Effect of temperature on subsite map of Bacillus licheniformis alpha-amylase.
To elucidate how temperature effects subsite mapping of a thermostable alpha-amylase from Bacillus licheniformis (BLA), a comparative study was performed by using 2-chloro-4-nitrophenyl (CNP) beta-maltooligosides with degree of polymerisation (DP) 4-10 as model substrates. Action patterns, cleavage frequencies and subsite binding energies were determined at 50 degrees C, 80 degrees C and 100 degrees C. Subsite map at 80 degrees C indicates more favourable bindings compared to the hydrolysis at 50 degrees C. Hydrolysis at 100 degrees C resulted in a clear shift in the product pattern and suggests significant differences in the active site architecture. Two preferred cleavage modes were seen for all substrates in which subsite (+2) and (+3) were dominant, but CNP-G1 was never formed. In the preferred binding mode of shorter oligomers, CNP-G2 serves as the leaving group (79%, 50%, 59% and 62% from CNP-G4, CNP-G5, CNP-G6 and CNP-G7, respectively), while CNP-G3 is the dominant hydrolysis product from CNP-G8, CNP-G9, and CNP-Gl0 (62%, 68% and 64%, respectively). The high binding energy value (-17.5 kJ/mol) found at subsite (+2) is consistent with the significant formation of CNP-G2. Subsite mapping at 80 degrees C and 100 degrees C confirms that there are no further binding sites despite the presence of longer products.
Kandra
Lili
L
Department of Biochemistry, Faculty of Sciences, University of Debrecen, P.O. Box 55, Debrecen, Hungary.
Remenyik
Judit
J
Gyémánt
Gyöngyi
G
Lipták
A
A
D001407:Bacillus / Q000201:enzymology*; D001665:Binding Sites; D001671:Biochemistry / Q000379:methods; D002845:Chromatography; D006868:Hydrolysis; D008956:Models, Chemical; D009596:Nitrophenols / Q000494:pharmacology; D009844:Oligosaccharides / Q000737:chemistry; D011485:Protein Binding; D013696:Temperature; D000516:alpha-Amylases / Q000737:chemistry*
D009596:Nitrophenols; D009844:Oligosaccharides; C021705:maltooligosaccharides; C068311:4-chloro-2-nitrophenol; D000516:alpha-Amylases; C501919:alpha-amylase, Bacillus licheniformis