Production of Alkaline Protease by Free and Immobilised Cells of VIBRIO sp. Under Different Fermentation Systems and Its Application on Deproteinisation of Prawn Shell Waste for Chitin Recovery

This thesis presents a detailed account of a cost - effective approach towards enhanced production of alkaline protease at profitable levels using different fermentation designs employing cheap agro-industrial residues. It involves the optimisation of process parameters for the production of a thermostable alkaline protease by Vibrio sp. V26 under solid state, submerged and biphasic fermentations, production of the enzyme using cell immobilisation technology and the application of the crude enzyme on the deproteinisation of crustacean waste.The present investigation suggests an economic move towards Improved production of alkaline protease at gainful altitudes employing different fermentation designs utilising inexpensive agro-industrial residues. Moreover, the use of agro-industrial and other solid waste substrates for fermentation helps to provide a substitute in conserving the already dwindling global energy resources. Another alternative for accomplishing economically feasible production is by the use of immobilisation technique. This method avoids the wasteful expense of continually growing microorganisms. The high protease producing potential of the organism under study ascertains their exploitation in the utilisation and management of wastes. However, strain improvement studies for the production of high yielding variants using mutagens or by gene transfer are required before recommending them to Industries.Industries, all over the world, have made several attempts to exploit the microbial diversity of this planet. For sustainable development, it is essential to discover, develop and defend this natural prosperity. The Industrial development of any country is critically dependent on the intellectual and financial investment in this area. The need of the hour is to harness the beneficial uses of microbes for maximum utilisation of natural resources and technological yields. Owing to the multitude of applications in a variety of industrial sectors, there has always been an increasing demand for novel producers and resources of alkaline proteases as well as for innovative methods of production at a commercial altitude. This investigation forms a humble endeavour towards this perspective and bequeaths hope and inspiration for inventions to follow.

lncidence of enterotoxigenic Staphylococcus aureus in relation to the microbial safety of seafood

A detailed study was made on the microbial quality, with special reference to food safety, of the fish and fishery products in the retail trade in Cochin and around. Also, farmed molluscan shellfishes like mussels and oysters were investigated for the microbial quality including the presence of pathogenic bacteria. Special stress has been given to monitor the incidence of coagulase positive as well as coagulase negative Staphylococcus in these products and their relative incidence have been recorded.In the next part, the investigation was centered mainly on toxigenic S.aureus. This is because among the Gram positive toxigenic bacteria, the Saureus with potential to produce thermostable enterotoxins are more relavent in food safety conceming seafoods in comparison with the Gram-negative pathogens like Salmonella and V.cholerae.The incidence, toxigenic potential and conditions of toxin production by S.aureus have been investigated in detail. An attempt has also been made to relate the toxigenisis with the presence of the concerned toxigenic genes in the genomes of S. aureus strains.

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.

Thermdstable alpha amylase production by bacillus coagulans

Microbial enzymes are in great demand owing to their importance in several industries such as brewing, baking, leather, laundry detergent, dairy. starch processing and textiles besides pharmaceuticals. About 80% of the enzymes produced through fermentation and sold in the industrial scale are hydrolytic enzymes. Due to recognition of new and new applications, an intensive screening of different kinds of enzymes with novel properties, from various microorganisms, is being pursued all over the world. Bacillus sp are largely known to produce a-amylase, among the different groups of microoganisms, at industrial level. They are known to produce both saccharifying and liquefying a-amylases (Fukumoto 1963; walker and Campbell, 1967a). which are distinguishable by their mechanisms of starch degradation by the fact that the saccharifying asamylases produce an increase in reducing power about twice that of the liquefying enzyme (Fukumoto, 1963; Pazur and Okada, 1966). Under this circumstances, the present study was undertaken, with a View to utilise a fast growing B.coagu1ans isolated from soil, for production of thermostable and alkaline oz-amylase under different fermentation processes

Molecular cloning and homology modelling of a subtilisin-like serine protease from the marine fungus, Engyodontium album BTMFS10

An alkaline protease gene (Eap) was isolated for the first time from a marine fungus, Engyodontium album. Eap consists of an open reading frame of 1,161 bp encoding a prepropeptide consisting of 387 amino acids with a calculated molecular mass of 40.923 kDa. Homology comparison of the deduced amino acid sequence of Eap with other known proteins indicated that Eap encode an extracellular protease that belongs to the subtilase family of serine protease (Family S8). A comparative homology model of the Engyodontium album protease (EAP) was developed using the crystal structure of proteinase K. The model revealed that EAP has broad substrate specificity similar to Proteinase K with preference for bulky hydrophobic residues at P1 and P4. Also, EAP is suggested to have two disulfide bonds and more than two Ca2? binding sites in its 3D structure; both of which are assumed to contribute to the thermostable nature of the protein.

Molecular cloning and homology modelling of a subtilisin-like serine protease from the marine fungus, Engyodontium album BTMFS10

An alkaline protease gene (Eap) was isolated for the first time from a marine fungus, Engyodontium album. Eap consists of an open reading frame of 1,161 bp encoding a prepropeptide consisting of 387 amino acids with a calculated molecular mass of 40.923 kDa. Homology comparison of the deduced amino acid sequence of Eap with other known proteins indicated that Eap encode an extracellular protease that belongs to the subtilase family of serine protease (Family S8). A comparative homology model of the Engyodontium album protease (EAP) was developed using the crystal structure of proteinase K. The model revealed that EAP has broad substrate specificity similar to Proteinase K with preference for bulky hydrophobic residues at P1 and P4. Also, EAP is suggested to have two disulfide bonds and more than two Ca2? binding sites in its 3D structure; both of which are assumed to contribute to the thermostable nature of the protein.

Detergent compatible alkaline lipase produced by marine Bacillus smithii BTMS 11

Bacillus smithii BTMS 11, isolated from marine sediment, produced alkaline and thermostable lipase. The enzyme was purified to homogeneity by ammonium sulfate precipitation and ion exchange chromatography which resulted in 0.51 % final yield and a 4.33 fold of purification. The purified enzyme was found to have a specific activity of 360 IU/mg protein. SDS-PAGE analyses, under non-reducing and reducing conditions, yielded a single band of 45 kDa indicating the single polypeptide nature of the enzyme and zymogram analysis using methylumbelliferyl butyrate as substrate confirmed the lipolytic activity of the protein band. The enzyme was found to have 50 C and pH 8.0 as optimum conditions for maximal activity. However, the enzyme was active over wide range of temperatures (30–80 C) and pH (7.0–10.0). Effect of a number of metal salts, solvents, surfactants, and other typical enzyme inhibitors on lipase activity was studied to determine the novel characteristics of the enzyme. More than 90 % of the enzyme activity was observed even after 3 h of incubation in the presence of commercial detergents Surf, Sunlight, Ariel, Henko, Tide and Ujala indicating the detergent compatibility of B. smithii lipase. The enzyme was also found to be efficient in stain removal from cotton cloths. Further it was observed that the enzyme could catalyse ester synthesis between fatty acids of varying carbon chain lengths and methanol with high preference for medium to long chain fatty acids showing 70 % of esterification. Results of the study indicated scope for application of this marine bacterial lipase in various industries

Trypsin Inhibitor from Edible Mushroom Pleurotus floridanus Active against Proteases of Microbial Origin

Protease inhibitors can be versatile tools mainly in the fields of medicine, agriculture and food preservative applications. Fungi have been recognized as sources of protease inhibitors, although there are only few such reports on mushrooms. This work reports the purification and characterization of a trypsin inhibitor from the fruiting body of edible mushroom Pleurotus floridanus (PfTI) and its effect on the activity of microbial proteases. The protease inhibitor was purified up to 35-fold by DEAE-Sepharose ion exchange column, trypsin-Sepharose column and Sephadex G100 column. The isoelectric point of the inhibitor was 4.4, and its molecular mass was calculated as 37 kDa by SDS-PAGE and 38.3 kDa by MALDI-TOF. Inhibitory activity confirmation was by dot-blot analysis and zymographic activity staining. The specificity of the inhibitor toward trypsin was with Ki of 1.043×10−10 M. The inhibitor was thermostable up to 90 °C with maximal stability at 30 °C, active over a pH range of 4–10 against proteases from Aspergillus oryzae, Bacillus licheniformis, Bacillus sp. and Bacillus amyloliquefaciens. Results indicate the possibility of utilization of protease inhibitor from P. floridanus against serine proteases

Barley oxalate oxidase is a hexameric protein related to seed storage proteins: evidence from X-ray crystallography

The oxalate oxidase enzyme expressed in barley roots is a thermostable, protease-resistant enzyme that generates H2O2. It has great medical importance because of its use to assay plasma and urinary oxalate, and it has also been used to generate transgenic, pathogen-resistant crops. This protein has now been purified and three types of crystals grown. X-ray analysis shows that the symmetry present in these crystals is consistent with a hexameric arrangement of subunits, probably a trimer of dimers. This structure may be similar to that found in the related seed storage proteins.

Molecular Basis of the Thermostability and Thermophilicity of Laminarinases: X-ray Structure of the Hyperthermostable Laminarinase from Rhodothermus marinus and Molecular Dynamics Simulations

Glycosyl hydrolases are enzymes capable of breaking the glycosidic linkage of polysaccharides and have considerable industrial and biotechnological applications. Driven by the later applications, it is frequently desirable that glycosyl hydrolases display stability and activity under extreme environment conditions, such as high temperatures and extreme pHs. Here, we present X-ray structure of the hyperthermophilic laminarinase from Rhodothermus marinus (RmLamR) determined at 1.95 angstrom resolution and molecular dynamics simulation studies aimed to comprehend the molecular basis, for the thermal stability of this class of enzymes. As most thermostable proteins, RmLamR contains a relatively large number of salt bridges, which are not randomly distributed on the structure. On the contrary, they form clusters interconnecting beta-sheets of the catalytic domain. Not all salt bridges, however, are beneficial for the protein thermostability: the existence of charge-charge interactions permeating the hydrophobic core of the enzymes actually contributes to destabilize the structure by facilitating water penetration into hydrophobic cavities, as can be seen in the case of mesophilic enzymes. Furthermore, we demonstrate that the mobility of the side-chains is perturbed differently in each class of enzymes. The side-chains of loop residues surrounding the catalytic cleft in the mesophilic laminarinase gain mobility and obstruct the active site at high temperature. By contrast, thermophilic laminarinases preserve their active site flexibility, and the active-site cleft remains accessible for recognition of polysaccharide substrates even at high temperatures. The present results provide structural insights into the role played by salt-bridges and active site flexibility on protein thermal stability and may be relevant for other classes of proteins, particularly glycosyl hydrolases.

Larvicidal activity of the water extract of Moringa oleifera seeds against Aedes aegypti and its toxicity upon laboratory animals

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Virulence factors of Escherichia coli isolated from diarrheic calves

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Cyclodextrin glycosyltransferase from Bacillus licheniformis: Optimization of production and its properties

Cyclodextrin glycosyltransferase (EC 2.4.1.19) is an enzyme that produces cyclodextrins from starch via an intramolecular transglycosylation reaction. An alkalophilic Bacillus strain, isolated from cassava peels, was identified as Bacillus licheniformis. CGTase production by this strain was better when potato starch was used as carbon source, followed by cassava starch and amylopectin. Glucose and amylose, on the other hand, acted as synthesis repressors. When the cultivation was supplemented with sodium ions and had the pH adjusted between 6.0 and 9.0, the microorganism maintained the growth and enzyme production capacity. This data is interesting because it contradicts the concept that alkalophilic microorganisms do not grow in this pH range. After ultrafiltration-centrifugation, one protein of 85.2 kDa with CGTase activity was isolated. This protein was identified in plates with starch and phenolphthalein. Determination of the optimum temperature showed higher activities at 25 degrees C and 55 degrees C, indicating the possible presence of more than one CGTase in the culture filtrate. Km and Vmax values were 1.77 mg/mL and 0.0263 U/mg protein, respectively, using potato starch as substrate.

Contribution to the study of diarrhea etiology in neonate dairy calves in São Paulo state, Brazil

Amostras fecais de 203 bezerros com diarréia, idade inferior a 30 dias, de ambos os sexos e de diferentes propriedades do Estado de São Paulo foram examinadas num período de dois anos. Cultivos para pesquisa bacteriana foram feitos em agar acrescido de 10% de sangue bovino e agar Levine. As placas foram incubadas por até 96 horas, em condições aeróbias, a 37°C, com observação dos aspectos de colônia e estudos morfológicos, bioquímicos e realização de outros testes, quando pertinentes. O teste de ELISA foi aplicado para pesquisa de Rotavirus. Cryptosporidium spp. também foi pesquisado e identificado. Resultados revelaram envolvimento de vários patógenos de forma isolada, assim como associados. Rotavirus foi encontrado em 51 (25,1%) das amostras, sendo em 58,8% só, em 41.7% associado a outros microrganismos. Cryptosporydium spp foi isolado em 43 (21.3%) das amostras, sendo só em 65,1% delas e associado a outros enteropatógenos em 34,9%. No exame parasitológico foram encontrados ovos de estrongilídeos em 5 (2,5%) das amostras, não excedendo mais de dois ovos por campo examinado. Ao exame microbiológico, um ou mais microrganismos foram isolados. Escherichia coli foi encontrada em 100% das amostras. As pesquisas de toxina termoestável e do antígeno de aderência K99 realizada nas 73 amostras de E.coli foram negativas, e o grupo sorológico das mesmas foi determinado, sendo 34,2%, 17,8% e 47,9% das amostras pertencentes aos sorogrupos O8, O11 e O101, respectivamente. Salmonella Dublin e Salmonella typhimurium foram isoladas em 5,4% e 6,1% das amostras examinadas, respectivamente.