E on the host E. coli. When the recombinant TK-PUL was analyzed by size exclusion chromatography foraem.asm.orgApplied and Environmental MicrobiologyThermoacidophilic Pullulanase from T. kodakarensisFIG 1 Regions conserved amongst pullulanases and other amylolytic enzymes. Three amino acid residues critical for catalytic activity are marked by a hash symbol (#), identical residues are shown in white with a black background, and similar residues are shown in black having a gray background. The sequences, identified by their UniProt accession numbers, are as follows: Q5JID9, TK-PUL; Q9P9A0, pullulan hydrolase type III from T. aggregans; Q9HHB0, pullulanases from D. mucosus; P32818, maltogenic -amylase from B. cidopullulyticus; P29964, cyclomaltodextrin hydrolase from Thermoanaerobacter ethanolicus; Q08751, neopullulanase from Thermoactinomyces vulgaris; P38940, neopullulanase from B. stearothermophilus; Q57482, neopullulanase from Bacillus species; Q45490, maltogenic amylase from G. stearothermophilus; and Q819G8, neopullulanase from Bacillus cereus.Oxibendazole manufacturer molecular mass determination, it eluted at a retention volume of 13.Dynorphin A site 7 ml, corresponding to a molecular mass of 84 kDa, indicating that TK-PUL existed inside a monomeric kind. Biochemical characteristics of TK-PUL. Examination in the enzyme activity of TK-PUL at numerous pH values revealed that it was active more than a broad pH variety (3.0 to eight.five). The highest pullulanase and -amylase activities had been observed at pH three.5 in acetate buffer and 4.2 in citrate buffer (Fig. two). Regardless of showing the highest activity in acidic pH, TK-PUL was additional steady at alkaline pH,and it displayed 84 , 77 , and 57 from the maximal activity just after 56 h of incubation at 4 and pH values of 8.five, six.5, and 4.two, respectively (data not shown). When the enzyme activity was examined at different temperatures together with the pH kept continuous either at 4.2 (in citrate buffer) or 6.5 (in acetate buffer), TK-PUL exhibited the highest activities at 95 at pH 4.two and 100 at pH six.five. A lot more than 50 activity was observed even at 120 (Fig. three). Prolonged incubation at elevated temperatures demonstrated that TK-PUL was particularly thermo-FIG two Comparison of pullulanase and -amylase activities of recombinant TK-PUL at numerous pH values in sodium citrate (), sodium acetate (OE), and sodiumphosphate (OE) buffers.PMID:25558565 Every single buffer was used at a final concentration of 50 mM. Pullulanase activity is shown by solid lines, although dotted lines represent amylase activity.February 2014 Volume 80 Numberaem.asm.orgAhmad et al.FIG three Comparison of pullulanase and -amylase activities of recombinant TK-PUL at many temperatures. (a) Activity in sodium citrate buffer, pH four.2. (b)Activity in sodium acetate buffer, pH 6.five. Pullulanase activity is shown by strong lines, though dotted lines represent amylase activity.steady, displaying far more than 90 activity even right after 10 h of incubation at 90 in sodium acetate buffer of pH 6.5. The half-life of TK-PUL was 45 min at one hundred when incubated either at pH six.five or 8.5. Beneath optimal circumstances, TK-PUL displayed a certain activity of 70.five mol min 1 mg 1. The Km worth toward pullulan was calculated to be 2 mg ml 1, plus the Vmax was 109 U mg 1. Effects of metal ions and other reagents on TK-PUL activity. There was no important difference inside the enzyme activity of TKPUL inside the presence or absence of Ca two, which indicated that, unlike other amylolytic enzymes, recombinant TK-PUL doesn’t rely on calcium for its activity. The activity of TK-PUL wa.
