Research Interest: Plant microbe insect interaction, DNA Fingerprinting and microbial diversity, EnzymologyReviewer: Biosciences, Biotechnology Research AsiaTechnical Skills: I got expertise in PCR, DGGE, TRFLP, Electrophoresis, Gel X-ray Contact Print method, UV-Vis spectrophotometer, I am also aware with DNA Sequencing
H. armigera is one of the constraints known to reduce yield of economically important crops, whic... more H. armigera is one of the constraints known to reduce yield of economically important crops, which harbor rich bacterial diversity in the gut. Hence present work deals with the microbial community study from gut of H. armigera when shifted from one to another hosts. By using PCR 16S rDNA was amplified and analyzed by denaturing gradient gel electrophoresis (DGGE). The resulting PCR-DGGE band number was counted, and the banding patterns were analyzed by calculating the Jaccard pairwise similarity coefficients (Cs). Shannon indices showed highest diversity when larvae fed on pigeonpea. But the shifting from pigeon pea to other host resulted in to the decrease in the diversity. On the contrary diversity was found to be increased when larvae shifted from other hosts to pigeonpea. This study concludes that shift of the larvae from one host to another result into the change in gut microbial diversity. Detail understanding of this change in diversity may help to design rational strategy fo...
Exploration of Amylases Producing Competency of Helicoverpa armigera Gut Bacterial Strain, Bacillus subtilis RTSBA6 6.00, 2019
The Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) is a polyphagous insect pest of agricu... more The Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) is a polyphagous insect pest of agriculturally important crops. The alkaline gut of this insect pest possesses diverse bacterial communities which may assist in digestive physiology. As part our investigations of understanding the role of gut bacterial communities in insect gut, here amylase producing competency of earlier identified H. armigera gut bacterial strain, i.e., Bacillus subtilis RTSBA6 6.00 is reported. Initial screening for amylase activity was assessed by starch agar plate. Upon 7% sodium dodecyl sulfate polyacrylamide gel electrophoresis amylase zymography, bacterial culture supernatant produced seven amylase bands on the gel. The observed molecular weights of amylases were 191.2 KDa, 158.0 KDa, 131.7 KDa, 54.0 KDa, 31.3 KDa, 67.2 KDa, and 44.6 KDa, respectively. Considerable amylase activity was observed in neutral to alkaline pH with optimum at pH 6.8. The optimal activity temperature of amylases was found to be 50°C, and the activity decreased dramatically at temperatures above 75°C.
The Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) is a polyphagous insect pest of agricu... more The Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) is a polyphagous insect pest of agriculturally important crops. The alkaline gut of this insect pest possesses diverse bacterial communities which may assist in digestive physiology. As part our investigations of understanding the role of gut bacterial communities in insect gut, here amylase producing competency of earlier identified H. armigera gut bacterial strain, i.e., Bacillus subtilis RTSBA6 6.00 is reported. Initial screening for amylase activity was assessed by starch agar plate. Upon 7% sodium dodecyl sulfate polyacrylamide gel electrophoresis amylase zymography, bacterial culture supernatant produced seven amylase bands on the gel. The observed molecular weights of amylases were 191.2 KDa, 158.0 KDa, 131.7 KDa, 54.0 KDa, 31.3 KDa, 67.2 KDa, and 44.6 KDa, respectively. Considerable amylase activity was observed in neutral to alkaline pH with optimum at pH 6.8. The optimal activity temperature of amylases was found t...
Protease inhibitors (PIs) are deployed in the plant kingdom as storage proteins or peptides, regu... more Protease inhibitors (PIs) are deployed in the plant kingdom as storage proteins or peptides, regulators of endogenous proteases, and plant protection agents against insect pests and pathogen attack. In humans, they are identified as chemopreventive agents against a range of cancers and have potential as drug to treat an array of disease associated with aberrant activity of proteases. The present investigation reports PIs activity data from 30 medicinal plants. The screening for PIs activity was done by dot blot assay using X-ray film coated with gelatin. Among screened seed extracts, Albizia lebbeck, Raphanus sativus, Mucuna pruriens, Achyranthes aspera, and Coffea arabica showed high inhibitory activities with trypsin protease. Most of seed extracts exhibited moderate activity, whereas Ocimum sanctum showed moderate to low activity against trypsin. The presence of varied protein content is reported from all seed extracts with highest in A. lebbeck (50.0 ± 3.4 mg/ml). The data produ...
ABSTRACT Proteinaceous inhibitors of digestive a-amylase occur naturally in leguminous seeds and ... more ABSTRACT Proteinaceous inhibitors of digestive a-amylase occur naturally in leguminous seeds and find applications in agriculture and clinical studies. We have detected and isolated eight novel a-amylase inhibitor isoforms in the seed extract of Albizia lebbeck. They are designated as ALaAI- 1 to AL-aAI-8. These isoforms specifically inhibit human salivary a-amylase and porcine pancreatic a-amylase. The occurrence and profile of a-amylase inhibitor isoforms were revealed by 7 % native-PAGE containing 0.1 % starch. The apparent molecular weights of native bands of AL-aAIs were 97.4, 68.6, 61.0, 57.2, 56.0, 54.7, 51.1, and 47.7 kDa, respectively. Partial purification of potent a-amylase inhibitor was achieved using ammonium sulfate fractionation and gel filtration chromatography on G-100 Sephadex column followed by preparative gel electrophoresis. SDS-PAGE analysis of partially purified AL-aAI showed two polypeptide bands of *35.8 and *32.6 kDa. All these isoforms showed effective resistance to in vitro proteolysis by pepsin, trypsin, and chymotrypsin. These inhibitors are stable over a wide range of pH and temperature and have optimum activity at pH 7 and at 37 �C. The finding and information obtained in the present investigation about novel isoforms of a-amylase inhibitors from A. lebbeck could be important and may find applications in clinical studies to modulate starch digestion and glycemic index.
ABSTRACT Here, we report multiple molecular forms of Albizia lebbeck trypsin inhibitors (AlTIs) b... more ABSTRACT Here, we report multiple molecular forms of Albizia lebbeck trypsin inhibitors (AlTIs) by using a simple and sensitive gel X-ray film contact print technique. About 17 AlTIs were detected in the seed extracts of A. lebbeck. Two groups of AlTIs-1 major (10 AlTIs; slow migration on the gel) and 1 minor (7 AlTIs; fast migration on the gel) were identified. The former was specific only toward trypsin. However, the latter was specific toward both trypsin and Helicoverpa armigera gut proteinases (HaGPs). The most potent AlTI (AlTI13) was purified to assess its in vivo bioefficacy toward HaGPs. Purification was achieved using (NH4)(2)SO4 fractionation, Sephadex G-100 column chromatography, and preparative native-polyacrylamide gel electrophoresis (PAGE). The dose dependent bioefficacies of AlTIs in the (NH4)(2)SO4 F-3 fractions (0.1%, 0.5%, and 1%) were approximately 79%, 83%, and 90%, respectively, resulting in reductions in the average larval weight of H. armigera. Artificial diet containing a single dose of AlTI13 (5 mu g/g diet) reduced the larval weight by about 76%, with 60% mortality. The half-maximal inhibitory concentrations (IC50) of AlTI13 for trypsin and HaGPs were 0.14 and 0.17 mu mol/ml, respectively. The optimum conditions for AlTI13 were pH 8 and temperatures ranging from 35 to 40 C. Reducing sodium dodecyl sulfate-PAGE analysis indicated that similar to 28 kDa Kunitz-like trypsin inhibitor was present. Thus, we showed that particularly, AlTI13 of A. lebbeck could be used as a transgene macromolecule to markedly increase insect resistance in genetically engineered plants.
H. armigera is one of the constraints known to reduce yield of economically important crops, whic... more H. armigera is one of the constraints known to reduce yield of economically important crops, which harbor rich bacterial diversity in the gut. Hence present work deals with the microbial community study from gut of H. armigera when shifted from one to another hosts. By using PCR 16S rDNA was amplified and analyzed by denaturing gradient gel electrophoresis (DGGE). The resulting PCR-DGGE band number was counted, and the banding patterns were analyzed by calculating the Jaccard pairwise similarity coefficients (Cs). Shannon indices showed highest diversity when larvae fed on pigeonpea. But the shifting from pigeon pea to other host resulted in to the decrease in the diversity. On the contrary diversity was found to be increased when larvae shifted from other hosts to pigeonpea. This study concludes that shift of the larvae from one host to another result into the change in gut microbial diversity. Detail understanding of this change in diversity may help to design rational strategy fo...
Exploration of Amylases Producing Competency of Helicoverpa armigera Gut Bacterial Strain, Bacillus subtilis RTSBA6 6.00, 2019
The Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) is a polyphagous insect pest of agricu... more The Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) is a polyphagous insect pest of agriculturally important crops. The alkaline gut of this insect pest possesses diverse bacterial communities which may assist in digestive physiology. As part our investigations of understanding the role of gut bacterial communities in insect gut, here amylase producing competency of earlier identified H. armigera gut bacterial strain, i.e., Bacillus subtilis RTSBA6 6.00 is reported. Initial screening for amylase activity was assessed by starch agar plate. Upon 7% sodium dodecyl sulfate polyacrylamide gel electrophoresis amylase zymography, bacterial culture supernatant produced seven amylase bands on the gel. The observed molecular weights of amylases were 191.2 KDa, 158.0 KDa, 131.7 KDa, 54.0 KDa, 31.3 KDa, 67.2 KDa, and 44.6 KDa, respectively. Considerable amylase activity was observed in neutral to alkaline pH with optimum at pH 6.8. The optimal activity temperature of amylases was found to be 50°C, and the activity decreased dramatically at temperatures above 75°C.
The Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) is a polyphagous insect pest of agricu... more The Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) is a polyphagous insect pest of agriculturally important crops. The alkaline gut of this insect pest possesses diverse bacterial communities which may assist in digestive physiology. As part our investigations of understanding the role of gut bacterial communities in insect gut, here amylase producing competency of earlier identified H. armigera gut bacterial strain, i.e., Bacillus subtilis RTSBA6 6.00 is reported. Initial screening for amylase activity was assessed by starch agar plate. Upon 7% sodium dodecyl sulfate polyacrylamide gel electrophoresis amylase zymography, bacterial culture supernatant produced seven amylase bands on the gel. The observed molecular weights of amylases were 191.2 KDa, 158.0 KDa, 131.7 KDa, 54.0 KDa, 31.3 KDa, 67.2 KDa, and 44.6 KDa, respectively. Considerable amylase activity was observed in neutral to alkaline pH with optimum at pH 6.8. The optimal activity temperature of amylases was found t...
Protease inhibitors (PIs) are deployed in the plant kingdom as storage proteins or peptides, regu... more Protease inhibitors (PIs) are deployed in the plant kingdom as storage proteins or peptides, regulators of endogenous proteases, and plant protection agents against insect pests and pathogen attack. In humans, they are identified as chemopreventive agents against a range of cancers and have potential as drug to treat an array of disease associated with aberrant activity of proteases. The present investigation reports PIs activity data from 30 medicinal plants. The screening for PIs activity was done by dot blot assay using X-ray film coated with gelatin. Among screened seed extracts, Albizia lebbeck, Raphanus sativus, Mucuna pruriens, Achyranthes aspera, and Coffea arabica showed high inhibitory activities with trypsin protease. Most of seed extracts exhibited moderate activity, whereas Ocimum sanctum showed moderate to low activity against trypsin. The presence of varied protein content is reported from all seed extracts with highest in A. lebbeck (50.0 ± 3.4 mg/ml). The data produ...
ABSTRACT Proteinaceous inhibitors of digestive a-amylase occur naturally in leguminous seeds and ... more ABSTRACT Proteinaceous inhibitors of digestive a-amylase occur naturally in leguminous seeds and find applications in agriculture and clinical studies. We have detected and isolated eight novel a-amylase inhibitor isoforms in the seed extract of Albizia lebbeck. They are designated as ALaAI- 1 to AL-aAI-8. These isoforms specifically inhibit human salivary a-amylase and porcine pancreatic a-amylase. The occurrence and profile of a-amylase inhibitor isoforms were revealed by 7 % native-PAGE containing 0.1 % starch. The apparent molecular weights of native bands of AL-aAIs were 97.4, 68.6, 61.0, 57.2, 56.0, 54.7, 51.1, and 47.7 kDa, respectively. Partial purification of potent a-amylase inhibitor was achieved using ammonium sulfate fractionation and gel filtration chromatography on G-100 Sephadex column followed by preparative gel electrophoresis. SDS-PAGE analysis of partially purified AL-aAI showed two polypeptide bands of *35.8 and *32.6 kDa. All these isoforms showed effective resistance to in vitro proteolysis by pepsin, trypsin, and chymotrypsin. These inhibitors are stable over a wide range of pH and temperature and have optimum activity at pH 7 and at 37 �C. The finding and information obtained in the present investigation about novel isoforms of a-amylase inhibitors from A. lebbeck could be important and may find applications in clinical studies to modulate starch digestion and glycemic index.
ABSTRACT Here, we report multiple molecular forms of Albizia lebbeck trypsin inhibitors (AlTIs) b... more ABSTRACT Here, we report multiple molecular forms of Albizia lebbeck trypsin inhibitors (AlTIs) by using a simple and sensitive gel X-ray film contact print technique. About 17 AlTIs were detected in the seed extracts of A. lebbeck. Two groups of AlTIs-1 major (10 AlTIs; slow migration on the gel) and 1 minor (7 AlTIs; fast migration on the gel) were identified. The former was specific only toward trypsin. However, the latter was specific toward both trypsin and Helicoverpa armigera gut proteinases (HaGPs). The most potent AlTI (AlTI13) was purified to assess its in vivo bioefficacy toward HaGPs. Purification was achieved using (NH4)(2)SO4 fractionation, Sephadex G-100 column chromatography, and preparative native-polyacrylamide gel electrophoresis (PAGE). The dose dependent bioefficacies of AlTIs in the (NH4)(2)SO4 F-3 fractions (0.1%, 0.5%, and 1%) were approximately 79%, 83%, and 90%, respectively, resulting in reductions in the average larval weight of H. armigera. Artificial diet containing a single dose of AlTI13 (5 mu g/g diet) reduced the larval weight by about 76%, with 60% mortality. The half-maximal inhibitory concentrations (IC50) of AlTI13 for trypsin and HaGPs were 0.14 and 0.17 mu mol/ml, respectively. The optimum conditions for AlTI13 were pH 8 and temperatures ranging from 35 to 40 C. Reducing sodium dodecyl sulfate-PAGE analysis indicated that similar to 28 kDa Kunitz-like trypsin inhibitor was present. Thus, we showed that particularly, AlTI13 of A. lebbeck could be used as a transgene macromolecule to markedly increase insect resistance in genetically engineered plants.
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