Effect of Dietary Proteins on Growth, Food Conversion and Digestive Enzyme Activity of Juvenile Macrobrachium Rosenbergii

School of Industrial Fisheries, Cochin University of Science and Technology

Glucoamylase immobilized on montmorillonite: influence of nature of binding on surface properties of clay-support and activity of enzyme

Glucoamylase was immobilized on acid activated montmorillonite clay via two different procedures namely adsorption and covalent binding. The immobilized enzymes were characterized by XRD, NMR and N2 adsorption measurements and the activity of immobilized glucoamylase for starch hydrolysis was determined in a batch reactor. XRD shows intercalation of enzyme into the clay matrix during both immobilization procedures. Intercalation occurs via the side chains of the amino acid residues, the entire polypeptide backbone being situated at the periphery of the clay matrix. 27Al NMR studies revealed the different nature of interaction of enzyme with the support for both immobilization techniques. N2 adsorption measurements indicated a sharp drop in surface area and pore volume for the covalently bound glucoamylase that suggested severe pore blockage. Activity studies were performed in a batch reactor. The adsorbed and covalently bound glucoamylase retained 49% and 66% activity of the free enzyme respectively. They showed enhanced pH and thermal stabilities. The immobilized enzymes also followed Michaelis–Menten kinetics. Km was greater than the free enzyme that was attributed to an effect of immobilization. The immobilized preparations demonstrated increased reusability as well as storage stability.

Dopamine Receptor Subtypes,IP3, cAMP and cGMP Functional Regulation in Parkinsonism Induced by Unilateral Infusion of Rotenone in Rats: Recovery with Bone Marrow Cells,Serotonin and GABA Supplementation

In the present study, the effects of 5-HT, GABA and Bone Marrow Cells infused intranigrally to substantia nigra individually and in combinations on unilateral rotenone infused Parkinsonism induced rats. Scatchard analysis of DA, DA D1 and D2 receptors in the corpus striatum, cerebral cortex, cerebellum, brain stem and hippocampus showed a significant increase in the Brain regions of rotenone infused rat compared to control. Real Time PCR amplification of DA D1, D2, Bax and ubiquitin carboxy-terminal hydrolase were up regulated in the brain regions of rotenone infused rats compared to control. Gene expression studies of -Synuclien, cGMP and Cyclic AMP response element-binding protein showed a significant down regulation in Rotenone infused rats compared to control. Behavioural studies were carried out to confirm the biochemical and molecular studies.Our study demonstrated that BMC administration alone cannot reverse the above said molecular changes occurring in PD rat. 5-HT and GABA acting through their specific receptors in combination with bone marrow cells play a crucial role in the functional recovery of PD rats. 5-HT, GABA and Bone marrow cells treated PD rats showed significant reversal to control in DA receptor binding and gene expression. 5-HT and GABA have co-mitogenic property. Proliferation and differentiation of cells re-establishing the connections in Parkinson's disease facilitates the functional recovery. Thus, it is evident that 5-HT and GABA along with BMC to rotenone infused rats renders protection against oxidative, related motor and cognitive deficits which makes them clinically significant for cellbased therapy. The BMC transformed to neurons when co-transplanted with 5-HT and GABA which was confirmed with PKH2GL and nestin. These newly formed neurons have functional significance in the therapeutic recovery of Parkinson’s disease.

Alkaline Protease from a Non-toxigenic Vibrio sp.(V26) and its Applications

The thesis presents a detailed account of the alkaline protease produced by Vibrio sp.(V26) a mangrove isolate,and the application of this enzyme in different fields.The protease producer strain was identified on the basis of biochemical characteristice,putative virulence traits and 16S rRNA gene sequencing.The purification and characterization of the protease has been carried out. Along with this, an attempt has been made to identifiy the protease gene. The physical parameters as well as the media components influencing protease production were optimized using Response Surfce Methodology(RSM).The scale up of the application of the protease from Vibrio sp.(V26) in the dissociation of cells in animal cell culture,in the recovery of silver from used X-ray films as well as an ingredient in commercial detergents were investigated.

Biochemical effects of the pesticide Chlorpyrifos on the fish Oreochromis mossambicus (Peters)

Man uses a variety of synthetic material for his comfortable materialistic life. Thus human interactions may become harmful for various terrestrial and aquatic lives. This is by contaminating their habitat and by becoming a threat to organisms itself. Thus the application and dispersal of several organic pollutants can lead to the development of several mutated forms of the species when exposed to sublethal concentrations of the pollutants. Otherwise, a decrease in number or extinction of these exposed species from earth's face may happen. Pesticides, we use for the benefit of crop yield, but its persistence may become havoc to non-target organism. Pesticides reaching a reservoir can subsequently enter the higher trophic levels. Organophosphorus compounds have replaced all other pesticides, due to its acute toxicity and non-persistent nature.Hence the present study has concentrated on the toxicity of the largest market-selling and multipurpose pesticide, chlorpyrifos on the commonly edible aquatic organism, fish. The euryhaline cichlid Oreochromis mossambicus was selected as animal model. The study has concentrated on investigating biochemical parameters like tissue-specific enzymes, antioxidant and lipid-peroxidation parameters, haematological and histological observations and pesticide residue analysis.Major findings of this work have indicated the possibility of aquatic toxicity to the fish on exposure to the insecticide chlorpyrifos. The insecticide was found as effective to induce structural alteration, depletion in protein content, decrease in different metabolic enzyme levels and to progress lipid peroxidation on a prolonged exposure of 21 days. The ion-transport mechanism was found to be adversely affected. Electrophoretic analysis revealed the disappearance of several protein bands after 21days of exposure to chlorpyrifos. Residue, analysis by gas chromatography explored the levels of chlorpyrifos retaining on the edible tissue portions during exposure period of 21days and also on a recovery period of 10 days.

Characterization of laccase from Streptomyces psammoticus: an enzyme for eco-friendly treatment of environmental pollutants

The present study has identified an actinomycete culture (S. psammoticus) which was capable of producing all the three major ligninolytic enzymes. The study revealed that least explored mangrove regions are potential sources for the isolation of actinomycetes with novel characteristics. The laccase production by the strain in SmF and SSF was found to be much higher than the reported values. The growth of the organism was favoured by alkaline pH and salinity of the medium. The enzyme also exhibited novel characteristics such as activity and stability at alkaline pH and salt tolerance. These two characters are quite significant from the industrial point of view making the enzyme an ideal candidate for industrial applications. Many of the application studies to date are focused on enzymes from fungal sources. However, the fungal laccases, which are mostly acidic in nature, could not be used universally for all application purposes especially, for the treatment of effluents from different industries, largely due to the alkaline nature of the effluents. Under such situations the enzymes from organisms like S. psammoticus with wide pH range could play a better role than the fungal counterparts. In the present study, the ability of the isolated strain and laccase in the degradation of dyes and phenolic compounds was successfully proved. The reusability of the immobilized enzyme system made the entire treatment process inexpensive. Thus it can be concluded from the present study that the laccase from this organism could be hopefully employed for the eco-friendly treatment of dye or phenol containing industrial effluents from various sources.

α-Galactosidase from Streptomyces griseoloalbus: an enzyme with versatile applications

The present study is focused on the production, purification and characterization of multiple thermostable α-galactosidases from a novel actinomycete strain Streptomyces griseoloalbus. The Chapter I of the thesis covers the wide literature regarding α-galactosidases from various sources and their potential applications. The Chapter 11 deals with the isolation of α-galactosidase- producing actinomycetes and selection of the best strain. The Chapters III and IV describe the optimization of α-galactosidase production under submerged fermentation and solid-state fermentation respectively. The Chapter V describes the purification and characterization of multiple α-galactosidases and also the obvious existence of a novel galactose-tolerant enzyme. The Chapter VI illustrates the potential applications of α-galactosidases from S. griseoloalbus followed by the Chapter VII summarizing and concluding the results of the present investigation.

Microbial enzyme production utilizing banana wastes

A critical survey of the fruits and vegetable markets of the towns and cities in South India reveals that banana fruit stalk wastes share a dominant proportion among the solid wastes generated. In the light of the review of literature presented in the foregoing section, few reports are available on the utilisation of banana waste for the production of alcoholic beverages, biogas, and single cell protein. However, it is not yet tried for the production of industrial enzymes. Moreover, preliminary fermentation studies conducted under uncontrolled conditions revealed that banana fruit stalk could be aptly utilised as solid substrate? for the industrial production of microbial amylases and cellulases at a cheaper cost. Therefore, it was proposed to conduct a detailed study towards the development of a suitable fermentation process for the production of industrial enzymes using banana fruit stalk wastes, which is rich in carbohydrate, as solid substrate, employing bacteria, under SSF.

Enzyme reactions as index of freshness of Fish and Shellfish

An attempt has been made in this study to screen some fish muscle enzymes to assess their potential worth in testing the degree of freshness of fish. A problem with routine enzyme activity determinations is the complexity of the method of enzyme assay. Hence, in the present study as far as possible simple assay techniques were adopted. Several species were screened to assess the possibility of employing this procedure on a large scale. It is hoped that findings of this study will lead to the development of meaningful criteria in testing the freshness of fish. This thesis has been divided into five chapters

Studies on the enzyme α-glucan phosphorylase

ac-qlncnn phosphorylsse is en ilportent enzyme in glycoiysis. It is the first used knows to exhibit ellosteric properties and lance its inhibition end ectivetion have significant effect on the rete ot qlycolysis. The thesis deals with 11 detailed study of the structure. inhibition and control or this snlrlls from rabbit uncle and troll e merino eninelo ‘the thesis is divided into two parts. Port 1 deals with studies on rabbit uncle glycogen phospherylese. After e review of the relevant literetnre (Chapter 1) the inhibition and chancel sodiiicetion studies on rabbit ensyle ere discussed in chepters 2 to 5. Chapter 6. gives the methods used for the study

“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

“Studies on glucose tolerant β-glucosidases from a novel Byssochlamys fulva and their applications in biomass to ethanol conversion”

Beta-glucosidases are critical enzymes in biomass hydrolysis process and is important in creating highly efficient enzyme cocktails for the bio-ethanol industry. Among the two strategies proposed for overcoming the glucose inhibition of commercial cellulases, one is to use heavy dose of BGL in the enzyme blends and the second is to do simultaneous saccharification and fermentation where glucose is converted to alcohol as soon as it is being generated. While the former needs extremely high quantities of enzyme, the latter is inefficient since the conditions for hydrolysis and fermentation are different. This makes the process technically challenging and also in this case, the alcohol generation is lesser, making its recovery difficult. A third option is to use glucose tolerant β-glucosidases which can work at elevated glucose concentrations. However, there are very few reports on such enzymes from microbial sources especially filamentous fungi which can be cultivated on cheap biomass as raw material. There has been very less number of studies directed at this, though there is every possibility that filamentous fungi that are efficient degraders of biomass may harbor such enzymes. The study therefore aimed at isolating a fungus capable of secreting glucose tolerant β- glucosidase enzyme. Production, characterization of β-glucosidases and application of BGL for bioethanol production were attempted.

Lignin recovery, Biochar Production and Decolourisation of Coir pith Black Liquor

Coir pith black liquor obtained as a dark brown filtrate from oxidative delignification needs to be decolourised before releasing to open environment. From this liquor industrially valuable lignin was recovered using acid precipitation method. ‘Biochar’ was produced by slow pyrolysis of coir pith at 500oC and 600oC. Water holding capacity and pH of the biochar were estimated. CHNS analysis was carried out to identify the nutrient profile. Structural characterization was done using FTIR and SEM Studies. Biochar produced at 600oC was found to be more suitable for decolourisation of the coir pith black liquor. FTIR analysis indicated peak changes while SEM analysis indicated surface area and porosity changes. Biochar decolourisation experiments were carried out on crude coir pith black liquor and also on lignin recovered coir pith black liquor.

Purification and Characterization of an Anti-cancer Enzyme Produced by Marine Vibrio Costicola Under A Novel Solid State Fermentation Process

L - Glutaminase, a therapeutically and industrially important enzyme, was produced from marine Vibrio costicola by a novel solid state fermentation process using polystyrene beads as inert support. The new fermentation system offered several advantages over the conventional systems, such as the yield of leachate with minimum viscosity and high specific activity for the target product besides facilitating the easy estimation of biomass. The enzyme thus produced was purified and characterised. It was active at physiological pH, showed high substrate specificity towards L - glutamine and had a Km value of 7.4 x 10-2 M. It also exhibited high salt and temperature tolerance indicating good scope for its industrial and therapeutic applications

Protease Production by haloarchaea Natrinema sp. BTSH10 isolated from salt pan of South India

The present study led to the recognition of Natrinema sp. BTSH 10 isolated from saltern ponds, as an ideal candidate species for production of gelatinase, which was noted as a halozyme capable of showing enzyme activity in the presence of 15% NaCl. Results obtained during the course of the present study indicated potential for application of this enzyme in industrial catalysis that are performed in the presence of high concentrations of salt. The enzyme characteristics noted with this gelatinase also indicate the scope for probable applications in leather industry, meat tenderization, production of fish sauce and soy sauce. Since halophilic proteases are tolerant to organic solvents, they could be used in antifouling coating preparations used to prevent biofouling of submarine equipments. The gelatinase from haloarchaea could be considered as a probable candidate for peptide synthesis. However, further studies are warranted on this haloarcheal gelatinase particularly on structure elucidation and enzyme engineering to suit a wide range of applications. There is immense scope for developing this halozyme as an industrial enzyme once thorough biochemistry of this gelatinase is studied and a pilot scale study is conducted towards industrial production of this enzyme under fermentation is facilitated. Based on the present study it is concluded that haloarchaea Natrinema sp. that inhabit solar saltern ponds are ideal source for deriving industrially important halozymes and molecular studies on enzymes are prerequisite for their probable industrial applications. This is the first time this species of archaea is recognized as a source of gelatinase enzyme that has potential for industrial applications.