Article Figures & Data
Figures
- FIG. 1.
Comparison of amino acid residues in the conserved regions (I, II, III, and IV) of P. furiosus TA (PFTA) and various amylolytic enzymes. Invariant or highly conserved amino acids are shaded light gray. The amino acids corresponding to those investigated in this study are shown in bold. Invariant amino acids are shaded dark gray, and the residues in CD-hydrolyzing enzymes are boxed. For amylolytic enzymes, the catalytic amino acid residues are indicated by asterisks and substrate binding amino acids are indicated by circles. ThMA, Thermus sp. strain IM6501 maltogenic amylase (GenBank accession no. AAC15072); BSMA, Bacillus stearothermophilus maltogenic amylase (GenBank accession no. AAC46346); CD I-5, alkalophilic Bacillus sp. strain I-5 cyclodextrinase (GenBank accession no. AAA92925); BsCD, Bacillus sphaericus cyclodextrinase (GenBank accession no. CAA44454); BsNPL, Bacillus stearothermophilus neopullulanase (GenBank accession no. AAK15003); TVAII, Thermoactinomyces vulgaris R-47 α-amylase II (GenBank accession no. 1911217A); BSTA, Bacillus stearothermophilus α-amylase (GenBank accession no. 1713273A); BLTA, Bacillus licheniformis α-amylase (GenBank accession no. P06278); TAA, Aspergillus oryzae α-amylase (GenBank accession no. CAA31218); BcCGT, Bacillus circulans CGTase (GenBank accession no. CAA48401); BsCGT, Bacillus stearothermophilus CGTase (GenBank accession no. P31797); PfCGT, P. furiosus CGTase (GenBank accession no. AAL80602).
- FIG. 2.
Analyses of the reaction products formed from β-CD by P. furiosus TA mutant enzymes with amino acid changes H414N, G415E, and H414N and G415E. Each enzyme (0.7 U) was reacted with 0.5% (wt/vol) β-CD in 50 mM sodium acetate buffer (pH 4.5) at 75°C for 1 h. (A) TLC analyses of the reaction mixtures containing TA(H414N), TA(G415E), and TA(H414N/G415E). Lane M, maltooligosaccharide standards; lane 1, reaction mixture with TA(H414N); lane 2, reaction mixture with TA(G415E); lane 3, reaction mixture with TA(H414N/G415E). (B) HPAEC analysis of the reaction mixture containing P. furiosus TA(H414N/G415E). MO standard, maltooligosaccharide standard; pullulan hydrolysate, products of the partial hydrolysis of pullulan by a pullulanase; after β-amylase, β-amylase-treated reaction mixture with TA(H414N/G415E).
- FIG. 3.
TLC analyses of the reaction products formed from various substrates by wild-type P. furiosus TA (A) and P. furiosus TA(H414N/G415E) (B). Lanes M, maltooligosaccharide standards (glucose to maltoheptaose); lanes 1, maltose; lanes 2, maltotriose; lanes 3, maltotetraose; lanes 4, maltopentaose; lanes 5, maltohexaose; lanes 6, maltoheptaose, lanes 7, α-CD; lanes 8, β-CD; lanes 9, γ-CD. Each enzyme (1.5 U) was reacted with each substrate at a concentration of 0.5% (wt/vol) in 50 mM sodium acetate buffer (pH 4.5) at 75°C for 2 h.
- FIG. 4.
TLC analyses of the reaction products of wild-type P. furiosus TA (PFTA WT), P. furiosus TA(D440H) (PFTA-D440H), and P. furiosus TA(M439W/D440H) (PFTA-M439W/D440H). Lane M contains maltooligosaccharide standards (glucose to maltoheptaose). Each aliquot was taken at different reaction times of 0, 2, 10, 20, and 60 min. Each enzyme (0.5 U) was reacted with β-CD at a concentration of 0.5% (wt/vol) in 50 mM sodium acetate buffer (pH 4.5) at 75°C.
Tables
- TABLE 1.
Oligonucleotides used for site-directed mutagenesis
Oligonucleotide DNA sequence (5′ to 3′)a H414N-f TGGATGTTGCTAATGGGGTTCCTCCAGAA H414N-r TTCTGGAGGAACCCCATTAGCAACATCCA G415E-f TGGATGTTGCTCATGAGGTTCCTCCAGAA G415E-r TTCTGGAGGAACCTCATGAGCAACATCCA H414N/G415E-f ATGGATGTTGCTAATGAGGTTCCTCCAGAA H414N/G415E-r TTCTGGAGGAACCTCATTAGCAACATCCAT M439W-f AATTGGAGAAGTTTGGGATGATGCAAGGT M439W-r ACCTTGCATCATCCCAAACTTCTCCAATT D440H-f AATTGGAGAAGTTATGCATGATGCAAGGT D440H-r ACCTTGCATCATGCATAACTTCTCCAATT M439W/D440H-f AATTGGAGAAGTTTGGCATGATGCAAGGT M439W/D440H-r ACCTTGCATCATGCCAAACTTCTCCAATT ↵ a Mutated codons are underlined.
- TABLE 2.
Kinetic parameters of wild-type P. furiosus TA and its variant formsa
Form of TA β-Cyclodextrin Maltoheptaose kcat (s−1) Km (mM) kcat/Km (s−1 mM−1) kcat (s−1) Km (mM) kcat/Km (s−1 mM−1) Wild type 610 6 100 160 14 11 D440H variant 64 4 16 170 13 13 M439W/D440H variant 23 2 11 55 1.8 31 ↵ a Kinetic parameters were determined by using 50 mM sodium acetate buffer (pH 4.5) at 85°C. Errors for the data in this table range from 5 to 15%.
