Isolation, purification, characterization and application of proteinaceous protease inhibitor from marine bacterium Pseudomonas mendocina BTMW 301

The microorganisms are recognized as important sources of protease inhibitors which are valuable in the fields of medicine, agriculture and biotechnology. The protease inhibitors of microbial origin are found to be versatile in their structure and mode of inhibition that vary from those of other sources. Although surplus of low molecular weight non-protein protease inhibitors from microorganisms have been reported, there is a dearth of reports on proteinaceous protease inhibitors. The search for new metabolites from marine organisms has resulted in the isolation of more or less 10,000 metabolites (Fuesetani and Fuesetani, 2000) many of which are gifted with pharmacodynamic properties. The existence of marine microorganisms was reported earlier, and they were found to be metabolically and physiologically dissimilar from terrestrial microorganisms. Marine microorganisms have potential as important new sources of enzyme inhibitors and consequently a detailed study of new marine microbial inhibitors will provide the basis for future research (Imada, 2004).

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

Studies on Halocin production by haloarchaea Natrinema sp. BTSH10

This study on halocin production by Natrinema sp.BTSH10 indicate the prospects for intensive research which could lead to discovery of novel halocins which could have far reaching impact in biopharmaceutical industry particularly as anticancer drug. It is also anticipated that further research on this halocin could lead towards development of novel anticancer drug and new era in pharmaceutical biotechnology. There is no doubt that haloarchaea from saltern ponds have immense potential to return novel and valuable drugs and bioactive substances.

“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.

Depth wise variation of microbial load in the soils of midland region of Kerala: a function of important soil physicochemical characteristics and nutrients

Soil microorganisms play a main part in organic matter decomposition and are consequently necessary to soil ecosystem processes maintaining primary productivity of plants. In light of current concerns about the impact of cultivation and climate change on biodiversity and ecosystem performance, it is vital to expand a complete understanding of the microbial community ecology in our soils. In the present study we measured the depth wise profile of microbial load in relation with important soil physicochemical characteristics (soil temperature, soil pH, moisture content, organic carbon and available NPK) of the soil samples collected from Mahatma Gandhi University Campus, Kottayam (midland region of Kerala). Soil cores (30 cm deep) were taken and the cores were separated into three 10-cm depths to examine depth wise distribution. In the present study, bacterial load ranged from 141×105 to 271×105 CFU/g (10cm depth), from 80×105 to 131×105 CFU/g (20cm depth) and from 260×104 to 47×105 CFU/g (30cm depth). Fungal load varies from 124×103 to 27×104 CFU/g, from 61×103 to110×103 CFU/g and from 16×103 to 49×103 CFU/g at 10, 20 and 30 cm respectively. Actinomycetes count ranged from 129×103 to 60×104 CFU/g (10cm), from 70×103 to 31×104 CFU/g (20cm) and from 14×103 to 66×103 CFU/g (30cm). The study revealed that there was a significant difference in the depthwise distribution of microbial load and soil physico-chemical properties. Bacterial, fungal and actinomycetes load showed a decreasing trend with increasing depth at all the sites. Except pH all other physicochemical properties showed decreasing trend with increasing depth. The vertical profile of total microbial load was well matched with the depthwise profiles of soil nutrients and organic carbon that is microbial load was highest at the soil surface where organics and nutrients were highest

Microbial Biosurfactants – Review

Biosurfactants are surface active compounds released by microorganisms. They are biodegradable non-toxic and eco-friendly materials. In this review we have updated the information about different microbial surfactants. The biosurfactant production depends on the fermentation conditions, environmental factors and nutrient availability. The extraction of the biosurfactants from the cell-free supernatant using the solvent extraction procedure and the qualitative and quantitative analysis has been discussed with appropriate equipment details. The application of the biosurfactant includes biomedical, cosmetic and bioremediation. The type of microbial biosurfactants include trehalose lipids, rhamnolipids, sophorolipids, glycolipids, cellobiose lipids, polyol lipids, diglycosyl diglycerides, lipoloysaccharides, arthrofactin, lichensyn A and B, surfactin, viscosin, phospholipids, sulphonyl lipids and fatty acids. Rhamnolipid biosurfactants produced by Pseudomonas aeruginosa DS10-129 showed significant applications in the bioremediation of hydrocarbons in gasoline spilled soil and petroleum oily sludge. Rhamnolipid biosurfactant enhanced the bioremediation process by releasing the weathered oil from the soil matrices and enhanced the bioavailability of hydrocarbons for microbial degradation. It is having potential applications in the remediation of hydrocarbon contaminated sites. Biosurfactants from marine microorganisms also offer great potential in bioremediation of oil contaminated oceanic environments

Spatial and Temporal Variation of Microbial Population in the Grassland Soils of Tropical Montane Forest: Influence of Soil Physico-Chemical Characteristics and Nutrients

Present study is focused on the spatiotemporal variation of the microbial population (bacteria, fungus and actinomycetes) in the grassland soils of tropical montane forest and its relation with important soil physico-chemical characteristics and nutrients. Different physico-chemical properties of the soil such as temperature, moisture content, organic carbon, available nitrogen, available phosphorous and available potassium have been studied. Results of the present study revealed that both microbial load and soil characteristics showed spatiotemporal variation. Microbial population of the grassland soils were characterized by high load of bacteria followed by fungus and actinomycetes. Microbial load was high during pre monsoon season, followed by post monsoon and monsoon. The microbial load varied with important soil physico-chemical properties and nutrients. Organic carbon content, available nitrogen and available phosphorous were positively correlated with bacterial load and the correlation is significant at 0.05 and 0.01 levels respectively. Available nitrogen and available phosphorous were positively correlated with fungus at 0.05 level significance. Moisture content was negatively correlated with actinomycetes at 0.01 level of significance. Organic carbon negatively correlated with actinomycetes load at 0.05 level of significance

Microbial quality of shrimp products of export trade produced from aquacultured shrimp

Bacteriological quality of individually quick frozen (IQF) shrimp products produced from aquacultured tiger shrimp (Penaeus monodon) has been analysed in terms of aerobic plate count (APC), coliforms, Escherichia coli, coagulase-positive staphylococci, Salmonella, and Listeria monocytogenes. Eight hundred forty-six samples of raw, peeled, and deveined tail-on (RPTO), 928 samples of cooked, peeled, and deveined tail-on (CPTO), 295 samples of headless, undeveined shell-on (HLSO), and 141 samples of raw, peeled, and deveined tail-off (RPND) shrimps were analysed for the above bacteriological parameters. Salmonella was isolated in only one sample of raw, peeled tail-on. Serotyping of the strain revealed that it was S. typhimurium. While none of the cooked, peeled tail-on shrimp samples exceeded the aerobic plate count (APC) of 105 colony forming units per gram (cfu/g), 2.5% of raw, peeled, tail-on, 6.4% of raw, peeled tail-off, and 7.5% of headless shell-on shrimp samples exceeded that level. Coliforms were detected in all the products, though at a low level. Prevalence of coliforms was higher in headless shell-on (26%) shrimps followed by raw, peeled, and deveined tail-off (19%), raw, peeled tail-on (10%), and cooked, peeled tail-on (3.8%) shrimps. While none of the cooked, peeled tail-on shrimp samples were positive for coagulase-positive staphylococci and E. coli, 0.6–1.3% of the raw, peeled tail-on were positive for staphylococci and E. coli, respectively. Prevalence of staphylococci was highest in raw, peeled tail-off (5%) shrimps and the highest prevalence of E. coli (4.8%) was noticed in headless shell-on shrimps. L. monocytogenes was not detected in any of the cooked, peeled tail-on shrimps. Overall results revealed that the plant under investigation had exerted good process control in order to maintain superior bacteriological quality of their products

"Alkaline Lipase Production by Marine Bacillus smithii BTMS 11"

Considering the potential of marine environment present study was designed for the screening and isolation of a potential salt tolerant. alkaline and thennotolerant lipase producing bacteria from the costal belts of South India and consequent development of ideal bioprocess for industrial production, purification characterisation and evaluation of the potential of the lipase enzyme for various industrial applications 1. Screening and isolation of a potential lipase producing bacteria. 2. Optimization of various physicochemical factors in Submerged fennentation for the production of alkaline lipase 3. Purification ofthe lipase enzyme 4. Characterisation of the enzyme 5. Evaluation of the enzyme for various industrial applications

“Effect of spice oleoresins in microbial decontamination and their potential application in quality stabilization of Tuna (Euthynnus affinis) during storage”

Unprocessed seafood harbor high number of bacteria, hence are more prone to spoilage. In this circumstance, the use of spice in fish for reduction of microorganism can play an important role in seafood processing. Many essential oils from herbs and spices are used widely in the food, health and personal care industries and are classified as GRAS (Generally regarded as safe) substances or are permitted food additives. A large number of these compounds have been the subject of extensive toxicological scrutiny. However, their principal function is to impart desirable flavours and aromas and not necessarily to act as antimicrobial agents. Given the high flavour and aroma impact to plant essential oils, the future for using these compound as food preservatives lies in the careful selection and evaluation of their efficacy at low concentrations but in combination with other chemical preservatives or preservation processes. For this reason they are worth of study alone or in combination with processing methods in order to establish if they could extend the shelf-life of foods. In this study, the effect of the spices, clove, turmeric, cardamom, oregano, rosemary and garlic in controlling the spoilage and pathogenic bacteria is investigated. Their effect on biogenic amine formation in tuna especially, histamine, as a result of bacterial control is also studied in detail. The contribution of spice oleoresin in the sensory and textural parameters is investigated using textural profile analysis and sensory panel. Finally, the potential of spices in quality stabilization and in increasing the shelf–life of tuna during frozen storage is analysed

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

Integron study, molecular typing and characterization of Salmonella serotypes isolated from seafood and poultry

The primary habitat of Salmonella is the gastrointestinal tract of animals and they are discharged into the water bodies through the feces. Aquatic animals act as asymptomatic reservoirs of a wide range of Salmonella serotypes. The inevitable delay in the detection of Salmonella contamination and the low sensitivity of the conventional methods is a serious issue in the seafood industry. Due to the indiscriminate use, the antibiotics are finally accumulated in the aquatic environment which provides the required antibiotic stress for the emergence of more and more antibiotic resistant phenotypes ofSalmonella. Several genetic determinants like integrons, genomic islands etc. play their role in acquisition and reshuffling of antibiotic resistance genes. A large number of virulence determinants are required for Salmonella pathogenicity. The virulence potential of Salmonella is determined, to some extent, by the presence of phages or phage mediated genes in the bacterial genome. There is much intra-serotype polymorphism in Salmonella and epidemiological studies rely on genetic resemblance of the isolated strains. Proper identification of the strain employing the traditional and molecular techniques is a prerequisite for accurate epidemiological studies (Soto et al., 2000). In this context, a study was undertaken to determine the prevalence of different Salmonella serotypes in seafood and to characterize them

Differential induction, isolation, physicochemical and molecular characterization of temperate phages of environmental Vibrio cholerae as evidence of phage mediated Horizontal Gene Transfer

The resurgence of the enteric pathogen Vibrio cholerae, the causative organism of epidemic cholera, remains a major health problem in many developing countries like India. The southern Indian state of Kerala is endemic to cholera. The outbreaks of cholera follow a seasonal pattern in regions of endemicity. Marine aquaculture settings and mangrove environments of Kerala serve as reservoirs for V. cholerae. The non-O1/non-O139 environmental isolates of V. cholerae with incomplete ‘virulence casette’ are to be dealt with caution as they constitute a major reservoir of diverse virulence genes in the marine environment and play a crucial role in pathogenicity and horizontal gene transfer. The genes coding cholera toxin are borne on, and can be infectiously transmitted by CTXΦ, a filamentous lysogenic vibriophages. Temperate phages can provide crucial virulence and fitness factors affecting cell metabolism, bacterial adhesion, colonization, immunity, antibiotic resistance and serum resistance. The present study was an attempt to screen the marine environments like aquafarms and mangroves of coastal areas of Alappuzha and Cochin, Kerala for the presence of lysogenic V. cholerae, to study their pathogenicity and also gene transfer potential. Phenotypic and molecular methods were used for identification of isolates as V. cholerae. The thirty one isolates which were Gram negative, oxidase positive, fermentative, with or without gas production on MOF media and which showed yellow coloured colonies on TCBS (Thiosulfate Citrate Bile salt Sucrose) agar were segregated as vibrios. Twenty two environmental V. cholerae strains of both O1 and non- O1/non-O139 serogroups on induction with mitomycin C showed the presence of lysogenic phages. They produced characteristic turbid plaques in double agar overlay assay using the indicator strain V. cholerae El Tor MAK 757. PCR based molecular typing with primers targeting specific conserved sequences in the bacterial genome, demonstrated genetic diversity among these lysogen containing non-O1 V. cholerae . Polymerase chain reaction was also employed as a rapid screening method to verify the presence of 9 virulence genes namely, ctxA, ctxB, ace, hlyA, toxR, zot,tcpA, ninT and nanH, using gene specific primers. The presence of tcpA gene in ALPVC3 was alarming, as it indicates the possibility of an epidemic by accepting the cholera. Differential induction studies used ΦALPVC3, ΦALPVC11, ΦALPVC12 and ΦEKM14, underlining the possibility of prophage induction in natural ecosystems, due to abiotic factors like antibiotics, pollutants, temperature and UV. The efficiency of induction of prophages varied considerably in response to the different induction agents. The growth curve of lysogenic V. cholerae used in the study drastically varied in the presence of strong prophage inducers like antibiotics and UV. Bacterial cell lysis was directly proportional to increase in phage number due to induction. Morphological characterization of vibriophages by Transmission Electron Microscopy revealed hexagonal heads for all the four phages. Vibriophage ΦALPVC3 exhibited isometric and contractile tails characteristic of family Myoviridae, while phages ΦALPVC11 and ΦALPVC12 demonstrated the typical hexagonal head and non-contractile tail of family Siphoviridae. ΦEKM14, the podophage was distinguished by short non-contractile tail and icosahedral head. This work demonstrated that environmental parameters can influence the viability and cell adsorption rates of V. cholerae phages. Adsorption studies showed 100% adsorption of ΦALPVC3 ΦALPVC11, ΦALPVC12 and ΦEKM14 after 25, 30, 40 and 35 minutes respectively. Exposure to high temperatures ranging from 50ºC to 100ºC drastically reduced phage viability. The optimum concentration of NaCl required for survival of vibriophages except ΦEKM14 was 0.5 M and that for ΦEKM14 was 1M NaCl. Survival of phage particles was maximum at pH 7-8. V. cholerae is assumed to have existed long before their human host and so the pathogenic clones may have evolved from aquatic forms which later colonized the human intestine by progressive acquisition of genes. This is supported by the fact that the vast majority of V. cholerae strains are still part of the natural aquatic environment. CTXΦ has played a critical role in the evolution of the pathogenicity of V. cholerae as it can transmit the ctxAB gene. The unusual transformation of V. cholerae strains associated with epidemics and the emergence of V. cholera O139 demonstrates the evolutionary success of the organism in attaining greater fitness. Genetic changes in pathogenic V. cholerae constitute a natural process for developing immunity within an endemically infected population. The alternative hosts and lysogenic environmental V. cholerae strains may potentially act as cofactors in promoting cholera phage ‘‘blooms’’ within aquatic environments, thereby influencing transmission of phage sensitive, pathogenic V. cholerae strains by aquatic vehicles. Differential induction of the phages is a clear indication of the impact of environmental pollution and global changes on phage induction. The development of molecular biology techniques offered an accessible gateway for investigating the molecular events leading to genetic diversity in the marine environment. Using nucleic acids as targets, the methods of fingerprinting like ERIC PCR and BOX PCR, revealed that the marine environment harbours potentially pathogenic group of bacteria with genetic diversity. The distribution of virulence associated genes in the environmental isolates of V. cholerae provides tangible material for further investigation. Nucleotide and protein sequence analysis alongwith protein structure prediction aids in better understanding of the variation inalleles of same gene in different ecological niche and its impact on the protein structure for attaining greater fitness of pathogens. The evidences of the co-evolution of virulence genes in toxigenic V. cholerae O1 from different lineages of environmental non-O1 strains is alarming. Transduction studies would indicate that the phenomenon of acquisition of these virulence genes by lateral gene transfer, although rare, is not quite uncommon amongst non-O1/non-O139 V. cholerae and it has a key role in diversification. All these considerations justify the need for an integrated approach towards the development of an effective surveillance system to monitor evolution of V. cholerae strains with epidemic potential. Results presented in this study, if considered together with the mechanism proposed as above, would strongly suggest that the bacteriophage also intervenes as a variable in shaping the cholera bacterium, which cannot be ignored and hinting at imminent future epidemics.