Sequence analysis and biochemical properties of an acidophilic and hyperthermophilic amylopullulanase from Thermofilum pendens.

The acidophilic and thermophilic pullulanases have many potential applications in the processes of starch liquefaction and saccharification. In this study, a gene encoding an amylopullulanase from Thermofilum pendens (TPApu) was heterologously expressed in Escherichia coli. Although TPApu possessed the same continuous GH57N_Apu domain and the succeeding α-helical region as other two amylopullulanases from Staphylothermus marinus (SMApu) and Caldivirga maquilingensis (CMApu), it only showed maximal amino acid identities of 25.7-28.7% with CMApu and SMApu. The purified TPApu appeared as a single band of SDS-PAGE with a molecular mass of 65.5kDa and exhibited the maximal activity at pH3.5 and 95-100°C. TPApu had the highest catalytic efficiency towards pullulan (kcat/km, 8.79s-1mLmg-1) and α-cyclodextrin (kcat/km, 0.36s-1mM-1). In the initial stages, the ring-opening reactions of γ-cyclodextrin, 6-O-glucosyl-ß-cyclodextrin, 6-O-maltosyl-ß-cyclodextrin and the debranching reactions of 6-O-maltooctaosyl-ß-cyclodextrin were firstly catalyzed. In the subsequent reactions, a serial of maltooligosaccharides were produced. As the most acidophilic amylopullulanase among thermophilic pullulanases reported to date, TPApu preferred to debranch the DP6-12 side chains of amylopectin at pH4.5 and 100°C.

Characterization and application of an acidophilic and thermostable ß-glucosidase from Thermofilum pendens.

The gene encoding a ß-glucosidase from the archaeon Thermofilum pendens (Tpbgl) was cloned and expressed in Escherichia coli. The purified recombinant enzyme had a molecular mass of 77.8 kDa and released glucose or mannose from p-nitrophenyl-ß-d-glucopyranoside (pNPG), cellobiose, mannobiose, and genistin. Peak Tpbgl activity was detected at 90°C, and 50% activity remained after incubation for 60 min at 95°C. The optimal pH for pNPG hydrolysis was 3.5. When the enzyme was incubated with pNPG in the presence of ethanol and propanol, the glucose moiety was transferred to acceptor alcohols. Tpbgl is the archaeal ß-glucosidase from glucoside hydrolase family 3 and found to be most heat stable under extremely acidic conditions (pH 3.5). The kinetic parameters revealed that Tpbgl had the highest catalytic efficiency toward pNPG (kcat/Km = 3.05) with strong substrate affinity for such natural substrates as cellobiose (Km = 0.149) and mannobiose (Km = 0.147). Genistin solubilized in 10-40% DMSO was hydrolyzed to genistein with nearly 99% conversion, indicating that high concentrations of the water-insoluble isoflavone glycoside can be treated by the enzyme. Our results indicate that Tpbgl has great potential in cellulose saccharification and the glucoside hydrolysis of natural compounds.

Characterization of a recombinant amylolytic enzyme of hyperthermophilic archaeon Thermofilum pendens with extremely thermostable maltogenic amylase activity.

A gene (Tpen_1458) encoding a putative alpha amylase from hyperthermophilic archaeon Thermofilum pendens (TfMA) was cloned and expressed in Escherichia coli. The recombinant amylolytic enzyme was purified by Ni-NTA affinity chromatography and its catalytic properties were examined. Purified TfMA was extremely thermostable with a half-life of 60 min at an optimal temperature of 95 degrees C. TfMA activity increased to 136% in the presence of 5 mM CaCl(2). Maximal activity was measured toward gamma-cyclodextrin with a specific activity of 56 U/mg using copper bicinchoninate method. TfMA catalyzed the ring-opening reaction by cleaving one alpha-1,4-glycosidic linkage of cyclodextrin to produce corresponding single maltooligosaccharide at the initial time. The final products from cyclodextrins, linear maltooligosaccharides, and starch were glucose and maltose, and TfMA could also degrade pullulan and amylase inhibitor acarbose to panose and acarviosine-glucose, respectively. These results revealed that TfMA is a novel maltogenic amylase.

Hydrogenases of the model hyperthermophiles.

Hydrogenases are enzymes found in all domains of life that catalyze a remarkably simple chemistry, the reversible oxidation of molecular hydrogen to protons and electrons. In order to perform this chemistry, cells have evolved, several different times, intricate organometal complexes built around a binuclear Ni-Fe or Fe-Fe center, with bound CO and CN(-) groups, as well as multiple FeS centers. These complicated enzymes have been an area of intense study for many decades, with interest peaking on the occasions of major increases in national energy costs. Interest in biologically generated hydrogen as a potential substitute for fossil fuels is again at the forefront, and the new tools of the postgenomic world available for manipulating these enzymes make it a truly viable possibility. Hydrogenases from hyperthermophilic microorganisms such as Pyrococcus furiosus and Thermotoga maritima, with optimal growth temperatures near 100 degrees C, are of particular interest and promise for elucidating and manipulating these enzymatic mechanisms.

Properties of an amylase from thermophilic Bacillus SP

Alpha-Amylase production by thermophilic Bacillus sp strain SMIA-2 cultivated in liquid cultures containing soluble starch as a carbon source and supplemented with 0.05 percent whey protein and 0.2 percent peptone reached a maximum activity at 32 h, with levels of 37 U/mL. Studies on the amylase characterization revealed that the optimum temperature of this enzyme was 90ºC. The enzyme was stable for 1 h at temperatures ranging from 40-50ºC while at 90ºC, 66 percent of its maximum activity was lost. However, in the presence of 5 mM CaCl2, the enzyme was stable at 90ºC for 30 min and retained about 58 percent residual activity after 1 h. The optimum pH of the enzyme was found to be 8.5. After incubation of enzyme for 2 h at pH 9.5 and 11.0 was observed a decrease of about 6.3 percent and 16.5 percent of its original activity. At pH 6.0 the enzyme lost about 36 percent of its original activity. The enzyme was strongly inhibited by Co2+, Cu2+ and Ba2+, but less affected by Mg2+, Na+ and K+. In the presence of 2.0 M NaCl, 63 percent of amylase activity was retained after 2 h incubation at 45ºC. The amylase exhibited more than 70 percent activity when incubated for 1 h at 50ºC with sodium dodecyl sulphate. However, very little residual activity was obtained with sodium hypochlorite and with hydrogen peroxide the enzyme was completely inhibited. The compatibility of Bacillus sp SMIA-2 amylase with certain commercial detergents was shown to be good as the enzyme retained 86 percent, 85 percent and 75 percent of its activity after 20 min incubation at 50ºC in the presence of the detergent brands Omo®, Campeiro® and Tide®, respectively.

A produção de alfa-amilase por um termofilico, Bacillus sp SMIA-2, cultivado em meio l¨ªquido contendo amido sol¨²vel como fonte de carbono, alcançou uma atividade m¨¢xima de 37 U/mL em 32 horas. Estudos sobre a caracterização da amilase revelaram que a temperatura ¨®tima desta enzima foi 90ºC. A enzima foi est¨¢vel por 1 hora a temperaturas de 40 e 50ºC enquanto a 90ºC, 66 por cento da atividade m¨¢xima foi perdida. Entretanto, na presença de 5 mM de CaClð2, a enzima foi est¨¢vel a 90ºC por 30 minutos e manteve cerca de 58 por cento de sua atividade residual por 1 hora. O pH ¨®timo da enzima encontrado foi de 8.5. Ap¨®s a incubação da enzima por 2 horas a pH 9.5 e 11.0 foi observado um decr¨¦scimo de aproximadamente 6.3 por cento e 16.5 por cento da atividade original. Em pH 6.0 a enzima perdeu cerca de 36 por cento de sua atividade original. A enzima foi fortemente inibida por Co2+, Cu2+, e Ba2+, por¨¦m pouco afetada por Mg2+, Na+ e K+. Na presença de 2.0 M de NaCl, 63 por cento da atividade da amilase foi mantida ap¨®s 2 horas de incubação a temperatura de 45ºC. A amilase exibiu atividade acima de 70 por cento quando incubada por 1 hora a 50ºC em presença de s¨®dio dodecil sufato (SDS). Entretanto, uma baixa atividade residual foi obtida quando na presença do hipoclorito de s¨®dio e uma completa inibição quando a enzima foi incubada em per¨®xido de hidrog¨ºnio. A compatibilidade da amilase produzida pelo Bacillus sp SMIA-2, em relação a alguns detergentes comerciais mostrou que a enzima manteve 86 por cento, 85 por cento, e 75 por cento da atividade ap¨®s 20 minutos de incubação a 50ºC na presença dos detergentes Omo®, Campeiro® e Tide®, respectivamente.