Measurement of Cellulolytic Potential of Cellulase Producing Bacteria

The bioconversion of cellulose and hemicellulose to soluble sugars is important for global stabilization and for a sustainable human society. Here, hundreds of cellulolytic bacteria were found in soil, compost and animal waste slurry of our environment. Bacillus spp. are aerobic cellulolytic bacteria. Here, two Bacillus strains 2414, 2579 (T) and their mixed culture utilized for measuring the cellulolytic potential. The capability of cellulolytic potential was analyzed by enriching the basal salt media with Whatman no.1 filter paper as a substrate for cellulose degradation. Here, Cellulose-degrading potential of Bacillus strains was measured by measuring the diameter of a clear zone around the colony and its hydrolytic value on cellulose Congo-Red agar media. The extracellular cellulase activities ranged from 0.08233 to 0.44 IU/mL for FPase and 0.243 to 0.595 IU/mL for endoglucanase assay. The maximum activities range of β-glucosidase or cellobiase activity was 0.6 to1.5 1U/ml. The maximum xylanase activities value Bacillus cellulolysticus 2579 (T), Bacillus subtilis 2414 and their mixed culture were 12.0,11.5 and 12.5 unit/mL, respectively. All the enzymes were stable at an optimum pH range value of 3.0-7.0 and temperature range of 30˚C-50˚C. The maximum filter paper degradation percentage was estimated to be 71.76% by mixed culture after 48hrs of incubation period, it was observed that the maximum filter paper degradation was done by mixed culture than Bacillus strains. Biodiesel production was estimated by following the EN-14103 method and ester content was calculated on the basis of response factor with a minimum set value of ester content will be 96.5%.

Statistical and evolutionary optimization for enhanced production of an anti-leukemic enzyme, L-asparaginase, in a protease-deficient Bacillus aryabhattai ITBHU02 isolated from the soil contaminated with hospital waste.

Over the past few decades, L-asparaginase has emerged as an excellent anti-neoplastic agent. In present study, a new strain ITBHU02, isolated from soil site near degrading hospital waste, was investigated for the production of extracellular L-asparaginase. Further, it was renamed as Bacillus aryabhattai ITBHU02 based on its phenotypical features, biochemical characteristics, fatty acid methyl ester (FAME) profile and phylogenetic similarity of 16S rDNA sequences. The strain was found protease-deficient and its optimal growth occurred at 37 °C and pH 7.5. The strain was capable of producing enzyme L-asparaginase with maximum specific activity of 3.02±0.3 Umg-1 protein, when grown in un-optimized medium composition and physical parameters. In order to improve the production of L-asparaginase by the isolate, response surface methodology (RSM) and genetic algorithm (GA) based techniques were implemented. The data achieved through the statistical design matrix were used for regression analysis and analysis of variance studies. Furthermore, GA was implemented utilizing polynomial regression equation as a fitness function. Maximum average L-asparaginase productivity of 6.35 Umg-1 was found at GA optimized concentrations of 4.07, 0.82, 4.91, and 5.2 gL‑1 for KH2PO4, MgSO4.7H2O, L-asparagine, and glucose respectively. The GA optimized yield of the enzyme was 7.8% higher in comparison to the yield obtained through RSM based optimization.

Comparative analysis of protein profiles of wild virulent (E156) and aroA-htrA double deletion mutant vaccine strain (S30) of Salmonella enterica subsp. enterica serovar Abortusequi under in vivo and in vitro growth conditions.

In the present study, cell lysate and cell supernatant of the both strains i.e., virulent wild type (E156) and mutant (S30) vaccine strains of Salmonella enterica subspecies enterica serovar Abortusequi (S. Abortusequi), grown under varied in vivo and in vitro conditions were subjected to SDS PAGE and western blotting (using rabbit hyperimmune serum). Variation in growth conditions did not have any significant effect on expression of different proteins. SDS PAGE of E156 and S30 cell lysate (CL) revealed 26 and 28 bands, respectively with 3 prominent proteins of 71, 46 and 42 kDa in cell lysate of E 156 and 4 prominent proteins 71, 65, 46 and 40 kDa in S30 strain. The cell supernatant (CS) from both the strains, subjected to SDS PAGE, exhibited similarity in protein profile among these strains, however three bands of 65, 53 and 40 kDa were more prominent in CS preparation of S30, whereas a 56 kDa protein was prominent in CS of E156. Western blotting of E156 and S30 revealed 3 unique proteins of 65, 53 and 40 kDa present in CS preparation of S30 strains which could be used for differentiation of mutant and wild strains and also in development of test for differentiating vaccinated animals from naturally infected.

A non-invasive technique for rapid extraction of DNA from fish scales.

DNA markers are being increasingly used in studies related to population genetics and conservation biology of endangered species. DNA isolation for such studies requires a source of biological material that is easy to collect, non-bulky and reliable. Further, the sampling strategies based on non-invasive procedures are desirable, especially for the endangered fish species. In view of above, a rapid DNA extraction method from fish scales has been developed with the use of a modified lysis buffer that require about 2 hr duration. This methodology is non-invasive, less expensive and reproducible with high efficiency of DNA recovery. The DNA extracted by this technique, have been found suitable for performing restriction enzyme digestion and PCR amplification. Therefore, the present DNA extraction procedure can be used as an alternative technique in population genetic studies pertaining to endangered fish species. The technique was also found equally effective for DNA isolation from fresh, dried and ethanol preserved scales.

Detection of pathogenic leptospires in animals by PCR based on lipL21 and lipL32 genes.

Efficacy of primers capable of amplifying conserved outer membrane protein (OMP) genes i.e., lipL21 and lipL32 of Leptospira strains was tested for rapid and early diagnosis of the leptospirosis using a polymerase chain reaction (PCR). These OMP genes were found to be conserved in various leptospiral serovars viz., Canicola, Pomona, Icterohaemorrhagiae, Pyrogenes, Sejroe, Grippotyphosa, Ballum and Tarassovi as PCR products of 561 bp and 756 bp were obtained by PCR employing lipL21 and lipL32 based primers, respectively, in all these serovars. Absence of such amplicons in DNA extracted from Pasteurella, Campylobacter and Brucella confirmed the specificity of the primers. Serum and tissue samples collected from cattle, buffaloes and experimentally infected guinea pigs and calves were subjected to PCR using above primers as well as conventionally used primers G1/G2. All the sera and tissue samples, whether field samples or collected from experimentally infected animals, found positive for G1/G2 specific PCR were also positive for lipL21 and lipL32 specific PCR. The present study indicated that lipL21 and lipL32 based primers could be used for PCR based diagnosis of leptospirosis. Since G1/G2 primers are known not to amplify the DNA of Grippotyphosa, the use of primers employed in the present study could have an additional advantage in detection of cases of the disease.

Immunoreactive outer membrane proteins of Leptospira interrogans serovar Canicola strain Hond Utrecht IV.

BACKGROUND & OBJECTIVES: Leptospirosis is a severe and complex zoonotic disease prevalent in many countries including India. Current leptospiral research is focussed on the identification of the outer membrane proteins (OMPs) of the organism that could be used in developing diagnostic assays for leptospirosis. METHODS: The Leptospira interrogans serovar Canicola was grown in EMJH medium and the cells were subjected to sarcosyl detergent treatment. The sarcosyl soluble (SS) and sarcosyl insoluble (SI) fractions were analyzed by SDS-PAGE and immunoblotting to deduce their protein profile and identifying various immunodominant antigens. RESULTS: The protein profile of SS fractions indicated the presence of three major bands of 41, 32 and 25 kDa and minor bands of 85 and 46 kDa. The SI fraction in serovar Canicola revealed the presence of 112, 93, 77, 43, 36, 29 and 22.5 kDa as major bands and minor bands of 102 and 53 kDa. In immunoblotting, the SS proteins of 41, 32 and 25 kDa and SI proteins of 112, 77, 36 and 22.5 kDa were detected to be major immunogenic proteins. INTERPRETATION & CONCLUSION: In our study immunogenic proteins were extracted from SS and SI fractions and OMPs were similar to those reported in other pathogenic Leptospira strains. These OMPs being unique to all the pathogenic leptospires, can be targeted for diagnostic purpose. Further analysis of the cellular location and expression of leptospiral proteins will be useful in the annotation of genomic sequence data and in providing insight into the biology of Leptospira cells.

Detection of Pasteurella multocida in experimentally infected embryonated chicken eggs by PCR assay.

Applicability of polymerase chain reaction (PCR) assay to detect Pasteurella multocida in experimentally infected embryonated chicken egg was assessed in the present study. PCR assay rapidly and specifically detected the genome of P. multocida in amniotic fluid, allantoic fluid and homogenates of infected embryo and its membranes. The sensitivity of detection was as low as 20 bacterial cells/ml of allantoic or amniotic fluids. Detection of P. multocida in dead embryos by PCR was possible up to 6 and 30 days or more following storage of dead embryos at 37 degrees C, and at 4 degrees C as well as at -20 degrees C, respectively. The study revealed that PCR assays could be employed directly for detection and confirmation of P. multocida infection in experimentally infected chicken embryos.

Cloning and sequencing of 28 kDa outer membrane protein gene of Brucella melitensis Rev. 1.

Brucella melitensis is an organism of paramount zoonotic importance. The 28 kDa outer membrane protein (OMP) is one of the immunodominant antigens of B. melitensis. The gene encoding 28 kDa OMP (omp28) has been amplified from B. melitensis Rev. 1 strain. A PCR product of 753 bp, encoding complete omp28 gene of B. melitensis, was obtained. The gene was further cloned and sequenced. The nucleotide sequence of B. melitensis Rev. 1 strain showed substitution of 2 nucleotides from that of 16M strain.

Heat modifiability of outer membrane protein of Pasteurella multocida serotype B:2.

Outer membrane proteins (OMP) are generally porins, functioning as molecular sieves assisting in the transmembrane transportation. Heat modifiable characteristics of OMP from P. multocida B: 2 have been explored to know their basic characteristics on event of temperature rise. A major band of 32 kDa and two minor bands of approximately 39 and approximately 28 kDa were found to be heat modifiable. It is suggested that boiling at 100 degrees C in presence of beta mercaptoethanol for 5 min is sufficient for characterisation of OMP by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis.

Inhibitory activity in buffalo follicular fluid and its elution pattern during different season.

Two pools of whole buffalo follicular fluid collected in winter (December, January) and spring (March, April) season were fractionated by Sephadex G-200 column chromatography. Follicular fluid collected in winter and spring eluted into different pattern resulted into four and three peaks respectively. Both the pools of follicular fluid were salted out with 18.5% ammonium sulphate. The salted out fraction of winter and spring season follicular fluid was again subjected to sephadex column chromatography which eluted into two and single peak respectively. Whole follicular fluid (0.6 ml) was administered into ovariectomized mice to see its inhibitory effect. The percentage of compensatory ovarian hypertrophy was 16.3 +/- 4.4 which was significantly different (P < 0.01) as compared to control. 200 micrograms material from peak 1 and peak 2 obtained after fractionation of salted out winter follicular fluid also had inhibitory effect on compensatory ovarian hypertrophy as compared to control group. It was 35.6 +/- 9.3 and 15.9 +/- 4.3% respectively. Thus, the variation in nature of buffalo follicular fluid and its inhibitory effect in ovariectomised mice have significant relation with anoestrus condition in summer and humid months in this species.

Stiff man's syndrome.

Myositis ossificans progressiva is an autosomal dominant disease resulting in progressive ossification and skeletal deformities, mainly in the connective tissue of muscle. The diagnosis is based on clinical and radiological findings and demonstration of skeletal malformations. We report a singular case of advanced myositis ossificans progressiva in a fourteen year old male and analyse the natural history, etiopathology, treatment alternatives and prognosis of this crippling disorder.

Outer membrane protein of Pasteurella multocida serotype B:2 is immunogenic and antiphagocytic.

Outer membrane protein (OMP) from Pasteurella multocida serotype B:2 was extracted and studied for its ability to immunize animals against P. multocida infection and to resist phagocytosis by murine peritoneal macrophage. Inoculation of OMP in rabbits resulted in the production of agglutinating antibodies which passively protected mice against P. multocida challenge and caused lysis of virulent P. multocida cells in vitro. Mice vaccinated with OMP vaccine resisted the challenge showing a satisfactory survival rate (67%) similar to mice given commercial whole cell vaccine (84%). The OMP was also found to be antiphagocytic, interfering with the phagocytosis of opsonized Candida albicans by murine peritoneal cells in vivo. The study suggested the role of OMP in conferring protection in animals against P. multocida infection and enhancing the virulence in infected animals through the anti-phagocytic mechanism.