Mirzaei Ali, M. Irshad, Z. Anwar, M. Zafar, M. Imran
Xylanases are mostly produced through submerged fermentation; nonetheless solid-state fermentation has increased profound attention and consideration of scholars having high conversion level biomass to energy conservation. This study depicted the purification of xylanases and their possible utilization in industry. The present study was carried out to examine the culture influence of fungal strain Fomes fomentarius (F. fomentarius) using different agro-industrial residues (wheat straw, rice husk, sugarcane bagasse and siris pods). F. fomentarius showed maximum enzyme production after 72 h of fermentation, when grown on wheat straw in solid state fermentation process while maximum activity showed on pH 6.0 at 30°C. The other parameters optimized by statistical design (RSM) showed maximum xylanase activity (146 ± 8 IU/mL) at 65% moisture content, 4 mL inoculums size, 175 mg Ammonium sulphate, 200 mg Calcium carbonate and 1.4 grams of glucose. Xylanase was salted out at 60% ammonium sulphate concentration and enzyme was further purified by Sephadex G-100 gel filtration chromatography with 2.2 fold increase in activity. The purified xylanase from F. fomentarius had optimum pH 6.0 and 40°C. Xylanase showed higher specificity for oat spelt xylan with kinetic constants Km 1.25 mg/mL and Vmax 54 mM/min. Xylanases have an industrial important enzyme used extensively in food, feed and paper industry.
{"title":"Screening and Statistical Optimization of Physiochemical Parameters for the Production of Xylanases from Agro-Industrial Wastes","authors":"Mirzaei Ali, M. Irshad, Z. Anwar, M. Zafar, M. Imran","doi":"10.4236/AER.2016.41003","DOIUrl":"https://doi.org/10.4236/AER.2016.41003","url":null,"abstract":"Xylanases are mostly produced through submerged fermentation; nonetheless solid-state fermentation has increased profound attention and consideration of scholars having high conversion level biomass to energy conservation. This study depicted the purification of xylanases and their possible utilization in industry. The present study was carried out to examine the culture influence of fungal strain Fomes fomentarius (F. fomentarius) using different agro-industrial residues (wheat straw, rice husk, sugarcane bagasse and siris pods). F. fomentarius showed maximum enzyme production after 72 h of fermentation, when grown on wheat straw in solid state fermentation process while maximum activity showed on pH 6.0 at 30°C. The other parameters optimized by statistical design (RSM) showed maximum xylanase activity (146 ± 8 IU/mL) at 65% moisture content, 4 mL inoculums size, 175 mg Ammonium sulphate, 200 mg Calcium carbonate and 1.4 grams of glucose. Xylanase was salted out at 60% ammonium sulphate concentration and enzyme was further purified by Sephadex G-100 gel filtration chromatography with 2.2 fold increase in activity. The purified xylanase from F. fomentarius had optimum pH 6.0 and 40°C. Xylanase showed higher specificity for oat spelt xylan with kinetic constants Km 1.25 mg/mL and Vmax 54 mM/min. Xylanases have an industrial important enzyme used extensively in food, feed and paper industry.","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":"70485941","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}
The recombinant exopolyphosphatase PPX1 with a specific activity of ~300 U/mg protein was purified from the strain of Saccharomyces cerevisiae with the inactivated PPN1 gene transformed by the expression vector carrying the yeast PPX1 gene. The recombinant PPX1 was similar to the PPX1 of wild strains in its substrate specificity and requirement for cations. PPX1 had the high substrate specificity to polyphosphates. The preparation was suitable for polyphosphate assay in the presence of orthophosphate and nucleoside phosphates not hydrolyzed by PPX1. The yield of the enzyme preparation was 250 assays per 1 g of the biomass. The recombinant PPX1 was successfully used in polyphosphate assay in different yeast species and some foodstuffs.
{"title":"Polyphosphatase PPX1 of Saccharomyces cerevisiae as a Tool for Polyphosphate Assay","authors":"L. Lichko, T. Kulakovskaya","doi":"10.4236/AER.2015.34010","DOIUrl":"https://doi.org/10.4236/AER.2015.34010","url":null,"abstract":"The recombinant exopolyphosphatase PPX1 with a specific activity of ~300 U/mg protein was purified from the strain of Saccharomyces cerevisiae with the inactivated PPN1 gene transformed by the expression vector carrying the yeast PPX1 gene. The recombinant PPX1 was similar to the PPX1 of wild strains in its substrate specificity and requirement for cations. PPX1 had the high substrate specificity to polyphosphates. The preparation was suitable for polyphosphate assay in the presence of orthophosphate and nucleoside phosphates not hydrolyzed by PPX1. The yield of the enzyme preparation was 250 assays per 1 g of the biomass. The recombinant PPX1 was successfully used in polyphosphate assay in different yeast species and some foodstuffs.","PeriodicalId":65616,"journal":{"name":"酶研究进展(英文)","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2015-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"70485464","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}
Sabrina Pacheco, Américo Cruz Júnior, Ayres Ferreira Morgado, A. F. Júnior, O. Amadi, J. Guisán, B. Pessela
Nineteen fungal strains were isolated from a chicken slaughterhouse effluent and within those, only one showed high values of lipolytic activity in submerged cultures. This fungus was identified as Trametes hirsuta. The crude extract was immobilized in chitosan/clay beads, with an immobilization yield of 80.9%. The analyses of the crude extract and the immobilized derivative at different temperatures, pH (s), solvents, metallic ions and storage showed that the immobilization process increased the enzyme life span. Ethyl esters were obtained in solvent free systems using chicken viscera oil and the enzyme crude extract. For effective comparison, a reaction using viscera oil and commercial lipase Novozym 435 was carried out. The result revealed 35% and 28% esters conversion in the reactions containing chicken viscera oil, using Novozym 435 and the crude extract respectively. The extract was also used in a reaction with soybean oil, traditionally used as starting substrate for biodiesel production.
{"title":"Isolation and Screening of Filamentous Fungi Producing Extracellular Lipase with Potential in Biodiesel Production","authors":"Sabrina Pacheco, Américo Cruz Júnior, Ayres Ferreira Morgado, A. F. Júnior, O. Amadi, J. Guisán, B. Pessela","doi":"10.4236/AER.2015.34011","DOIUrl":"https://doi.org/10.4236/AER.2015.34011","url":null,"abstract":"Nineteen fungal strains were isolated from a chicken slaughterhouse effluent and within those, only one showed high values of lipolytic activity in submerged cultures. This fungus was identified as Trametes hirsuta. The crude extract was immobilized in chitosan/clay beads, with an immobilization yield of 80.9%. The analyses of the crude extract and the immobilized derivative at different temperatures, pH (s), solvents, metallic ions and storage showed that the immobilization process increased the enzyme life span. Ethyl esters were obtained in solvent free systems using chicken viscera oil and the enzyme crude extract. For effective comparison, a reaction using viscera oil and commercial lipase Novozym 435 was carried out. The result revealed 35% and 28% esters conversion in the reactions containing chicken viscera oil, using Novozym 435 and the crude extract respectively. The extract was also used in a reaction with soybean oil, traditionally used as starting substrate for biodiesel production.","PeriodicalId":65616,"journal":{"name":"酶研究进展(英文)","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2015-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"70485883","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}
Isolating cellulase-secreting microbes followed-by screening their cellulolytic activities has been an essential approach to discover novel and potential cellulases for cellulolytic industrial applications. This study was aimed to explore competitive exoglucanases by screening avicelase activities for 92 fungal strains isolated from environmental airborne-fungal-spore samples. Results showed that an isolated fungal strain numbered 58 exhibited the best avicelase activity of 0.209 U/mL when cultured for six days at pH 5.0 - 5.3 and 25℃ - 27℃, and was lately identified as a yeast strain of Meyerozyma sp. (96% ITS fragment similar with Meyerozyma caribbica, HG970748). Based on amino acid sequences revealed from LC/MS/MS, the target exoglucanase was identical to 1,4-beta-D-glucan cellobiohydrolases and was named Mc-CBHI which had optimal avicelase reaction conditions of pH 5 and 70℃ and could remain fairly stable after 4hr incubation at acid conditions (pH 3 - 5) or wide temperature ranges (30℃ - 80℃). Additionally, the Mc-CBHI (~70 kDa and ~3.6% of crude enzyme) had specific FPase and avicelase activities of 0.179 U/mg and 0.126 U/mg, respectively (which were about 40% - 50% activities of a commercial cellulase Accellerase-1000). These results demonstrated that the newly-found Mc-CBHI could become one of potential exoglucanase resources for related cellulolytic industrial applications.
{"title":"A Novel Exo-Glucanase Explored from a Meyerozyma sp. Fungal Strain","authors":"H. Kuo, J. Zeng, Pinyi Wang, Wen-Chin Chen","doi":"10.4236/AER.2015.33006","DOIUrl":"https://doi.org/10.4236/AER.2015.33006","url":null,"abstract":"Isolating cellulase-secreting microbes followed-by screening their cellulolytic activities has been an essential approach to discover novel and potential cellulases for cellulolytic industrial applications. This study was aimed to explore competitive exoglucanases by screening avicelase activities for 92 fungal strains isolated from environmental airborne-fungal-spore samples. Results showed that an isolated fungal strain numbered 58 exhibited the best avicelase activity of 0.209 U/mL when cultured for six days at pH 5.0 - 5.3 and 25℃ - 27℃, and was lately identified as a yeast strain of Meyerozyma sp. (96% ITS fragment similar with Meyerozyma caribbica, HG970748). Based on amino acid sequences revealed from LC/MS/MS, the target exoglucanase was identical to 1,4-beta-D-glucan cellobiohydrolases and was named Mc-CBHI which had optimal avicelase reaction conditions of pH 5 and 70℃ and could remain fairly stable after 4hr incubation at acid conditions (pH 3 - 5) or wide temperature ranges (30℃ - 80℃). Additionally, the Mc-CBHI (~70 kDa and ~3.6% of crude enzyme) had specific FPase and avicelase activities of 0.179 U/mg and 0.126 U/mg, respectively (which were about 40% - 50% activities of a commercial cellulase Accellerase-1000). These results demonstrated that the newly-found Mc-CBHI could become one of potential exoglucanase resources for related cellulolytic industrial applications.","PeriodicalId":65616,"journal":{"name":"酶研究进展(英文)","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2015-08-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"70485426","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}
Peroxidases (POXs) are the key extracellular enzymes produced by crude oil degrading microbes. Knowledge of optimum conditions for POXs activity is crucial for providing effective environment for bioremediation. In this study, physicochemical properties of POXs produced by Actinomyces israelii and Actinomyces viscosus during growth on crude oil were studied. The POXs exhibited similarities in activity and stability with striking differences in response to two divalent metal ions. The POXs from both species had optimum pH of 7.0 and were very stable over a narrow pH range (6.0 - 8.0). The POXs demonstrated similar thermostability exhibiting relative residual activity of 62% at 50°C after 30 min incubation and 45% residual activity at the same temperature after 60 min despite the fact that POXs from A. viscosus and A. israelii had optimum temperatures of 50°C and 40°C, respectively. The POXs from A. viscosus and A. israelii were greatly activated by Fe2+ at 5.0 and 10.0 mM. The enzymes were both strongly inhibited by Cu2+, Mg2+ and Hg2+. Surprisingly, these congeneric POXs demonstrated striking differences in their response to Ca2+ and Mn2+. POX from A. viscosus was activated by Ca2+ and Mn2+ exhibiting relative activity of 136% and 106% at 5 mM, respectively. In contrast, POX from A. israelii was strongly inhibited by Ca2+ and Mn2+ exhibiting 62.5% relative activity in the presence of 5 mM of each metal ion. Increasing the concentration of Ca2+ and Mn2+ led to further activation of POX from A. viscosus and inhibition of POX from A. israelii. Results provide deeper insights into functional properties of studied POXs from closely related microbes. The physicochemical properties are very similar; however, notable differences provide a strong basis for structural characterization of these congeneric enzymes.
过氧化物酶(POXs)是原油降解微生物产生的关键胞外酶。了解pox活性的最佳条件对于提供有效的生物修复环境至关重要。本研究研究了以色列放线菌和粘放线菌在原油上生长过程中产生的pox的理化性质。pox在活性和稳定性方面表现出相似性，但对两种二价金属离子的反应存在显著差异。两种植物的pox最适pH均为7.0，在较窄的pH范围(6.0 ~ 8.0)内非常稳定。两种pox表现出相似的热稳定性，在50°C孵育30 min后的相对残留活性为62%，在相同温度下孵育60 min后的相对残留活性为45%，尽管粘胶蒿和以色列蒿的pox的最佳温度分别为50°C和40°C。在5.0和10.0 mM Fe2+作用下，黏豆和以色列豆的pox酶活性显著，而Cu2+、Mg2+和Hg2+对pox酶均有较强的抑制作用。令人惊讶的是，这些同源pox在对Ca2+和Mn2+的反应中表现出显著的差异。在5 mM处，Ca2+和Mn2+的相对活性分别为136%和106%。而在Ca2+和Mn2+浓度为5 mM的条件下，A. israelii的痘蛋白的相对活性为62.5%。增加Ca2+和Mn2+浓度可进一步激活粘草的痘，抑制以色列草的痘。研究结果为研究来自密切相关微生物的pox的功能特性提供了更深入的见解。物理化学性质非常相似;然而，这些显著的差异为这些同源酶的结构表征提供了强有力的基础。
{"title":"Preliminary Study towards Enhanced Crude Oil Biodegradation Reveals Congeneric Total Peroxidases with Striking Distinctions","authors":"F. M. Olajuyigbe, K. Ehiosun, K. F. Jaiyesimi","doi":"10.4236/AER.2015.33007","DOIUrl":"https://doi.org/10.4236/AER.2015.33007","url":null,"abstract":"Peroxidases (POXs) are the key extracellular enzymes produced by crude oil degrading microbes. Knowledge of optimum conditions for POXs activity is crucial for providing effective environment for bioremediation. In this study, physicochemical properties of POXs produced by Actinomyces israelii and Actinomyces viscosus during growth on crude oil were studied. The POXs exhibited similarities in activity and stability with striking differences in response to two divalent metal ions. The POXs from both species had optimum pH of 7.0 and were very stable over a narrow pH range (6.0 - 8.0). The POXs demonstrated similar thermostability exhibiting relative residual activity of 62% at 50°C after 30 min incubation and 45% residual activity at the same temperature after 60 min despite the fact that POXs from A. viscosus and A. israelii had optimum temperatures of 50°C and 40°C, respectively. The POXs from A. viscosus and A. israelii were greatly activated by Fe2+ at 5.0 and 10.0 mM. The enzymes were both strongly inhibited by Cu2+, Mg2+ and Hg2+. Surprisingly, these congeneric POXs demonstrated striking differences in their response to Ca2+ and Mn2+. POX from A. viscosus was activated by Ca2+ and Mn2+ exhibiting relative activity of 136% and 106% at 5 mM, respectively. In contrast, POX from A. israelii was strongly inhibited by Ca2+ and Mn2+ exhibiting 62.5% relative activity in the presence of 5 mM of each metal ion. Increasing the concentration of Ca2+ and Mn2+ led to further activation of POX from A. viscosus and inhibition of POX from A. israelii. Results provide deeper insights into functional properties of studied POXs from closely related microbes. The physicochemical properties are very similar; however, notable differences provide a strong basis for structural characterization of these congeneric enzymes.","PeriodicalId":65616,"journal":{"name":"酶研究进展(英文)","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2015-08-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"70485280","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}
T. Nascimento, A. E. Sales, C. S. Porto, Romero M P Brandão, Galba Maria C Takaki, J. Teixeira, T. S. Porto, A. Porto
Fibrinolytic enzymes have received attention regarding their medicinal potential for thrombolytic diseases, a leading cause of morbidity and mortality worldwide. Various natural enzymes purified from animal, plant and microbial sources have been extensively studied. The aim of this work was to produce fibrinolytic protease by solid state fermentation using agro industrial substrates. Rhizopus arrhizus var. arrhizus UCP 1295 and Mucor subtillissimus UCP 1262 filamentous fungi species isolated from soil of Caatinga-PE, Brasil, were used as producer microorganisms. Wheat bran was shown to be the best substrate for the production of the enzyme and by using a 23 full factorial design the main effects and interactions of the quantity of the substrate wheat bran, moisture and temperature on the fibrinolytic enzyme production and protease were evaluated. The best results for fibrinolytic and protease activities, 144.58 U/mL and 48.33 U/mL, respectively, were obtained with Mucor subtillissimus UCP 1262 using as culture medium 3 g wheat bran, 50% moisture at a temperature of 25°C for 72 hours. The optimum temperature for the produced enzyme was 45°C and most of its original activity was retained after being subjected to 80°C for 120 min. The protease activity was enhanced by K+, Ca+ and Mn+; but with Cu+ there was an inhibition. The specificity to chromogenic substrate and the inhibition by PMSF indicates that it is a chymotrypsin-like serine protease. Presented results suggest that this enzyme produced by solid-state fermentation is an interesting alternative as a candidate for thrombolytic therapy.
{"title":"Production and Characterization of New Fibrinolytic Protease from Mucor subtillissimus UCP 1262 in Solid-State Fermentation","authors":"T. Nascimento, A. E. Sales, C. S. Porto, Romero M P Brandão, Galba Maria C Takaki, J. Teixeira, T. S. Porto, A. Porto","doi":"10.4236/AER.2015.33009","DOIUrl":"https://doi.org/10.4236/AER.2015.33009","url":null,"abstract":"Fibrinolytic enzymes have received attention regarding their medicinal potential for thrombolytic diseases, a leading cause of morbidity and mortality worldwide. Various natural enzymes purified from animal, plant and microbial sources have been extensively studied. The aim of this work was to produce fibrinolytic protease by solid state fermentation using agro industrial substrates. Rhizopus arrhizus var. arrhizus UCP 1295 and Mucor subtillissimus UCP 1262 filamentous fungi species isolated from soil of Caatinga-PE, Brasil, were used as producer microorganisms. Wheat bran was shown to be the best substrate for the production of the enzyme and by using a 23 full factorial design the main effects and interactions of the quantity of the substrate wheat bran, moisture and temperature on the fibrinolytic enzyme production and protease were evaluated. The best results for fibrinolytic and protease activities, 144.58 U/mL and 48.33 U/mL, respectively, were obtained with Mucor subtillissimus UCP 1262 using as culture medium 3 g wheat bran, 50% moisture at a temperature of 25°C for 72 hours. The optimum temperature for the produced enzyme was 45°C and most of its original activity was retained after being subjected to 80°C for 120 min. The protease activity was enhanced by K+, Ca+ and Mn+; but with Cu+ there was an inhibition. The specificity to chromogenic substrate and the inhibition by PMSF indicates that it is a chymotrypsin-like serine protease. Presented results suggest that this enzyme produced by solid-state fermentation is an interesting alternative as a candidate for thrombolytic therapy.","PeriodicalId":65616,"journal":{"name":"酶研究进展(英文)","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2015-08-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"70485345","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}
2-ketoisovalerate ferredoxin oxidoreductase (VOR) is a key enzyme in hyperthermophiles catalyzing the coenzyme A-dependent oxidative decarboxylation of aliphatic amino acid-derived 2-keto acids. The enzyme purified under anaerobic conditions from a hyperthermophilic archaeon, Thermococcus profundus, is a hetero-octamer (αβγδ)2 consisting of four different subunits, α = 45 kDa, β = 31 kDa, γ = 22 kDa and δ = 13 kDa, respectively, and it has three [4Fe-4S] clusters per αβγδ-protomer, similar to other ferredoxin oxidoreductases. In the present study, the native enzyme was purified from this strain and crystallized to give rod-like crystals that were suitable for X-ray diffraction experiments. The crystals belonged to space group P41212, with unit-cell parameters a = b = 136.20 A, c = 221.07 A. Diffraction images were processed to a resolution of 3.0 A. The data collected so far indicate the approximate molecular boundaries and a partial main-chain trace of the enzyme.
2-酮异戊酸铁氧还蛋白氧化还原酶(VOR)是嗜热菌催化辅酶a依赖性脂肪氨基酸衍生的2-酮酸氧化脱羧的关键酶。该酶在厌氧条件下从嗜热古菌深热球菌(Thermococcus proundus)中纯化得到，是一个异八聚体(αβγδ)2，分别由α = 45 kDa、β = 31 kDa、γ = 22 kDa和δ = 13 kDa组成，每个αβγδ原聚体具有3个[4Fe-4S]簇，类似于其他铁氧还蛋白氧化还原酶。在本研究中，从该菌株中纯化了天然酶并结晶为适合x射线衍射实验的棒状晶体。晶体属于空间群P41212，晶胞参数a = b = 136.20 a, c = 221.07 a。衍射图像处理至3.0 a分辨率。迄今为止收集的数据表明了该酶的大致分子边界和部分主链痕迹。
{"title":"Subunit Arrangement of a 2-Ketoisovalerate Ferredoxin Oxidoreductase from Thermococcus profundus Revealed by a Low Resolution X-Ray Analysis","authors":"Y. Ozawa, Y. Umena, T. Imai, Y. Morimoto","doi":"10.4236/AER.2015.33008","DOIUrl":"https://doi.org/10.4236/AER.2015.33008","url":null,"abstract":"2-ketoisovalerate ferredoxin oxidoreductase (VOR) is a key enzyme in hyperthermophiles catalyzing the coenzyme A-dependent oxidative decarboxylation of aliphatic amino acid-derived 2-keto acids. The enzyme purified under anaerobic conditions from a hyperthermophilic archaeon, Thermococcus profundus, is a hetero-octamer (αβγδ)2 consisting of four different subunits, α = 45 kDa, β = 31 kDa, γ = 22 kDa and δ = 13 kDa, respectively, and it has three [4Fe-4S] clusters per αβγδ-protomer, similar to other ferredoxin oxidoreductases. In the present study, the native enzyme was purified from this strain and crystallized to give rod-like crystals that were suitable for X-ray diffraction experiments. The crystals belonged to space group P41212, with unit-cell parameters a = b = 136.20 A, c = 221.07 A. Diffraction images were processed to a resolution of 3.0 A. The data collected so far indicate the approximate molecular boundaries and a partial main-chain trace of the enzyme.","PeriodicalId":65616,"journal":{"name":"酶研究进展(英文)","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2015-08-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"70485334","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}
Phosphatases are diverse groups of enzymes that deserve special attention because of their significant roles in organic phosphorus (OP) mineralization to inorganic available forms (Pi). This work 1) compared the catalytic potentials of commercially acid phosphatase from wheat germ, sweet potato, and potato, and alkaline phosphatase from E. coli; 2) demonstrated that the rate of hydrolysis, catalytic efficiency, thermal stability, and optimal pH of these enzymes depended on enzyme sources and the stereochemical or stereoisomeric structures of the substrates; 3) revealed that both acid and alkaline phosphatases exhibited broad range of substrate hydrolysis with high affinity for p-nitrophenyl phosphate bis (cyclohexylammonium) than the widely used p-nitrophenyl phosphate disodium hexahydrate for phosphatase assay. Sweet potato had relatively higher reaction kinetics (Vmax, Km, Kcat, Kcat/Km) values with most substrates tested. The order of catalytic activity was in the order: sweet potato > wheat germ > potato, while the order of substrate hydrolyzed was: PNPBC > PNP > PNP2A2E > DG6P2Na > DG6PNa > Bis-PNP > phytate. The optimum pH for the acid phosphatase was observed to be 5.0. Generally, the activity of alkaline phosphatase was similar to that of acid phosphatase with optimal pH between 10 and 13, depending on the substrates. Knowledge derived from this work would be helpful in enzyme catalysis in soils and water environments.
{"title":"Phosphatase Hydrolysis of Organic Phosphorus Compounds","authors":"I. Tazisong, Z. Senwo, Zhongqi He","doi":"10.4236/AER.2015.32005","DOIUrl":"https://doi.org/10.4236/AER.2015.32005","url":null,"abstract":"Phosphatases are diverse groups of enzymes that deserve special attention because of their significant roles in organic phosphorus (OP) mineralization to inorganic available forms (Pi). This work 1) compared the catalytic potentials of commercially acid phosphatase from wheat germ, sweet potato, and potato, and alkaline phosphatase from E. coli; 2) demonstrated that the rate of hydrolysis, catalytic efficiency, thermal stability, and optimal pH of these enzymes depended on enzyme sources and the stereochemical or stereoisomeric structures of the substrates; 3) revealed that both acid and alkaline phosphatases exhibited broad range of substrate hydrolysis with high affinity for p-nitrophenyl phosphate bis (cyclohexylammonium) than the widely used p-nitrophenyl phosphate disodium hexahydrate for phosphatase assay. Sweet potato had relatively higher reaction kinetics (Vmax, Km, Kcat, Kcat/Km) values with most substrates tested. The order of catalytic activity was in the order: sweet potato > wheat germ > potato, while the order of substrate hydrolyzed was: PNPBC > PNP > PNP2A2E > DG6P2Na > DG6PNa > Bis-PNP > phytate. The optimum pH for the acid phosphatase was observed to be 5.0. Generally, the activity of alkaline phosphatase was similar to that of acid phosphatase with optimal pH between 10 and 13, depending on the substrates. Knowledge derived from this work would be helpful in enzyme catalysis in soils and water environments.","PeriodicalId":65616,"journal":{"name":"酶研究进展(英文)","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2015-06-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"70485415","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}
K. N. Unni, P. Faisal, P. Priji, S. Sajith, S. Sreedevi, E. S. Hareesh, Trikariyoor Asokan Nidheesh Roy, S. Benjamin
This study explored the utility of flours of rubber seed, coconut and groundnut kernels, and de-oiled cakes of coconut and groundnut as solid substrate for the production of lipase by Pseudomonas aeruginosa strain BUP2 (MTCC No. 5924), a novel bacterium reported from the rumen of Malabari goat. Various proportions (10%, 20%, 30%, 40% or 50%) of flours or cakes were prepared (w/v) with BUP medium (pH 4, 5, 6, 7 or 8), and incubated at different temperature (25°C, 28°C, 30°C or 32°C) for 24 to 96 h. The samples were assayed for lipase activity at 24 h intervals. The rubber seed flour (20%)-BUP medium supported the maximum lipase production (871 U/gds) at 48h incubation (pH 6, 28°C), followed by ground nut flour (398 U/gds), while ground nut cake supported the least lipase production (244 U/gds). From this, it is evident that the cheaply available rubber seed is an efficient substrate for the production of lipase, irrespective of its known demerit that it contains the limarin, a toxin; in fact, we could not detect limarin in the fermented matter. Thus, the utility of rubber seed for the production of a costly enzyme is reported from a novel rumen bacterium, which would be advantageous for rubber farmers.
{"title":"Rubber Seed Kernel as Potent Solid Substrate for the Production of Lipase by Pseudomonas aeruginosa Strain BUP2","authors":"K. N. Unni, P. Faisal, P. Priji, S. Sajith, S. Sreedevi, E. S. Hareesh, Trikariyoor Asokan Nidheesh Roy, S. Benjamin","doi":"10.4236/AER.2015.32004","DOIUrl":"https://doi.org/10.4236/AER.2015.32004","url":null,"abstract":"This study explored the utility of flours of rubber seed, coconut and groundnut kernels, and de-oiled cakes of coconut and groundnut as solid substrate for the production of lipase by Pseudomonas aeruginosa strain BUP2 (MTCC No. 5924), a novel bacterium reported from the rumen of Malabari goat. Various proportions (10%, 20%, 30%, 40% or 50%) of flours or cakes were prepared (w/v) with BUP medium (pH 4, 5, 6, 7 or 8), and incubated at different temperature (25°C, 28°C, 30°C or 32°C) for 24 to 96 h. The samples were assayed for lipase activity at 24 h intervals. The rubber seed flour (20%)-BUP medium supported the maximum lipase production (871 U/gds) at 48h incubation (pH 6, 28°C), followed by ground nut flour (398 U/gds), while ground nut cake supported the least lipase production (244 U/gds). From this, it is evident that the cheaply available rubber seed is an efficient substrate for the production of lipase, irrespective of its known demerit that it contains the limarin, a toxin; in fact, we could not detect limarin in the fermented matter. Thus, the utility of rubber seed for the production of a costly enzyme is reported from a novel rumen bacterium, which would be advantageous for rubber farmers.","PeriodicalId":65616,"journal":{"name":"酶研究进展(英文)","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2015-06-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"70485363","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}
Valon Ejupi, Shpend Dragusha, T. Kabashima, Qinchang Zhu, A. EL-Mahdy, Sheng Yin, T. Shibata, M. Kai
A selective, sensitive, and convenient assay for human collagenase is required because of its implication in diseases such as rheumatoid arthritis, osteoarthritis, and tumors. Here, a novel assay for human collagenase activity is described in which enzymatic degradation of native collagen or acetyl peptide is determined by using a fluorogenic reaction for oligopeptides. The oligopeptides are quantified spectrofluorometrically with either 3,4-dihydroxyphenylacetic acid or 1,2-dihydroxybenzen reaction in the presence of sodium periodate and sodium borate (pH 7 - 8). These reactions can selectively convert N-terminal Gly-containing oligopeptides and N-terminal Ile-containing oligopeptides to fluorescence (FL) compounds, respectively, but not proteins, acetyl peptides or amino acids. Under optimized conditions using 1.65 μM collagen IV or 1.5 mM Ac-GPQGI- AGQ as substrates, this assay exhibits a proportional relationship between FL intensities and human collagenase-3 (MMP-13) concentrations. It can assay endogenous collagenase activities in several biological samples, such as cultured human cells and cheek tissue.
由于胶原酶与类风湿关节炎、骨关节炎和肿瘤等疾病有关，因此需要一种选择性、敏感和方便的测定方法。在这里，一种新的测定人类胶原酶活性的方法被描述，其中天然胶原蛋白或乙酰肽的酶降解是通过对寡肽的荧光反应来确定的。用3,4-二羟基苯基乙酸或1,2-二羟基苯在高碘酸钠和硼酸钠(pH 7 - 8)存在下的反应对寡肽进行荧光定量。这些反应可以选择性地将n端含甘氨酸寡肽和n端含甘氨酸寡肽分别转化为荧光(FL)化合物，但不能将蛋白质、乙酰肽或氨基酸转化为荧光(FL)化合物。在以1.65 μM胶原IV或1.5 mM Ac-GPQGI- AGQ为底物的优化条件下，该实验显示FL强度与人胶原酶-3 (MMP-13)浓度成正比关系。它可以测定几种生物样品中的内源性胶原酶活性，如培养的人类细胞和脸颊组织。
{"title":"Spectrofluorometric Assays of Human Collagenase Activity Using Native Collagen and Acetyl-Peptide Substrates","authors":"Valon Ejupi, Shpend Dragusha, T. Kabashima, Qinchang Zhu, A. EL-Mahdy, Sheng Yin, T. Shibata, M. Kai","doi":"10.4236/AER.2015.31003","DOIUrl":"https://doi.org/10.4236/AER.2015.31003","url":null,"abstract":"A selective, sensitive, and convenient assay for human collagenase is required because of its implication in diseases such as rheumatoid arthritis, osteoarthritis, and tumors. Here, a novel assay for human collagenase activity is described in which enzymatic degradation of native collagen or acetyl peptide is determined by using a fluorogenic reaction for oligopeptides. The oligopeptides are quantified spectrofluorometrically with either 3,4-dihydroxyphenylacetic acid or 1,2-dihydroxybenzen reaction in the presence of sodium periodate and sodium borate (pH 7 - 8). These reactions can selectively convert N-terminal Gly-containing oligopeptides and N-terminal Ile-containing oligopeptides to fluorescence (FL) compounds, respectively, but not proteins, acetyl peptides or amino acids. Under optimized conditions using 1.65 μM collagen IV or 1.5 mM Ac-GPQGI- AGQ as substrates, this assay exhibits a proportional relationship between FL intensities and human collagenase-3 (MMP-13) concentrations. It can assay endogenous collagenase activities in several biological samples, such as cultured human cells and cheek tissue.","PeriodicalId":65616,"journal":{"name":"酶研究进展(英文)","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2015-03-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.4236/AER.2015.31003","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"70485323","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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