for 15 h, and the activated beads were packed into a packed-bed reactor. Enzyme production medium (pH 9.0) was pumped through the bed, and the effluent was collected from the top of the column. The effect of flow rate of the medium, substrate concentration, aeration, and bed height on continuous production of L-glutaminase was studied. Production was monitored for 5 h in each case, and the volumetric productivity was calculated. Under the optimized conditions for continuous production, the reactor gave a volumetric productivity of 4.048 U/(mL·h), which indicates that continuous production of the enzyme by Ca-alginate-immobilizedspores is well suited for B. bassiana and results in a higher yield of enzyme within a shorter time. The results indicate the scope of utilizing immobilized B. bassiana for continuous commercial production of L-glutaminase

Characterization of an extracellular alkaline serine protease from marine Engyodontium album BTMFS10

An alkaline protease from marine Engyodontium album was characterized for its physicochemical properties towards evaluation of its suitability for potential industrial applications. Molecular mass of the enzyme by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) analysis was calculated as 28.6 kDa. Isoelectric focusing yielded pI of 3–4. Enzyme inhibition by phenylmethylsulfonyl fluoride (PMSF) and aprotinin confirmed the serine protease nature of the enzyme.Km, Vmax, and Kcat of the enzyme were 4.727 9 10-2 mg/ml, 394.68 U, and 4.2175 9 10-2 s-1, respectively. Enzyme was noted to be active over a broad range of pH (6–12) and temperature (15–65 C), withmaximumactivity at pH 11 and 60 C. CaCl2 (1 mM), starch (1%), and sucrose (1%) imparted thermal stability at 65 C. Hg2?, Cu2?, Fe3?, Zn2?, Cd?, and Al3? inhibited enzyme activity, while 1 mMCo2? enhanced enzyme activity. Reducing agents enhanced enzyme activity at lower concentrations. The enzyme showed considerable storage stability, and retained its activity in the presence of hydrocarbons, natural oils, surfactants, and most of the organic solvents tested. Results indicate that the marine protease holds potential for use in the detergent industry and for varied applications.

Distribution, extracellular virulence factors and antibiogram of motile aeromonads in fresh water ornamental fishes and immune response of Cyprinus carpio against Aeromonas hydrophila infection

The thesis deals with the prevalence and distribution of motile aeromonads in selected ornamental fishes. The presence of motile aeromonads in ornamental fishes and associated carriage water is well documented. Though aeromonads are a part of autochthonous flora of natural waters, disease outbreak occurs as a result of environmental stress on the cultured species and virulence of the pathogens. While ornamental aquaculture in many parts of the world is highly organized and practiced scientifically, it is highly unorganized in India. The culture ponds/tanks are often maintained in very poor manner and the fishes are subjected to high degree of stress during transportation from the production facility to retail vendors. The situation is no better at retail outlets, where fishes are maintained in crowded condition without proper aeration or food. All these could result in high prevalence of diseases caused by motile aeromonads. No systematic study has been carried out to understand the prevalence of motile aeromonads in ornamental fishes and carriage water . It also gives an account of the production of extracellular virulence factors and the antibiogram of the different species of motile aeromonads isolated. The growth characteristics and virulence potential of a representative strain of Aeromonas hydrophila is also studied. The nucleotide sequencing of the strain was carried out and sequences deposited in Genbank. Survival and immune response of Cyprinus carpio under different stress conditions and on probiotic treatment with Bacillus NL110, when challenged with A. hydrophila is also dealt within this thesis.

A Study of Single Stage Semi‐Dry Anaerobic Digestion of Organic Fraction of Municipal Solid Waste

Solid waste generation is a natural consequence of human activity and is increasing along with population growth, urbanization and industrialization. Improper disposal of the huge amount of solid waste seriously affects the environment and contributes to climate change by the release of greenhouse gases. Practicing anaerobic digestion (AD) for the organic fraction of municipal solid waste (OFMSW) can reduce emissions to environment and thereby alleviate the environmental problems together with production of biogas, an energy source, and digestate, a soil amendment. The amenability of substrate for biogasification varies from substrate to substrate and different environmental and operating conditions such as pH, temperature, type and quality of substrate, mixing, retention time etc. Therefore, the purpose of this research work is to develop feasible semi-dry anaerobic digestion process for the treatment of OFMSW from Kerala, India for potential energy recovery and sustainable waste management. This study was carried out in three phases in order to reach the research purpose. In the first phase, batch study of anaerobic digestion of OFMSW was carried out for 100 days at 32°C (mesophilic digestion) for varying substrate concentrations. The aim of this study was to obtain the optimal conditions for biogas production using response surface methodology (RSM). The parameters studied were initial pH, substrate concentration and total organic carbon (TOC). The experimental results showed that the linear model terms of initial pH and substrate concentration and the quadratic model terms of the substrate concentration and TOC had significant individual effect (p < 0.05) on biogas yield. However, there was no interactive effect between these variables (p > 0.05). The optimum conditions for maximizing the biogas yield were a substrate concentration of 99 g/l, an initial pH of 6.5 and TOC of 20.32 g/l. AD of OFMSW with optimized substrate concentration of 99 g/l [Total Solid (TS)-10.5%] is a semi-dry digestion system .Under the optimized condition, the maximum biogas yield was 53.4 L/kg VS (volatile solid).. In the second phase, semi-dry anaerobic digestion of organic solid wastes was conducted for 45 days in a lab-scale batch experiment for substrate concentration of 100 g/l (TS-11.2%) for investigating the start-up performances under thermophilic condition (50°C). The performance of the reactor was evaluated by measuring the daily biogas production and calculating the degradation of total solids and the total volatile solids. The biogas yield at the end of the digestion was 52.9 L/kg VS for the substrate concentration of 100 g/l. About 66.7% of volatile solid degradation was obtained during the digestion. A first order model based on the availability of substrate as the limiting factor was used to perform the kinetic studies of batch anaerobic digestion system. The value of reaction rate constant, k, obtained was 0.0249 day-1. A laboratory bench scale reactor with a capacity of 36.8 litres was designed and fabricated to carry out the continuous anaerobic digestion of OFMSW in the third phase. The purpose of this study was to evaluate the performance of the digester at total solid concentration of 12% (semi-dry) under mesophlic condition (32°C). The digester was operated with different organic loading rates (OLRs) and constant retention time. The performance of the reactor was evaluated using parameters such as pH, volatile fatty acid (VFA), alkalinity, chemical oxygen demand (COD), TOC and ammonia-N as well as biogas yield. During the reactor’s start-up period, the process is stable and there is no inhibition occurred and the average biogas production was 14.7 L/day. The reactor was fed in continuous mode with different OLRs (3.1,4.2 and 5.65 kg VS/m3/d) at constant retention time of 30 days. The highest volatile solid degradation of 65.9%, with specific biogas production of 368 L/kg VS fed was achieved with OLR of 3.1 kg VS/m3/d. Modelling and simulation of anaerobic digestion of OFMSW in continuous operation is done using adapted Anaerobic Digestion Model No 1 (ADM1).The proposed model, which has 34 dynamic state variables, considers both biochemical and physicochemical processes and contains several inhibition factors including three gas components. The number of processes considered is 28. The model is implemented in Matlab® version 7.11.0.584(R2010b). The model based on adapted ADM1 was tested to simulate the behaviour of a bioreactor for the mesophilic anaerobic digestion of OFMSW at OLR of 3.1 kg VS/m3/d. ADM1 showed acceptable simulating results.