Osmar Soares da Silva, R. L. C. Oliveira, C. Souza-Motta, A. Porto, T. S. Porto
This study reports the protease production from Aspergillus tamarii using agroindustrial residues as substrate for solid-state fermentation (SSF) and biochemical characterization. The highest protease production was obtained using wheat bran as substrate at 72 h fermentation with maximum proteolytic activity of 401.42 U/mL, collagenase of 243.0 U/mL and keratinase of 19.1 U/mL. The protease exhibited KM = 18.7 mg/mL and Vmax = 28.5 mg/mL/min. The optimal pH was 8.0 and stable in a wide pH range (5.0 - 11.0) during 24 h. The optimum temperature was 40°C. The proteolytic activity was inhibited by Cu2+ (33.98%) and Hg2+ (22.69%). The enzyme was also inhibited by PMSF (65.11%), indicating that is a Serine Protease. These properties suggest that alkaline protease from A. tamarii URM4634 is suitable for application in food industries and leather processing. Additionally, the present findings opened new vistas in the utilization of wheat bran and other effective agroindustrial wastes as substrates for SSF.
{"title":"Novel Protease from Aspergillus tamarii URM4634: Production and Characterization Using Inexpensive Agroindustrial Substrates by Solid-State Fermentation","authors":"Osmar Soares da Silva, R. L. C. Oliveira, C. Souza-Motta, A. Porto, T. S. Porto","doi":"10.4236/AER.2016.44012","DOIUrl":"https://doi.org/10.4236/AER.2016.44012","url":null,"abstract":"This study reports the protease production from Aspergillus tamarii using agroindustrial residues as substrate for solid-state fermentation (SSF) and biochemical characterization. The highest protease production was obtained using wheat bran as substrate at 72 h fermentation with maximum proteolytic activity of 401.42 U/mL, collagenase of 243.0 U/mL and keratinase of 19.1 U/mL. The protease exhibited KM = 18.7 mg/mL and Vmax = 28.5 mg/mL/min. The optimal pH was 8.0 and stable in a wide pH range (5.0 - 11.0) during 24 h. The optimum temperature was 40°C. The proteolytic activity was inhibited by Cu2+ (33.98%) and Hg2+ (22.69%). The enzyme was also inhibited by PMSF (65.11%), indicating that is a Serine Protease. These properties suggest that alkaline protease from A. tamarii URM4634 is suitable for application in food industries and leather processing. Additionally, the present findings opened new vistas in the utilization of wheat bran and other effective agroindustrial wastes as substrates for SSF.","PeriodicalId":65616,"journal":{"name":"酶研究进展(英文)","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2016-11-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"70485697","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Shamim Mushtaq, Meraj Zehra, A. Khan, Mehwish Ahmed, R. Ghani, N. Ahmed
Aim: To evaluate the functional relationship between the nitric oxide synthase (NOS) and superoxide dismutase (SOD) enzymes in the pathogenesis of human senile cataract lenses of non-diabetic patients. Methods: Total solubilized proteins from human cataract lens were compared with normal lens (control) by 2-Dimenstional gel electrophoresis (2-DE). Proteins with different abundances were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Western blot analysis was used to verify the changes in expression of NOS3 and SOD2. A further functional association of NOS3 with SOD2 and other proteins was seen by STRING 8.3 databases. Results: In the 2-DE maps, the cataract and normal lens proteins migrated in the region of pH 3 - 10 with a relative molecular weight of 20 - 130 kDa. Approximately two protein spots with differential intensity were detected as NOS3 and SOD2 using MALDI-TOF-MS. Western blot analysis showed high expression of NOS3 in cataract and SOD2 in normal lens samples. String interaction network revealed strong interactions between NOS3 and SOD2 at high confidence score, which is helpful in characterization of functional abnormalities that may be a causative factor in the pathogenesis of cataract. Conclusion: This study will offer new avenues for mechanistic evaluation and future prevention of cataractogensis. However, large scale studies will be required to evaluate the effect of this interaction on the clinical outcome in human cataract.
{"title":"Biological Association and Expressions of NOS3 & SOD2 in Non-Diabetic Senile Cataractogenesis","authors":"Shamim Mushtaq, Meraj Zehra, A. Khan, Mehwish Ahmed, R. Ghani, N. Ahmed","doi":"10.4236/AER.2016.43009","DOIUrl":"https://doi.org/10.4236/AER.2016.43009","url":null,"abstract":"Aim: To evaluate the functional relationship between the nitric oxide synthase (NOS) and superoxide dismutase (SOD) enzymes in the pathogenesis of human senile cataract lenses of non-diabetic patients. Methods: Total solubilized proteins from human cataract lens were compared with normal lens (control) by 2-Dimenstional gel electrophoresis (2-DE). Proteins with different abundances were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Western blot analysis was used to verify the changes in expression of NOS3 and SOD2. A further functional association of NOS3 with SOD2 and other proteins was seen by STRING 8.3 databases. Results: In the 2-DE maps, the cataract and normal lens proteins migrated in the region of pH 3 - 10 with a relative molecular weight of 20 - 130 kDa. Approximately two protein spots with differential intensity were detected as NOS3 and SOD2 using MALDI-TOF-MS. Western blot analysis showed high expression of NOS3 in cataract and SOD2 in normal lens samples. String interaction network revealed strong interactions between NOS3 and SOD2 at high confidence score, which is helpful in characterization of functional abnormalities that may be a causative factor in the pathogenesis of cataract. Conclusion: This study will offer new avenues for mechanistic evaluation and future prevention of cataractogensis. However, large scale studies will be required to evaluate the effect of this interaction on the clinical outcome in human cataract.","PeriodicalId":65616,"journal":{"name":"酶研究进展(英文)","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2016-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"70486098","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Lluvia de Carolina Sánchez-Pérez, S. Rodríguez-Navarro, Víctor Hugo Marín-Cruz, M. Ramos-López, A. P. Ramos, J. E. Barranco-Florido
The application of enzymatic extracts and conidia of Beauveria bassiana in Metamasius spinolae and Cyclocephala lunulata was evaluated. The variables were mortality and time of death. In M. spinolae, mortality with extracts 29%, conidia 27% and the combination of both 31%, all had a time of death of four days. Although with different symptoms, used enzymatic extracts: contraction and softening of the joints; by conidia: mycelium in the joints; in the combination of conidia and enzymatic extracts: abundant aerial mycelium. In C. lunulata, 100% mortality in all treatments; Time of death: enzymatic extracts and extracts with conidia 1.2 days; conidia 2.8 days. Symptoms were different, enzymatic extracts: melanization and internal tissue lysis; enzymatic extract and conidia: mycelium emerged and melanization; conidia: mycelium emerged. Enzymatic extracts showed insecticidal activity in M. spinolae and C. lunulata. These results suggest the potential of enzymatic extracts as biocontrol agents to improve the use of entomopathogenic fungi.
{"title":"Assessment of Beauveria bassiana and Their Enzymatic Extracts against Metamasius spinolae and Cyclocephala lunulata in Laboratory","authors":"Lluvia de Carolina Sánchez-Pérez, S. Rodríguez-Navarro, Víctor Hugo Marín-Cruz, M. Ramos-López, A. P. Ramos, J. E. Barranco-Florido","doi":"10.4236/AER.2016.43010","DOIUrl":"https://doi.org/10.4236/AER.2016.43010","url":null,"abstract":"The application of enzymatic extracts and conidia of Beauveria bassiana in Metamasius spinolae and Cyclocephala lunulata was evaluated. The variables were mortality and time of death. In M. spinolae, mortality with extracts 29%, conidia 27% and the combination of both 31%, all had a time of death of four days. Although with different symptoms, used enzymatic extracts: contraction and softening of the joints; by conidia: mycelium in the joints; in the combination of conidia and enzymatic extracts: abundant aerial mycelium. In C. lunulata, 100% mortality in all treatments; Time of death: enzymatic extracts and extracts with conidia 1.2 days; conidia 2.8 days. Symptoms were different, enzymatic extracts: melanization and internal tissue lysis; enzymatic extract and conidia: mycelium emerged and melanization; conidia: mycelium emerged. Enzymatic extracts showed insecticidal activity in M. spinolae and C. lunulata. These results suggest the potential of enzymatic extracts as biocontrol agents to improve the use of entomopathogenic fungi.","PeriodicalId":65616,"journal":{"name":"酶研究进展(英文)","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2016-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"70485642","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
In energy deficient world, cellulases play a major role for the production of alternative energy resources utilizing lignocellulosic waste materials for bioethanol and biogas production. This study highlights fungal and bacterial strains for the production of cellulases and its industrial applications. Solid State Fermentation (SSF) is more suitable process for cellulase production as compared to submerge fermentation techniques. Fungal cellulosomes system for the production of cellulases is more desirable and resistant to harsh environmental conditions. Trichoderma species are considered as most suitable candidate for cellulase production and utilization in industry as compared to Aspergillus and Humicola species. However, genetically modified strains of Aspergillus have capability to produce cellulase in relatively higher amount. Bacterial cellulase are more resistant to alkaline and thermophile conditions and good candidate in laundries. Cellulases are used in variety of industries such as textile, detergents and laundries, food industry, paper and pulp industry and biofuel production. Thermally stable modified strains of fungi and bacteria are good future prospect for cellulase production.
{"title":"Cellulase Production from Species of Fungi and Bacteria from Agricultural Wastes and Its Utilization in Industry: A Review","authors":"M. Imran, Z. Anwar, M. Irshad, M. Asad, H. Ashfaq","doi":"10.4236/AER.2016.42005","DOIUrl":"https://doi.org/10.4236/AER.2016.42005","url":null,"abstract":"In energy deficient world, cellulases play a major role for the production of alternative energy resources utilizing lignocellulosic waste materials for bioethanol and biogas production. This study highlights fungal and bacterial strains for the production of cellulases and its industrial applications. Solid State Fermentation (SSF) is more suitable process for cellulase production as compared to submerge fermentation techniques. Fungal cellulosomes system for the production of cellulases is more desirable and resistant to harsh environmental conditions. Trichoderma species are considered as most suitable candidate for cellulase production and utilization in industry as compared to Aspergillus and Humicola species. However, genetically modified strains of Aspergillus have capability to produce cellulase in relatively higher amount. Bacterial cellulase are more resistant to alkaline and thermophile conditions and good candidate in laundries. Cellulases are used in variety of industries such as textile, detergents and laundries, food industry, paper and pulp industry and biofuel production. Thermally stable modified strains of fungi and bacteria are good future prospect for cellulase production.","PeriodicalId":65616,"journal":{"name":"酶研究进展(英文)","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2016-05-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"70485991","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
It is demonstrated that (3Z)-nonenal (NON) and (3Z)-hexenal (HEX) are oxidized in a cascade by lipoxygenase (LOX) and hydroperoxide peroxygenase (HP peroxygenase) into (2E)-4-hydroxy-2- nonenal (HNE) and (2E)-4-hydroxy-2-hexenal (HHE), respectively. In turn, HNE inactivates LOX terminating the cascade. The hydroxy-alkenals produced serve to inhibit plant pathogens, which initiated the cascade. In addition to LOX, other unknown oxygenases may be involved in the cascade.
{"title":"Formation of (2E)-4-Hydroxy-2-nonenal and (2E)-4-Hydroxy-2-hexenal by Plant Enzymes: A Review Suggests a Role in the Physiology of Plants","authors":"H. Gardner","doi":"10.4236/AER.2016.42006","DOIUrl":"https://doi.org/10.4236/AER.2016.42006","url":null,"abstract":"It is demonstrated that (3Z)-nonenal (NON) and (3Z)-hexenal (HEX) are oxidized in a cascade by lipoxygenase (LOX) and hydroperoxide peroxygenase (HP peroxygenase) into (2E)-4-hydroxy-2- nonenal (HNE) and (2E)-4-hydroxy-2-hexenal (HHE), respectively. In turn, HNE inactivates LOX terminating the cascade. The hydroxy-alkenals produced serve to inhibit plant pathogens, which initiated the cascade. In addition to LOX, other unknown oxygenases may be involved in the cascade.","PeriodicalId":65616,"journal":{"name":"酶研究进展(英文)","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2016-05-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"70486030","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Mezajoug Kenfack Laurette Blandine, N. Serge, Tchiegang Clergé
Crude enzyme extracts were prepared from leaves and stems of Linn. (Fabaceae) from Cameroon under optimized conditions. Proteolytic enzymes were precipitated with ammonium sulfate at 35% (w/v) saturation and assayed for enzyme activity. The effects of temperature, pH, incubation time and substrate specificity were studied. SDS-PAGE was used to determine molecular weight of precipitated protease. Results indicated that proteolytic activity of crude extract was 35.20 U/ml compared to 51.03 U/ml of partial purified extract. The optimum enzyme activity was found to be at 40°C, while 50% of activity was maintained at 60°C after 60 min incubation. Partial purified crude extract exhibited two optimum pH (2.75 and 9.0). The highest enzyme activity towards Bovine Serum Albumine (25.9 U/ml) was noted. SDS-PAGE gels exhibited molecular weight between 40 - 60 KDa. This result confirms that partial purified extract of A. precatorius contains proteases and could be a promising source for proteolytic enzyme extraction.
{"title":"Partial Purification and Characterization of Protease from Abrus precatorius Linn. (Fabaceae) from Cameroon","authors":"Mezajoug Kenfack Laurette Blandine, N. Serge, Tchiegang Clergé","doi":"10.4236/AER.2016.42004","DOIUrl":"https://doi.org/10.4236/AER.2016.42004","url":null,"abstract":"Crude enzyme extracts were prepared from leaves and stems of Linn. (Fabaceae) from Cameroon under optimized conditions. Proteolytic enzymes were precipitated with ammonium sulfate at 35% (w/v) saturation and assayed for enzyme activity. The effects of temperature, pH, incubation time and substrate specificity were studied. SDS-PAGE was used to determine molecular weight of precipitated protease. Results indicated that proteolytic activity of crude extract was 35.20 U/ml compared to 51.03 U/ml of partial purified extract. The optimum enzyme activity was found to be at 40°C, while 50% of activity was maintained at 60°C after 60 min incubation. Partial purified crude extract exhibited two optimum pH (2.75 and 9.0). The highest enzyme activity towards Bovine Serum Albumine (25.9 U/ml) was noted. SDS-PAGE gels exhibited molecular weight between 40 - 60 KDa. This result confirms that partial purified extract of A. precatorius contains proteases and could be a promising source for proteolytic enzyme extraction.","PeriodicalId":65616,"journal":{"name":"酶研究进展(英文)","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2016-05-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"70485983","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
A. Lewin, T. A. Strand, T. Haugen, G. Klinkenberg, H. Kotlar, S. Valla, F. Drabløs, A. Wentzel
With the aim of identifying novel thermostable esterases, comprehensive sequence databases and cloned fosmid libraries of metagenomes derived from an offshore oil reservoir on the Norwegian Continental Shelf were screened for enzyme candidates using both sequence-and function-based screening. From several candidates identified in both approaches, one enzyme discovered by the functional approach was verified as a novel esterase and subjected to a deeper characterization. The enzyme was successfully over-produced in Escherichia coli and was shown to be thermostable up to 90°C, with the highest esterase activity on short-chain ester substrates and with tolerance to solvents and metal ions. The fact that the thermostable enzyme was solely found by functional screening of the oil reservoir metagenomes illustrates the importance of this approach as a complement to purely sequence-based screening, in which the enzyme candidate was not detected. In addition, this example indicates the large potential of deep-sub-surface oil reservoir metagenomes as a source of novel, thermostable enzymes of potential relevance for industrial applications.
{"title":"Discovery and Characterization of a Thermostable Esterase from an Oil Reservoir Metagenome","authors":"A. Lewin, T. A. Strand, T. Haugen, G. Klinkenberg, H. Kotlar, S. Valla, F. Drabløs, A. Wentzel","doi":"10.4236/AER.2016.42008","DOIUrl":"https://doi.org/10.4236/AER.2016.42008","url":null,"abstract":"With the aim of identifying novel thermostable esterases, comprehensive sequence databases and cloned fosmid libraries of metagenomes derived from an offshore oil reservoir on the Norwegian Continental Shelf were screened for enzyme candidates using both sequence-and function-based screening. From several candidates identified in both approaches, one enzyme discovered by the functional approach was verified as a novel esterase and subjected to a deeper characterization. The enzyme was successfully over-produced in Escherichia coli and was shown to be thermostable up to 90°C, with the highest esterase activity on short-chain ester substrates and with tolerance to solvents and metal ions. The fact that the thermostable enzyme was solely found by functional screening of the oil reservoir metagenomes illustrates the importance of this approach as a complement to purely sequence-based screening, in which the enzyme candidate was not detected. In addition, this example indicates the large potential of deep-sub-surface oil reservoir metagenomes as a source of novel, thermostable enzymes of potential relevance for industrial applications.","PeriodicalId":65616,"journal":{"name":"酶研究进展(英文)","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2016-05-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"70486083","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Z. Suleimenova, N. Akhmetsadykov, A. Kalieva, K. Mustafin, Z. Saduyeva
In agriculture, phytase is one of the most important monogastric animal sources of nutrient components because it effectively catalyzes the release of phosphate from phytate and phosphorylated compounds. In present work, Aspergillus niger strain (own collection) was used. Various physical and chemical factors have been known to affect the growth and the production of phytase. The effect of carbon and nitrogen sources, temperature and pH for extra cellular phytase production was investigated. Maximal phytase activity of Aspergillus niger was detected in media with 1.0% sucrose as a carbon source. Among the inorganic and organic nitrogen sources, ammonium nitrate in concentration of 0.5% was found to be a favorable nitrogen source for phytase production in Aspergillus niger. Optimum temperature and pH for phytase production by Aspergillus niger were 30°C and 5.5.
{"title":"Effect of Different Cultural Conditions for Phytase Production by Aspergillus niger in Submerged Fermentation","authors":"Z. Suleimenova, N. Akhmetsadykov, A. Kalieva, K. Mustafin, Z. Saduyeva","doi":"10.4236/AER.2016.42007","DOIUrl":"https://doi.org/10.4236/AER.2016.42007","url":null,"abstract":"In agriculture, phytase is one of the most important monogastric animal sources of nutrient components because it effectively catalyzes the release of phosphate from phytate and phosphorylated compounds. In present work, Aspergillus niger strain (own collection) was used. Various physical and chemical factors have been known to affect the growth and the production of phytase. The effect of carbon and nitrogen sources, temperature and pH for extra cellular phytase production was investigated. Maximal phytase activity of Aspergillus niger was detected in media with 1.0% sucrose as a carbon source. Among the inorganic and organic nitrogen sources, ammonium nitrate in concentration of 0.5% was found to be a favorable nitrogen source for phytase production in Aspergillus niger. Optimum temperature and pH for phytase production by Aspergillus niger were 30°C and 5.5.","PeriodicalId":65616,"journal":{"name":"酶研究进展(英文)","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2016-05-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"70486047","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Xueke Zhou, Tingting Wang, Anjun Wang, Renqiang Li
SDS-PAGE was applied to determine trypsin activity and inhibition. After the hydrolysis by trypsin to substrate bovine serum albulnin (BSA) under different temperatures and pH, the hydrolysis degree of BSA was conducted using SDS-PAGE. From the quantitative analysis to the electrophoresis bands of BSA and its hydrolysis products in SDS-PAGE pattern, the change of trypsin activity was determined, and then the optimum temperature at 40°C and the optimum pH at pH 8.5 - 8.7 for trypsin activity were obtained. All the target bonds in BSA molecule could be hydrolyzed at the same time by trypsin. The inhibition was due to the binding of inhibitor to trypsin, which made it impossible for trypsin to touch the substrate protein. SDS-PAGE was demonstrated to be also an effect method for assaying the characteristics of trypsin activity and its inhibition.
{"title":"Optima of Trypsin-Catalyzed Hydrolysis and Its Inhibition Determined by SDS-PAGE","authors":"Xueke Zhou, Tingting Wang, Anjun Wang, Renqiang Li","doi":"10.4236/AER.2016.41001","DOIUrl":"https://doi.org/10.4236/AER.2016.41001","url":null,"abstract":"SDS-PAGE was applied to determine trypsin activity and inhibition. After the hydrolysis by trypsin to substrate bovine serum albulnin (BSA) under different temperatures and pH, the hydrolysis degree of BSA was conducted using SDS-PAGE. From the quantitative analysis to the electrophoresis bands of BSA and its hydrolysis products in SDS-PAGE pattern, the change of trypsin activity was determined, and then the optimum temperature at 40°C and the optimum pH at pH 8.5 - 8.7 for trypsin activity were obtained. All the target bonds in BSA molecule could be hydrolyzed at the same time by trypsin. The inhibition was due to the binding of inhibitor to trypsin, which made it impossible for trypsin to touch the substrate protein. SDS-PAGE was demonstrated to be also an effect method for assaying the characteristics of trypsin activity and its inhibition.","PeriodicalId":65616,"journal":{"name":"酶研究进展(英文)","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2016-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"70485921","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
P. R. Adalberto, C. C. Golfeto, A. Moreira, F. G. Almeida, D. Ferreira, Q. Cass, D. H. Souza
The present study aimed to purify and characterize one polygalacturonase from L. gongylophorus (PGaseLg), the symbiotic fungus of Atta sexdens. The enzyme was isolated by salting out of crude extract followed by two chromatographic steps. PGaseLG was identified with MS analysis and molecular exclusion chromatography revealed the monomeric nature of a protein with an estimated molecular weight of about 39 kDa. PGaseLg has an optimum temperature of 60°C and optimum pH activity at 5.0. Using polygalacturonate as a substrate, the calculations of KM, Vmax and kcat were 0.65 mg·mL-1, 1800 μmol·min-1·mg-1 and 35.97 s-1, respectively. The enzyme was stable for more than 3 h at 50°C at pH 5.0; otherwise, at lower or higher pH values, the PGaseLg was less stable. The influence of several metals, EDTA and β-mercaptoethanol on enzyme activity was also determined. Thin layer chromatography (TLC) analyses indicated that PGaseLg is an exopolygalacturonase.
{"title":"Characterization of an Exopolygalacturonase from Leucoagaricus gongylophorus, the Symbiotic Fungus of Atta sexdens","authors":"P. R. Adalberto, C. C. Golfeto, A. Moreira, F. G. Almeida, D. Ferreira, Q. Cass, D. H. Souza","doi":"10.4236/AER.2016.41002","DOIUrl":"https://doi.org/10.4236/AER.2016.41002","url":null,"abstract":"The present study aimed to purify and characterize one polygalacturonase from L. gongylophorus (PGaseLg), the symbiotic fungus of Atta sexdens. The enzyme was isolated by salting out of crude extract followed by two chromatographic steps. PGaseLG was identified with MS analysis and molecular exclusion chromatography revealed the monomeric nature of a protein with an estimated molecular weight of about 39 kDa. PGaseLg has an optimum temperature of 60°C and optimum pH activity at 5.0. Using polygalacturonate as a substrate, the calculations of KM, Vmax and kcat were 0.65 mg·mL-1, 1800 μmol·min-1·mg-1 and 35.97 s-1, respectively. The enzyme was stable for more than 3 h at 50°C at pH 5.0; otherwise, at lower or higher pH values, the PGaseLg was less stable. The influence of several metals, EDTA and β-mercaptoethanol on enzyme activity was also determined. Thin layer chromatography (TLC) analyses indicated that PGaseLg is an exopolygalacturonase.","PeriodicalId":65616,"journal":{"name":"酶研究进展(英文)","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2016-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"70485929","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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