Article Figures & Data
Figures
- FIG. 1.
Comparison of the primary structures and homologous regions of PFTA and related enzymes. (A) Schematic drawing of the primary structures and the four conserved regions of Bacillus stearothermophilus α-amylase, Thermus malogenic amylase, and PFTA. The N-terminal domains of Thermus malogenic amylase and PFTA are represented as dark boxes. Other boxes represent the four conserved regions in the amylolytic enzymes. (B) Comparison of amino acid residues in the conserved regions (I, II, III, and IV) of various amylolytic enzymes such as maltogenic amylase, cyclodextrinase, neopullulanase, and α-amylases. Invariant or highly conserved amino acids are emphasized by shaded boxes. In cyclodextrin- and pullulan-hydrolyzing enzymes, the catalytic amino acid residues are indicated by asterisks and substrate-binding amino acids are indicated by dots. BSTA, B. stearothermophilus α-amylase (1713273A); ThMA, Thermus sp. strain IM6501 maltogenic amylase (AAC15072); CD I-5, alkalophilic Bacillus sp. strain I-5 cyclodextrinase (AAA92925); TVAII, Thermoactinomyces vulgaris R-47 α-amylase II (1911217A); PwaA, Pyrococcus woesei α-amylase (AAD54338); TAA, Aspergillus oryzae α-amylase (CAA31218).
- FIG. 2.
SDS-PAGE analysis of recombinant PFTA at different stages of purification. Lane M, protein size standards; lane 1, cellular proteins from crude extract (pETPFTA-6h); lane 2, cellular proteins from crude extract (pETPFTA-6h and pRARE); lane 3, proteins after heat treatment; lane 4, purified PFTA after Ni-NTA column chromatography; lane 5, zymogram analysis with purified PFTA. PFTA was visualized in a zymogram developed by soaking the gel in 1% (wt/vol) soluble starch solution at 85°C followed by iodine solution (7). The enzyme activity was detected as a white band on the gel.
- FIG. 3.
Effect of pH on the activity and stability of PFTA. (A) For the determination of optimal pH, the following buffers were used: for pH 3.0 to 4.0, 50 mM sodium citrate (▪); for pH 4.0 to 6.0, 50 mM sodium acetate (▴); for pH 6.0 to 7.5, 50 mM sodium phosphate (•); and for pH 7.5 to 8.0, 50 mM Tris-HCl (♦). The values are shown as percentages of the maximum specific activity of PFTA observed at pH 4.5, which was taken as 100%. (B) To assess the pH stability of PFTA, the enzyme was incubated at the indicated pH in 0.1 M Britton-Robinson buffer and at 37°C for 24 h. The residual activity was measured at 90°C under the standard conditions of the assay. The values are shown as percentages of the original activity, which was taken as 100%.
- FIG. 4.
Effect of temperature on the activity and stability of PFTA. (A) Activity was measured at the temperatures indicated on the plot in the standard activity assay (in a water bath for measurements up to 90°C or in a silicon oil bath for measurements between 90 and 110°C). The values are shown as percentages of the specific activity of PFTA observed at 90°C, which was taken as 100%. (B) For determination of the thermostability of PFTA, purified enzyme (0.1 mg/ml) was incubated at 85°C (▪), 90°C (▴), 95°C (•), and 100°C (♦) in 50 mM sodium acetate buffer (pH 5.0). After various time intervals as indicated on the plot, samples were withdrawn, and the residual activity was measured at 90°C under the standard conditions of the assay.
- FIG. 5.
Differential scanning calorimetry of the recombinant PFTA. The recombinant PFTA was concentrated to 1 mg/ml in 50 mM sodium phosphate buffer (pH 7.0) with a Microcon filter (Millipore Corp.). The sample was scanned at temperatures from 25 to 125°C with a scan rate of 1°C/min.
- FIG. 6.
Hydrolysis pattern of PFTA on various substrates. Lane M, maltooligosaccharide standards (glucose to maltoheptaose); lane 1, maltotriose; lane 2, maltotetraose; lane 3, maltopentaose; lane 4, maltohexaose; lane 5, β-cyclodextrin; lane 6, pullulan; lane 7, soluble starch; lane 8, acarbose (Ac). Pan, panose; PTS, acarviosine-glucose. PFTA was reacted with various substrates at a concentration of 0.5% (wt/vol) at 90°C for 5 h.
- FIG. 7.
Change of the hydrolysis product of p-nitrophenyl-α-d-maltopentaoside (pNPG5) by PFTA as a function of reaction time. Lane M, maltooligosaccharide standards (glucose to maltoheptaose); lanes 1-6, hydrolysis product of p-nitrophenyl-α-d-maltopentaoside at different reaction times (0, 0.5, 1, 1.5, 2, and 4 h, respectively). PFTA was reacted with 0.5% (wt/vol) p-nitrophenyl-α-d-maltopentaoside at 90°C.
- FIG. 8.
Proposed modes of action of PFTA on various substrates.
Tables
- TABLE 2.
Kinetic parameters for hydrolysis of various substratesa
Substrate kcat (s−1) Km (mM) kcat/Km (s−1 mM−1) α-Cyclodextrin 241 ± 6 2.61 ± 0.17 92.3 β-Cyclodextrin 196 ± 5 2.16 ± 0.11 90.7 γ-Cyclodextrin 173 ± 4 5.12 ± 0.42 33.8 Maltotriose 268 ± 8 62.9 ± 3.8 4.3 Acarbose 228 ± 6 11.1 ± 1.1 20.5 Starchb 67 ± 1 0.52 ± 0.02 128.8 - TABLE 3.
Overall comparisons of cyclodextrin-hydrolyzing enzyme, PFTA, and α-amylasea
ThMA PFTA BSTA Action mode Maltose from reducing end Random Random Major product(s) from: Maltooligosaccharide G2 G3, G4 G2≈G5 Pullulan Panose Panose N.D. β-Cyclodextrin G2 G7 N.D. Acarbose PTS PTS N.D. Length of N-terminal domain (amino acids) 124 196 N.A. Oligomeric state Dimer Dimer Monomer Optimal temp (°C) 60 90 80 Optimal pH 6.0 4.5 5.5 ↵ a ThMA, maltogenic amylase from Thermus sp. strain IM6501; PFTA, thermostable amylase from P. furiosus, BSTA, thermostable α-amylase from B. stearothermophilus (29). N.D., not detected; N.A., not available.
