β-N-Acetyl-D-glucosaminidase （NAGase, EC.3.2.1.52） is chitinolytic enzymes and disintegrate dimmer and trimer a composition of oligomers of N-acetyl-β-D-glucosamine （NAG） into monomer. Prawn （P. vannamei） NAG...β-N-Acetyl-D-glucosaminidase （NAGase, EC.3.2.1.52） is chitinolytic enzymes and disintegrate dimmer and trimer a composition of oligomers of N-acetyl-β-D-glucosamine （NAG） into monomer. Prawn （P. vannamei） NAGase is involved in digestion and molting processes. Some pollutants in seawater affect the enzyme activity causing loss of the biological function of the enzyme, which affects the exuviating shell and threatens the survival of the animal. The effect of formaldehyde on prawn （P. vannamei） β-N-acetyl-D-glucosaminidase activity for the hydrolysis of pNP-NAG has been studied. The results show that formaldehyde, at appropriate concentrations, can lead to reversible inactivation of the enzyme, and the IC50 is estimated to be 1.05mol· L^-1. The inactivation mechanism obtained from Lineweaver-Burk plots shows that the inactivation of the enzyme by formaldehyde belongs to the competitive type. The inactivation kinetics of the enzyme by formaldehyde has been studied using the progress-of-substrate-reaction method described by Tsou, and the rate constants have been determined. The results show that k＋0 is much larger than k-0, indicating the free enzyme molecule is fragile in the formaldehyde solution.展开更多
Royal palm tree peroxidase (RPTP) has been isolated to homogeneity from leaves of Roystonea regia palm trees. The enzyme purification steps included homogenization, (NH4)SO4 precipitation, extraction of palm leaf colo...Royal palm tree peroxidase (RPTP) has been isolated to homogeneity from leaves of Roystonea regia palm trees. The enzyme purification steps included homogenization, (NH4)SO4 precipitation, extraction of palm leaf colored compounds and consecutive chromatography on Phenyl-Sepharose, TSK-Gel DEAE-5PW and Superdex-200. The novel peroxidase was characterized as having a molecular weight of 48.2 ± 3.0 kDa and an isoelectric point pI 5.4 ± 0.1. The enzyme forms dimers in solution with approximate molecular weight of 92 ± 2 kDa. Here we investigated the steady-state kinetic mechanism of the H2O2-supported oxidation of different organic substrates by RPTP. The results of the analysis of the initial rates vs. H2O2 and reducing substrate concentrations were seen to be consistent with a substrate-inhibited Ping-Pong Bi-Bi reaction mechanism. The phenomenological approach used expresses the peroxidase Ping-Pong mechanism in the form of the Michaelis-Menten equation and affords an interpretation of the effects in terms of the kinetic parameters KmH2O2, KmAH2, kcat, KSIH2O2, KSIAH2 and of the microscopic rate constants k1 and k3 of the shared three-step peroxidase catalytic cycle. Furthermore, the concentration and time-dependences and the mechanism of the suicide inactivation of RPTP by hydrogen peroxide were studied kinetically with guaiacol as co-substrate. The turnover number (r) of H2O2 required to complete the inactivation of the enzyme was 2154 ± 100 and the apparent rate constants of catalysis 185 s–1 and 18 s–1.展开更多
N-Acetyl-β-D-glucosaminidase（NAGase, EC 3.2.1.52）, which catalyzes the cleavage of N-acetylgluco- samine polymers, plays important roles in the molting, digestion of chitinous foods in green crab. In the study, the...N-Acetyl-β-D-glucosaminidase（NAGase, EC 3.2.1.52）, which catalyzes the cleavage of N-acetylgluco- samine polymers, plays important roles in the molting, digestion of chitinous foods in green crab. In the study, the efforts of urea on the activity of NAGase purified from the viscera of green crab（Scylla serrata） have been studied. The results show that appropriate concentrations of urea can lead to reversible inactivation of the enzyme, and the value of the inhibitor concentration leading to 50% of enzyme activity lost（IC50） is estimated to be 0.63 mol/L. The inactivation kinetics has been studied via the kinetic method of the substrate reaction. The rate constants of inactivation have been determined. The value of k＋0 is larger than that of k′＋0, indicating the free enzyme molecule is more fragile than the enzyme-substrate complex in urea solution. It is suggested that the presence of the substrate offers the marked protection of this enzyme against inactivation by urea.展开更多
A new enzyme (alkaline protease 894) obtained from the marine extremophile Flavobacterium yellowsea (YS-80-122) has exhibited strong substrate-binding and catalytic activity, even at low temperature, but the character...A new enzyme (alkaline protease 894) obtained from the marine extremophile Flavobacterium yellowsea (YS-80-122) has exhibited strong substrate-binding and catalytic activity, even at low temperature, but the characteristics of the hydrolysis with this enzyme are still unclear. The pearl oyster Pinctada martensii was used in this study as the raw material to illustrate the kinetic properties of protease 894. After investigating the intrinsic relationship between the degree of hydrolysis and several factors, including initial reaction pH, temperature, substrate concentration, enzyme concentration, and hydrolysis time, the kinetics model was established. This study showed that the optimal conditions for the enzymatic hydrolysis were an initial reaction pH of 5.0, temperature of 30°C, substrate concentration of 10% (w/v), enzyme concentration of 2 500 U/g, and hydrolysis time of 160 min. The kinetic characteristics of the protease for the hydrolysis of P. martensii were obtained. The inactivation constant was found to be 15.16/min, and the average relative error between the derived kinetics model and the actual measurement was only 3.04%, which indicated a high degree of fitness. Therefore, this study provides a basis for the investigation of the concrete kinetic characteristics of the new protease, which has potential applications in the food industry.展开更多
文摘β-N-Acetyl-D-glucosaminidase （NAGase, EC.3.2.1.52） is chitinolytic enzymes and disintegrate dimmer and trimer a composition of oligomers of N-acetyl-β-D-glucosamine （NAG） into monomer. Prawn （P. vannamei） NAGase is involved in digestion and molting processes. Some pollutants in seawater affect the enzyme activity causing loss of the biological function of the enzyme, which affects the exuviating shell and threatens the survival of the animal. The effect of formaldehyde on prawn （P. vannamei） β-N-acetyl-D-glucosaminidase activity for the hydrolysis of pNP-NAG has been studied. The results show that formaldehyde, at appropriate concentrations, can lead to reversible inactivation of the enzyme, and the IC50 is estimated to be 1.05mol· L^-1. The inactivation mechanism obtained from Lineweaver-Burk plots shows that the inactivation of the enzyme by formaldehyde belongs to the competitive type. The inactivation kinetics of the enzyme by formaldehyde has been studied using the progress-of-substrate-reaction method described by Tsou, and the rate constants have been determined. The results show that k＋0 is much larger than k-0, indicating the free enzyme molecule is fragile in the formaldehyde solution.
文摘Royal palm tree peroxidase (RPTP) has been isolated to homogeneity from leaves of Roystonea regia palm trees. The enzyme purification steps included homogenization, (NH4)SO4 precipitation, extraction of palm leaf colored compounds and consecutive chromatography on Phenyl-Sepharose, TSK-Gel DEAE-5PW and Superdex-200. The novel peroxidase was characterized as having a molecular weight of 48.2 ± 3.0 kDa and an isoelectric point pI 5.4 ± 0.1. The enzyme forms dimers in solution with approximate molecular weight of 92 ± 2 kDa. Here we investigated the steady-state kinetic mechanism of the H2O2-supported oxidation of different organic substrates by RPTP. The results of the analysis of the initial rates vs. H2O2 and reducing substrate concentrations were seen to be consistent with a substrate-inhibited Ping-Pong Bi-Bi reaction mechanism. The phenomenological approach used expresses the peroxidase Ping-Pong mechanism in the form of the Michaelis-Menten equation and affords an interpretation of the effects in terms of the kinetic parameters KmH2O2, KmAH2, kcat, KSIH2O2, KSIAH2 and of the microscopic rate constants k1 and k3 of the shared three-step peroxidase catalytic cycle. Furthermore, the concentration and time-dependences and the mechanism of the suicide inactivation of RPTP by hydrogen peroxide were studied kinetically with guaiacol as co-substrate. The turnover number (r) of H2O2 required to complete the inactivation of the enzyme was 2154 ± 100 and the apparent rate constants of catalysis 185 s–1 and 18 s–1.
基金Supported by the National Natural Science Foundation of China(No.40576066)the Science and Technology Foundation of Xiamen,China(No.3502Z20081143)
文摘N-Acetyl-β-D-glucosaminidase（NAGase, EC 3.2.1.52）, which catalyzes the cleavage of N-acetylgluco- samine polymers, plays important roles in the molting, digestion of chitinous foods in green crab. In the study, the efforts of urea on the activity of NAGase purified from the viscera of green crab（Scylla serrata） have been studied. The results show that appropriate concentrations of urea can lead to reversible inactivation of the enzyme, and the value of the inhibitor concentration leading to 50% of enzyme activity lost（IC50） is estimated to be 0.63 mol/L. The inactivation kinetics has been studied via the kinetic method of the substrate reaction. The rate constants of inactivation have been determined. The value of k＋0 is larger than that of k′＋0, indicating the free enzyme molecule is more fragile than the enzyme-substrate complex in urea solution. It is suggested that the presence of the substrate offers the marked protection of this enzyme against inactivation by urea.
基金Supported by the Comprehensive Strategic Cooperation Programs between Guangdong Province and Chinese Academy of Sciences(No.2011A090100008)the Knowledge Innovation Program of Chinese Academy of Sciences(No.KZCX2-EW-Q214)
文摘A new enzyme (alkaline protease 894) obtained from the marine extremophile Flavobacterium yellowsea (YS-80-122) has exhibited strong substrate-binding and catalytic activity, even at low temperature, but the characteristics of the hydrolysis with this enzyme are still unclear. The pearl oyster Pinctada martensii was used in this study as the raw material to illustrate the kinetic properties of protease 894. After investigating the intrinsic relationship between the degree of hydrolysis and several factors, including initial reaction pH, temperature, substrate concentration, enzyme concentration, and hydrolysis time, the kinetics model was established. This study showed that the optimal conditions for the enzymatic hydrolysis were an initial reaction pH of 5.0, temperature of 30°C, substrate concentration of 10% (w/v), enzyme concentration of 2 500 U/g, and hydrolysis time of 160 min. The kinetic characteristics of the protease for the hydrolysis of P. martensii were obtained. The inactivation constant was found to be 15.16/min, and the average relative error between the derived kinetics model and the actual measurement was only 3.04%, which indicated a high degree of fitness. Therefore, this study provides a basis for the investigation of the concrete kinetic characteristics of the new protease, which has potential applications in the food industry.
