Prafull Gadge - Academia.edu (original) (raw)
Papers by Prafull Gadge
American Journal of Biochemistry and Biotechnology, 2011
Problem statement: Lipase is one of the important enzymes in food, pharmaceutical, detergent and ... more Problem statement: Lipase is one of the important enzymes in food, pharmaceutical, detergent and biofuels industries. Search for the lipase with distinct features, possibly from germinating seeds, is of interest for industrial applications. Approach: The lipase produced by soybean oil seeds was partially purified and characterized in terms of the optimal pH and temperature for activity as well as substrate specificity. Results: The lipase was extracted and partially purified from germinating soybean seeds using chilled acetone and ammonium sulfate precipitation. Partially purified and dialyzed enzyme profile was observed on native-Polyacrylamide Gel Electrophoresis (PAGE). The lipase was optimally active at pH 8 and temperature of 24°C. In the presence of Ca 2+ and Mg 2+ enhance the activity at low concentration, while the Hg 2+ and Ethylene Diaminotetracetic Acid (EDTA) showed inhibitory effect. The enzyme was found to be metalloenzyme. Enzyme kinetics with olive oil emulsion substrate showed k m and v max of 7.67 mg and 0.0125 µm mL min −1 , respectively. Conclusion: The mettaloenzyme enzyme was able to attack specifically on oil in seeds to generate free fatty acids as the major end product. This understanding may help in devising efficient methods to overcome the problem of soybean seed oil in stability.
Cihan University-Erbil scientific journal, Aug 1, 2022
Proteases are one of the most widely used industrial enzymes. A thermo stable, extracellular, alk... more Proteases are one of the most widely used industrial enzymes. A thermo stable, extracellular, alkaline protease was isolated from bacteria. Bacterial protease was purified by using ammonium sulphate precipitation, membrane filtration, DEAE cellulose and Sephadex G-200 chromatography. Protease activity was assayed using the casein as a substrate. The molecular weight of purified protease was estimated approximately 45kDa using casein 12% SDS PAGE Zymography. The optimum temperature and pH of this enzyme was 45 0 C and 10 respectively. Purified enzyme was found detergent compatible.
![Research paper thumbnail of {"__content__"=>"Subtilisin inhibitor like protein 'LPI-1' from leaves of pigeonpea (, cv. BSMR 736) exhibits inhibition againstgut proteinases.", "i"=>[{"__content__"=>"pp"}, {"__content__"=>"Cajanus cajan"}, {"__content__"=>"Helicoverpa armigera"}]}](https://attachments.academia-assets.com/94492459/thumbnails/1.jpg)
](3 Biotech, 2018
is an orthodox rival of many crop plants affecting agricultural economy. Plant leaves found to ac... more is an orthodox rival of many crop plants affecting agricultural economy. Plant leaves found to accumulate proteinase inhibitors, although this insect pest chooses leaves for laying eggs. Plant defense response at this juncture is not fully explored. In this context, here we are reporting proteinase inhibitor (LPI-1) having significant homology with the I13 family from leaves of pigeonpea (cv. BSMR 736). The isolation ofLPI-1 was carried out from leaves of field-grown pigeonpea under an outbreak of. The acetone precipitatedLPI-1 (125 µg) displayed substantial inhibition potential towards bovine trypsin (56.5 ± 1.8%) and HaGPs (52.6 ± 1.7%) on solution assay. These results were corroborated with dot-blot analysis. The molecular form ofLPI-1 was characterized by reverse zymography and GXCP. The optimum condition was found to be pH 8 and temperature in the range of 30-40 °C. The protein identification via MASCOT-PMF and NCBI-BLAST search showed substantial homology with an inducible sub...
Probiotics and Antimicrobial Proteins, 2017
Peptidase therapy is suggested to be effective to minimize gliadin toxicity in celiac disease (CD... more Peptidase therapy is suggested to be effective to minimize gliadin toxicity in celiac disease (CD). Hence, present study deals with gliadin-hydrolysing peptidases. The efficient peptidase from the Bacillus tequilensis was purified using ammonium sulfate fractionation and preparative electrophoresis. Analysis of in-solution and in-gel hydrolysis of gliadin using one and two-dimensional SDS-PAGE revealed nearly complete hydrolysis of gliadin peptides after 180 min of incubation with B. tequilensis protease. Purified peptidase was found to be stable at acidic (pH 3.5) to neutral (pH 7.2) pH range. The molecular mass and isoelectric point of the peptidase were observed around 29 kDa and 5.2, respectively. The internal protein sequence obtained through mass spectrometric analysis suggested that peptidase might belong to peptidase S9 family known for prolyl-specific peptidases. This study recommends the possible applicability of this peptidase for elimination of immunotoxic gliadin peptides and may prove useful in CD treatment.
Sugarcane woolly aphid, Ceratovacuna lanigera is one of the important pests of the sugarcane whic... more Sugarcane woolly aphid, Ceratovacuna lanigera is one of the important pests of the sugarcane which is responsible for the low production of the sugarcane in India. The incidences of pest itself suggest importance of study on this pest. Carbohydrate metabolizing digestive enzyme, α-amylase was studied from this insect. Biochemical characterization of this enzyme and screening of natural inhibitors will prove vital strategies against infestation. As initial step to this goal here we report the detection of α-amylase of Ceratovacuna lanigera by starch agar well diffusion assay. Biochemical parameters like pH, temperature and effect of substrate concentration were studied and compared with α-amylases from other sources. The optimum pH of α-amylase of Ceratovacuna lanigera was found to be pH 8 in contrast to human salivary α-amylases and fungal α-amylases those shows optimum activity at pH 7 and pH 6 respectively. Maximum activity of α-amylase was found to be at 40°C. Protein profile by Native-PAGE showed banding pattern with varied intensities. The α-amylase of Ceratovacuna lanigera detected in the present study needs further exploration for its use in better management of sugarcane woolly aphid.
Pesticide Biochemistry and Physiology, 2015
This paper evaluates α-amylase inhibitor (α-AI) mediated defense of pigeonpea against Helicoverpa... more This paper evaluates α-amylase inhibitor (α-AI) mediated defense of pigeonpea against Helicoverpa armigera. A bifunctional α-amylase/trypsin inhibitor was purified from the seeds of pigeonpea by native liquid phase isoelectric focusing (N-LP-IEF), affinity chromatography and preparative electrophoresis. Its in-vivo and in-vitro interaction with midgut amylases of H. armigera was studied along with growth inhibitory activity. One and two dimensional (2D) zymographic analyses revealed that the purified inhibitor is dimeric glycoprotein (60.2kDa and 56kDa) exist in a multi-isomeric form with five pI variants (pI 5.5 to 6.3). It was found to be heat labile with complete inactivation up to 80°C and stable over a wide range of pH (4-11). The slow binding and competitive type of α-amylase inhibition was observed with 0.08μM of dissociation constant (Ki) for the enzyme-inhibitor complex (EI). The internal protein sequence of two subunits obtained by mass spectrometry matched with cereal-type α-AI, a conserved domain from AAI_LTSS superfamily and sialyltransferase-like protein respectively. In-vivo studies indicated up-regulation of total midgut α-amylase activity with negative effect on growth rate of H. armigera suggesting its suitability for pest control.
Journal of Asia-Pacific Entomology, 2014
Here, we report multiple molecular forms of Albizia lebbeck trypsin inhibitors (AlTIs) by using a... more 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 (AlTI 13) was purified to assess its in vivo bioefficacy toward HaGPs. Purification was achieved using (NH 4) 2 SO 4 fractionation, Sephadex G-100 column chromatography, and preparative native-polyacrylamide gel electrophoresis (PAGE). The dose dependent bioefficacies of AlTIs in the (NH 4) 2 SO 4 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 AlTI 13 (5 μg/g diet) reduced the larval weight by about 76%, with 60% mortality. The half-maximal inhibitory concentrations (IC 50) of AlTI 13 for trypsin and HaGPs were 0.14 and 0.17 μmol/ml, respectively. The optimum conditions for AlTI 13 were pH 8 and temperatures ranging from 35 to 40°C. Reducing sodium dodecyl sulfate-PAGE analysis indicated that~28 kDa Kunitz-like trypsin inhibitor was present. Thus, we showed that AlTIs, particularly, AlTI 13 of A. lebbeck could be used as a transgene macromolecule to markedly increase insect resistance in genetically engineered plants.
Acta Physiologiae Plantarum, 2012
Proteinaceous inhibitors of digestive a-amylase occur naturally in leguminous seeds and find appl... more 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 AL-aAI-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. Keywords Proteinaceous a-amylase inhibitor Á Albizia lebbeck Á Human salivary a-amylase Á Porcine pancreatic a-amylase Abbreviations a-AI a-Amylase inhibitor AL-aAI Albizia lebbeck a-amylase inhibitor BPB Bromophenol blue BSA Bovine serum albumin DNSA 3,5-Dinitrosalicylic acid HSA Human salivary a-amylase PAGE Polyacrylamide gel electrophoresis PPA Porcine pancreatic a-amylase PVP Polyvinylpyrrolidone Communicated by M. Stobiecki.
Journal of the Saudi Society of Agricultural Sciences, 2017
Sugarcane woolly aphid, Ceratovacuna lanigera is one of the important pests of the sugarcane whic... more Sugarcane woolly aphid, Ceratovacuna lanigera is one of the important pests of the sugarcane which is responsible for the low production of the sugarcane in India. The incidences of pest itself suggest importance of study on this pest. Carbohydrate metabolizing digestive enzyme, α-amylase was studied from this insect. Biochemical characterization of this enzyme and screening of natural inhibitors will prove vital strategies against infestation. As initial step to this goal here we report the detection of α-amylase of Ceratovacuna lanigera by starch agar well diffusion assay. Biochemical parameters like pH, temperature and effect of substrate concentration were studied and compared with α-amylases from other sources. The optimum pH of α-amylase of Ceratovacuna lanigera was found to be pH 8 in contrast to human salivary α-amylases and fungal α-amylases those shows optimum activity at pH 7 and pH 6 respectively. Maximum activity of α-amylase was found to be at 40°C. Protein profile by Native -PAGE showed banding pattern with varied intensities. The α-amylase of Ceratovacuna lanigera detected in the present study needs further exploration for its use in better management of sugarcane woolly aphid. 50 0.2 ml enzyme. After incubation at 37°C for 30 min, the liberated reducing sugars were estimated using DNSA (1% 3, 5-Dinitrosalisylic acid, 30% Sodium potassium tartarate, 0.2M NaoH) reagent. One unit of enzyme activity was defined as the quantity of enzyme producing 1μM reducing sugar (maltose) per min at defined assay condition.
Proteinaceous inhibitors of digestive a-amylase occur naturally in leguminous seeds and find appl... more 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 AL-aAI-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.
This paper evaluates α-amylase inhibitor (α-AI) mediated defense of pigeonpea against Helicoverpa... more This paper evaluates α-amylase inhibitor (α-AI) mediated defense of pigeonpea against Helicoverpa armigera. A bifunctional α-amylase/trypsin inhibitor was purified from the seeds of pigeonpea by native liquid phase isoelectric focusing (N-LP-IEF), affinity chromatography and preparative electrophoresis. Its in-vivo and in-vitro interaction with midgut amylases of H. armigera was studied along with growth inhibitory activity. One and two dimensional (2D) zymographic analyses revealed that the purified inhibitor is dimeric glycoprotein (60.2 kDa and 56 kDa) exist in a multi-isomeric form with five pI variants (pI 5.5 to 6.3). It was found to be heat labile with complete inactivation up to 80 °C and stable over a wide range of pH (4–11). The slow binding and competitive type of α-amylase inhibition was observed with 0.08 μM of dissociation constant (Ki) for the enzyme–inhibitor complex (EI). The internal protein sequence of two subunits obtained by mass spectrometry matched with cereal-type α-AI, a conserved domain from AAI_LTSS superfamily and sialyltransferase-like protein respectively. In-vivo studies indicated up-regulation of total midgut α-amylase activity with negative effect on growth rate of H. armigera suggesting its suitability for pest control.
Here, we report multiple molecular forms of Albizia lebbeck trypsin inhibitors (AlTIs) by using a... more 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)2SO4 fractionation, Sephadex G-100 column chromatography, and preparative native-polyacrylamide gel electrophoresis (PAGE). The dose dependent bioefficacies of AlTIs in the (NH4)2SO4 F3 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 μg/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 μmol/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 ~ 28 kDa Kunitz-like trypsin inhibitor was present. Thus, we showed that AlTIs, particularly, AlTI13 of A. lebbeck could be used as a transgene macromolecule to markedly increase insect resistance in genetically engineered plants.
Proteinaceous inhibitors of digestive α-amylase occur naturally in leguminous seeds and find appl... more Proteinaceous inhibitors of digestive α-amylase occur naturally in leguminous seeds and find applications in agriculture and clinical studies. We have detected and isolated eight novel α-amylase inhibitor isoforms in the seed extract of Albizia lebbeck. They are designated as AL-αAI-1 to AL-αAI-8. These isoforms specifically inhibit human salivary α-amylase and porcine pancreatic α-amylase. The occurrence and profile of α-amylase inhibitor isoforms were revealed by 7 % native-PAGE containing 0.1 % starch. The apparent molecular weights of native bands of AL-αAIs were 97.4, 68.6, 61.0, 57.2, 56.0, 54.7, 51.1, and 47.7 kDa, respectively. Partial purification of potent α-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-αAI 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 α-amylase inhibitors from A. lebbeck could be important and may find applications in clinical studies to modulate starch digestion and glycemic index.
Lipase is one of the important enzymes in food, pharmaceutical, detergent and biofuels industrie... more Lipase is one of the important enzymes in food, pharmaceutical, detergent and biofuels industries. Search for the lipase with distinct features, possibly from germinating seeds, is of interest for industrial applications. Approach: The lipase produced by soybean oil seeds was partially purified and characterized in terms of the optimal pH and temperature for activity as well as substrate specificity. Results: The lipase was extracted and partially purified from germinating soybean seeds using chilled acetone and ammonium sulfate precipitation. Partially purified and dialyzed enzyme profile was observed on native-Polyacrylamide Gel Electrophoresis (PAGE). The lipase was optimally active at pH 8 and temperature of 24°C. In the presence of Ca2+ and Mg2+ enhance the activity at low concentration, while the Hg2+ and Ethylene Diaminotetracetic Acid (EDTA) showed inhibitory effect. The enzyme was found to be metalloenzyme. Enzyme kinetics with olive oil emulsion substrate showed km and vmax of 7.67 mg and 0.0125 µm mL min-1, respectively. Conclusion: The mettaloenzyme enzyme was able to attack specifically on oil in seeds to generate free fatty acids as the major end product. This understanding may help in devising efficient methods to overcome the problem of soybean seed oil in stability.
American Journal of Biochemistry and Biotechnology, 2011
Problem statement: Lipase is one of the important enzymes in food, pharmaceutical, detergent and ... more Problem statement: Lipase is one of the important enzymes in food, pharmaceutical, detergent and biofuels industries. Search for the lipase with distinct features, possibly from germinating seeds, is of interest for industrial applications. Approach: The lipase produced by soybean oil seeds was partially purified and characterized in terms of the optimal pH and temperature for activity as well as substrate specificity. Results: The lipase was extracted and partially purified from germinating soybean seeds using chilled acetone and ammonium sulfate precipitation. Partially purified and dialyzed enzyme profile was observed on native-Polyacrylamide Gel Electrophoresis (PAGE). The lipase was optimally active at pH 8 and temperature of 24°C. In the presence of Ca 2+ and Mg 2+ enhance the activity at low concentration, while the Hg 2+ and Ethylene Diaminotetracetic Acid (EDTA) showed inhibitory effect. The enzyme was found to be metalloenzyme. Enzyme kinetics with olive oil emulsion substrate showed k m and v max of 7.67 mg and 0.0125 µm mL min −1 , respectively. Conclusion: The mettaloenzyme enzyme was able to attack specifically on oil in seeds to generate free fatty acids as the major end product. This understanding may help in devising efficient methods to overcome the problem of soybean seed oil in stability.
Cihan University-Erbil scientific journal, Aug 1, 2022
Proteases are one of the most widely used industrial enzymes. A thermo stable, extracellular, alk... more Proteases are one of the most widely used industrial enzymes. A thermo stable, extracellular, alkaline protease was isolated from bacteria. Bacterial protease was purified by using ammonium sulphate precipitation, membrane filtration, DEAE cellulose and Sephadex G-200 chromatography. Protease activity was assayed using the casein as a substrate. The molecular weight of purified protease was estimated approximately 45kDa using casein 12% SDS PAGE Zymography. The optimum temperature and pH of this enzyme was 45 0 C and 10 respectively. Purified enzyme was found detergent compatible.
3 Biotech, 2018
is an orthodox rival of many crop plants affecting agricultural economy. Plant leaves found to ac... more is an orthodox rival of many crop plants affecting agricultural economy. Plant leaves found to accumulate proteinase inhibitors, although this insect pest chooses leaves for laying eggs. Plant defense response at this juncture is not fully explored. In this context, here we are reporting proteinase inhibitor (LPI-1) having significant homology with the I13 family from leaves of pigeonpea (cv. BSMR 736). The isolation ofLPI-1 was carried out from leaves of field-grown pigeonpea under an outbreak of. The acetone precipitatedLPI-1 (125 µg) displayed substantial inhibition potential towards bovine trypsin (56.5 ± 1.8%) and HaGPs (52.6 ± 1.7%) on solution assay. These results were corroborated with dot-blot analysis. The molecular form ofLPI-1 was characterized by reverse zymography and GXCP. The optimum condition was found to be pH 8 and temperature in the range of 30-40 °C. The protein identification via MASCOT-PMF and NCBI-BLAST search showed substantial homology with an inducible sub...
Probiotics and Antimicrobial Proteins, 2017
Peptidase therapy is suggested to be effective to minimize gliadin toxicity in celiac disease (CD... more Peptidase therapy is suggested to be effective to minimize gliadin toxicity in celiac disease (CD). Hence, present study deals with gliadin-hydrolysing peptidases. The efficient peptidase from the Bacillus tequilensis was purified using ammonium sulfate fractionation and preparative electrophoresis. Analysis of in-solution and in-gel hydrolysis of gliadin using one and two-dimensional SDS-PAGE revealed nearly complete hydrolysis of gliadin peptides after 180 min of incubation with B. tequilensis protease. Purified peptidase was found to be stable at acidic (pH 3.5) to neutral (pH 7.2) pH range. The molecular mass and isoelectric point of the peptidase were observed around 29 kDa and 5.2, respectively. The internal protein sequence obtained through mass spectrometric analysis suggested that peptidase might belong to peptidase S9 family known for prolyl-specific peptidases. This study recommends the possible applicability of this peptidase for elimination of immunotoxic gliadin peptides and may prove useful in CD treatment.
Sugarcane woolly aphid, Ceratovacuna lanigera is one of the important pests of the sugarcane whic... more Sugarcane woolly aphid, Ceratovacuna lanigera is one of the important pests of the sugarcane which is responsible for the low production of the sugarcane in India. The incidences of pest itself suggest importance of study on this pest. Carbohydrate metabolizing digestive enzyme, α-amylase was studied from this insect. Biochemical characterization of this enzyme and screening of natural inhibitors will prove vital strategies against infestation. As initial step to this goal here we report the detection of α-amylase of Ceratovacuna lanigera by starch agar well diffusion assay. Biochemical parameters like pH, temperature and effect of substrate concentration were studied and compared with α-amylases from other sources. The optimum pH of α-amylase of Ceratovacuna lanigera was found to be pH 8 in contrast to human salivary α-amylases and fungal α-amylases those shows optimum activity at pH 7 and pH 6 respectively. Maximum activity of α-amylase was found to be at 40°C. Protein profile by Native-PAGE showed banding pattern with varied intensities. The α-amylase of Ceratovacuna lanigera detected in the present study needs further exploration for its use in better management of sugarcane woolly aphid.
Pesticide Biochemistry and Physiology, 2015
This paper evaluates α-amylase inhibitor (α-AI) mediated defense of pigeonpea against Helicoverpa... more This paper evaluates α-amylase inhibitor (α-AI) mediated defense of pigeonpea against Helicoverpa armigera. A bifunctional α-amylase/trypsin inhibitor was purified from the seeds of pigeonpea by native liquid phase isoelectric focusing (N-LP-IEF), affinity chromatography and preparative electrophoresis. Its in-vivo and in-vitro interaction with midgut amylases of H. armigera was studied along with growth inhibitory activity. One and two dimensional (2D) zymographic analyses revealed that the purified inhibitor is dimeric glycoprotein (60.2kDa and 56kDa) exist in a multi-isomeric form with five pI variants (pI 5.5 to 6.3). It was found to be heat labile with complete inactivation up to 80°C and stable over a wide range of pH (4-11). The slow binding and competitive type of α-amylase inhibition was observed with 0.08μM of dissociation constant (Ki) for the enzyme-inhibitor complex (EI). The internal protein sequence of two subunits obtained by mass spectrometry matched with cereal-type α-AI, a conserved domain from AAI_LTSS superfamily and sialyltransferase-like protein respectively. In-vivo studies indicated up-regulation of total midgut α-amylase activity with negative effect on growth rate of H. armigera suggesting its suitability for pest control.
Journal of Asia-Pacific Entomology, 2014
Here, we report multiple molecular forms of Albizia lebbeck trypsin inhibitors (AlTIs) by using a... more 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 (AlTI 13) was purified to assess its in vivo bioefficacy toward HaGPs. Purification was achieved using (NH 4) 2 SO 4 fractionation, Sephadex G-100 column chromatography, and preparative native-polyacrylamide gel electrophoresis (PAGE). The dose dependent bioefficacies of AlTIs in the (NH 4) 2 SO 4 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 AlTI 13 (5 μg/g diet) reduced the larval weight by about 76%, with 60% mortality. The half-maximal inhibitory concentrations (IC 50) of AlTI 13 for trypsin and HaGPs were 0.14 and 0.17 μmol/ml, respectively. The optimum conditions for AlTI 13 were pH 8 and temperatures ranging from 35 to 40°C. Reducing sodium dodecyl sulfate-PAGE analysis indicated that~28 kDa Kunitz-like trypsin inhibitor was present. Thus, we showed that AlTIs, particularly, AlTI 13 of A. lebbeck could be used as a transgene macromolecule to markedly increase insect resistance in genetically engineered plants.
Acta Physiologiae Plantarum, 2012
Proteinaceous inhibitors of digestive a-amylase occur naturally in leguminous seeds and find appl... more 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 AL-aAI-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. Keywords Proteinaceous a-amylase inhibitor Á Albizia lebbeck Á Human salivary a-amylase Á Porcine pancreatic a-amylase Abbreviations a-AI a-Amylase inhibitor AL-aAI Albizia lebbeck a-amylase inhibitor BPB Bromophenol blue BSA Bovine serum albumin DNSA 3,5-Dinitrosalicylic acid HSA Human salivary a-amylase PAGE Polyacrylamide gel electrophoresis PPA Porcine pancreatic a-amylase PVP Polyvinylpyrrolidone Communicated by M. Stobiecki.
Journal of the Saudi Society of Agricultural Sciences, 2017
Sugarcane woolly aphid, Ceratovacuna lanigera is one of the important pests of the sugarcane whic... more Sugarcane woolly aphid, Ceratovacuna lanigera is one of the important pests of the sugarcane which is responsible for the low production of the sugarcane in India. The incidences of pest itself suggest importance of study on this pest. Carbohydrate metabolizing digestive enzyme, α-amylase was studied from this insect. Biochemical characterization of this enzyme and screening of natural inhibitors will prove vital strategies against infestation. As initial step to this goal here we report the detection of α-amylase of Ceratovacuna lanigera by starch agar well diffusion assay. Biochemical parameters like pH, temperature and effect of substrate concentration were studied and compared with α-amylases from other sources. The optimum pH of α-amylase of Ceratovacuna lanigera was found to be pH 8 in contrast to human salivary α-amylases and fungal α-amylases those shows optimum activity at pH 7 and pH 6 respectively. Maximum activity of α-amylase was found to be at 40°C. Protein profile by Native -PAGE showed banding pattern with varied intensities. The α-amylase of Ceratovacuna lanigera detected in the present study needs further exploration for its use in better management of sugarcane woolly aphid. 50 0.2 ml enzyme. After incubation at 37°C for 30 min, the liberated reducing sugars were estimated using DNSA (1% 3, 5-Dinitrosalisylic acid, 30% Sodium potassium tartarate, 0.2M NaoH) reagent. One unit of enzyme activity was defined as the quantity of enzyme producing 1μM reducing sugar (maltose) per min at defined assay condition.
Proteinaceous inhibitors of digestive a-amylase occur naturally in leguminous seeds and find appl... more 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 AL-aAI-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.
This paper evaluates α-amylase inhibitor (α-AI) mediated defense of pigeonpea against Helicoverpa... more This paper evaluates α-amylase inhibitor (α-AI) mediated defense of pigeonpea against Helicoverpa armigera. A bifunctional α-amylase/trypsin inhibitor was purified from the seeds of pigeonpea by native liquid phase isoelectric focusing (N-LP-IEF), affinity chromatography and preparative electrophoresis. Its in-vivo and in-vitro interaction with midgut amylases of H. armigera was studied along with growth inhibitory activity. One and two dimensional (2D) zymographic analyses revealed that the purified inhibitor is dimeric glycoprotein (60.2 kDa and 56 kDa) exist in a multi-isomeric form with five pI variants (pI 5.5 to 6.3). It was found to be heat labile with complete inactivation up to 80 °C and stable over a wide range of pH (4–11). The slow binding and competitive type of α-amylase inhibition was observed with 0.08 μM of dissociation constant (Ki) for the enzyme–inhibitor complex (EI). The internal protein sequence of two subunits obtained by mass spectrometry matched with cereal-type α-AI, a conserved domain from AAI_LTSS superfamily and sialyltransferase-like protein respectively. In-vivo studies indicated up-regulation of total midgut α-amylase activity with negative effect on growth rate of H. armigera suggesting its suitability for pest control.
Here, we report multiple molecular forms of Albizia lebbeck trypsin inhibitors (AlTIs) by using a... more 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)2SO4 fractionation, Sephadex G-100 column chromatography, and preparative native-polyacrylamide gel electrophoresis (PAGE). The dose dependent bioefficacies of AlTIs in the (NH4)2SO4 F3 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 μg/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 μmol/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 ~ 28 kDa Kunitz-like trypsin inhibitor was present. Thus, we showed that AlTIs, particularly, AlTI13 of A. lebbeck could be used as a transgene macromolecule to markedly increase insect resistance in genetically engineered plants.
Proteinaceous inhibitors of digestive α-amylase occur naturally in leguminous seeds and find appl... more Proteinaceous inhibitors of digestive α-amylase occur naturally in leguminous seeds and find applications in agriculture and clinical studies. We have detected and isolated eight novel α-amylase inhibitor isoforms in the seed extract of Albizia lebbeck. They are designated as AL-αAI-1 to AL-αAI-8. These isoforms specifically inhibit human salivary α-amylase and porcine pancreatic α-amylase. The occurrence and profile of α-amylase inhibitor isoforms were revealed by 7 % native-PAGE containing 0.1 % starch. The apparent molecular weights of native bands of AL-αAIs were 97.4, 68.6, 61.0, 57.2, 56.0, 54.7, 51.1, and 47.7 kDa, respectively. Partial purification of potent α-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-αAI 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 α-amylase inhibitors from A. lebbeck could be important and may find applications in clinical studies to modulate starch digestion and glycemic index.
Lipase is one of the important enzymes in food, pharmaceutical, detergent and biofuels industrie... more Lipase is one of the important enzymes in food, pharmaceutical, detergent and biofuels industries. Search for the lipase with distinct features, possibly from germinating seeds, is of interest for industrial applications. Approach: The lipase produced by soybean oil seeds was partially purified and characterized in terms of the optimal pH and temperature for activity as well as substrate specificity. Results: The lipase was extracted and partially purified from germinating soybean seeds using chilled acetone and ammonium sulfate precipitation. Partially purified and dialyzed enzyme profile was observed on native-Polyacrylamide Gel Electrophoresis (PAGE). The lipase was optimally active at pH 8 and temperature of 24°C. In the presence of Ca2+ and Mg2+ enhance the activity at low concentration, while the Hg2+ and Ethylene Diaminotetracetic Acid (EDTA) showed inhibitory effect. The enzyme was found to be metalloenzyme. Enzyme kinetics with olive oil emulsion substrate showed km and vmax of 7.67 mg and 0.0125 µm mL min-1, respectively. Conclusion: The mettaloenzyme enzyme was able to attack specifically on oil in seeds to generate free fatty acids as the major end product. This understanding may help in devising efficient methods to overcome the problem of soybean seed oil in stability.