RESUMO
The polyphenol oxidase was extracted and purified from truffles (Terfezia arenaria) and it exhibited a molecular weight of 67 kDa. The truffle PPO was able to oxidize monophenolic, o-diphenolic and triphenolic substrates. Thus, the enzyme seems to be stable under wide range of pH and temperature. Best catalytic efficiency was observed for catechol as substrate (Kcat/km; 674.2 S-1 mM-1).The effect of detergents, chaotropic agents, metal ions and eleven different inhibitors on relative activity of Truffles PPO was also investigated. A latent form of enzyme was observed and its activity was stimulated using 4 mM of SDS. Likewise, the type of inhibition and the values of KI and IC50 were reported for L-cysteine, Sodium fluoride, sodium metabisulfite and kojic acid. Besides, the effect of four concentrations of kojic acid(0.05.,0.1.,0.2 and 0.3 mM) on thermal inactivation of PPO was performed in temperature range " 60-75° C". The use of Kojic acid increase the rate of inactivation process and disrupt enzymatic activity. Moreover, the combined effect of temperature and kojic acid prevent from enzymatic browning reaction and maintain high antioxidant activities including ABTS scavenging activity, FRAP, and total phenolic contents.
Assuntos
Ascomicetos/química , Catecol Oxidase/química , Antioxidantes/química , Catecóis/química , Cisteína/química , Concentração de Íons de Hidrogênio , Fenóis/química , Pironas/química , Fluoreto de Sódio/química , Especificidade por Substrato , Sulfitos/química , TemperaturaRESUMO
A highly thermostable amylopullulanase was purified to homogeneity from the culture filtrate of the Clostridium thermosulfurogenes SVM17. On SDS-PAGE, the purified fraction having both amylase and pullulanase activities were observed as a single band. The molecular weight of the purified amylopullulanase on SDS-PAGE was 97 kDa. The optimum temperature for both amylase and pullulanase was 70 °C. The enzyme was completely stable at 70 °C for 2 h. The presence of 5% starch increased the thermal stability of the enzyme at 100 °C up to 2 h. Both amylase and pullulanase activities were optimum at pH 5.5 to 6.0 and were stable over a pH range of 4.0 to 6.5. The TLC analysis of the reaction products on starch showed that maltose was the main product along with trace amounts of glucose. The analysis of hydrolysis product of pullulan showed that maltotriose was the main product. At 5 mM concentration, Mn2+ and Ag+ strongly stimulated both amylase and pullulanase activities, where as Mg2+, Ca2+, Cu2+, Fe3+, Zn2+, Hg2+, EDTA, Cd2+ and Li2+ inhibited both amylase and pullulanase activities. When the concentration of metal ions was increased from 5 to10 mM, a further increase in amylase activity was observed in the presence of Ni2+, Mn2+ and Co2+. Where as substantial decrease was observed at 10 mM concentration of Ag+, Pb2+ and Ca2+.
RESUMO
Armillariella tabescens EJLY2098 was induced to produce ?-mannanase with konjac fine flour (Amorphopallus rivieri) as single carbon source. This induced enzyme was then purified using DEAE ion exchange chromatography and named atMAN47. Zymologic analysis showed that the molecular weight of this ?-mannanase was approximately 47 kD. The enzyme was stable when pH ranged from 5.0 to 6.5 and could be activated by Na+ and Ba2+. With an optimal temperature of 50?C. Action mode analysis of TLC revealed that the enzyme belonged to the endo-?-mannanase family. Being a meta-acid endo-?-mannanase, it was suitable to be applied to feed industry with a promising future as an enzyme preparation.
RESUMO
One alkaline protease from Actinomucor elegans AS3.2778 was purified protein. The enzyme was purified using ammonium sulfate precipitation, ion exchange chromatography, hydrophobic chromatography and size exclusion chromatography method, and its properties were also investigated. The molecular weight of this enzyme is 32 kDa with SDS-PAGE method, optimum temperature is 60℃, optimum pH is 8.5 to 10.5, it is stable in the pH range of 6.0 to 9.0 at < 40℃ temperature, and being completely inhibited by the serine protease inhibitor, PMSF, indicated that it belongs to the serine protease family. Specificity test indicated this protease has extensive selectivity to peptide bones, especially to peptide bones composed of Leucine residue.
RESUMO
One alkaline protease from Actinomucor elegans AS3.2778 was purified protein. The enzyme was purified using ammonium sulfate precipitation, ion exchange chromatography, hydrophobic chromatography and size exclusion chromatography method, and its properties were also investigated. The molecular weight of this enzyme is 32 kDa with SDS-PAGE method, optimum temperature is 60℃, optimum pH is 8.5 to 10.5, it is stable in the pH range of 6.0 to 9.0 at < 40℃ temperature, and being completely inhibited by the serine protease inhibitor, PMSF, indicated that it belongs to the serine protease family. Specificity test indicated this protease has extensive selectivity to peptide bones, especially to peptide bones composed of Leucine residue.
RESUMO
One alkaline protease from Actinomucor elegans AS3.2778 was purified protein. The enzyme was purified using ammonium sulfate precipitation, ion exchange chromatography, hydrophobic chromatography and size exclusion chromatography method, and its properties were also investigated. The molecular weight of this enzyme is 32 kDa with SDS-PAGE method, optimum temperature is 60℃, optimum pH is 8.5 to 10.5, it is stable in the pH range of 6.0 to 9.0 at < 40℃ temperature, and being completely inhibited by the serine protease inhibitor, PMSF, indicated that it belongs to the serine protease family. Specificity test indicated this protease has extensive selectivity to peptide bones, especially to peptide bones composed of Leucine residue.
RESUMO
The dhaD gene encoding glycerol dehydrogenase(GDH) from Klebsiella sp.was amplified,and was inserted into expression vector pET-28a(+),the plasmid pET-28a-dhaD was constructed and was transformed into Escherichia coli BL21(DE3).SDS-PAGE showed that the gene dhaD was expressed successfully in recombinant E.coli BL21.Then GDH was purified by Ni-NTA affinity chromatography,the results showed a single band about 39kDa on SDS-PAGE gel,and the specified activity was about 156U/mg.The special activity of GDH is 4.6-fold higher than that of unpurified and the activity recovery is 67.4%.The optimum reaction pH was 11.0,and the GDH activity have little changed when incubated in the buffer of pH7.0～11.0.The optimum reactive temperature was 30℃,and the GDH was more stable on the temperature of 25℃～45℃.The Km value was 0.54mmol/L and Vmax was 0.49 ?mol/ml?min in the glycerol.
RESUMO
Arginine Deiminase(ADI) was purified to homogeneity using ammonium sulfate precipitation,Q-Sepharose Fast Flow anion exchange chromatography and SephadexG-75 gel filtration chromatography. This purification protocol resulted in a 34.5-fold purification of ADI with 31.4% final yield. A molecular weight of about 190 kD determined by native gradient polyacrylamide gel electrophoresis. The enzyme has only one kind of 46 kD subunit determined by SDS-PAGE. Combining the results from the two kinds of electrophoresis,the authors deduce that the enzyme may be a tetramer. The optimum pH and temperature for lipolytic activity of ADI was pH 6.5 and 50℃,respectively. It was extremely stable at 45℃ and retained 97.9% of its original activity for 30 min. The stability declined rapidly as soon as the temperature rose over 50℃. ADI was highly stable in the pH range from pH 5-8. ADI acted on L-arginine but not on D-arginine. ADI catabolism was dependent on metal ions. At their adequate concentration,Mn2+,Mg2+ and Co2+ were the effective promoter,while superfluous Zn2+and Co2+ inhibited ADI activity. L-citrulline did not act on ADI,but L-ornithine inhibited ADI activity. The degradation of L-arginine with ADI catalysis was according to simple Michaelis-Menten equation. The Michaelis constant was 3.2686 mmol/L and the maxi-mum velocity was 2.44 ?mol/min.