<b>Effect of temperature on subsite map of <i>Bacillus licheniformis</i></b><b>a</b><b>-amylase</b>
2006
Kandra, Lili | Remenyik, Judit | Gyémánt, Gyöngyi | Lipták, A. | Lipták, A. | Lipták, A.
To elucidate how temperature effects subsite mapping of a thermostable a-amylase from <i>Bacillus licheniformis</i> (BLA), a comparative study was performed by using 2-chloro-4-nitrophenyl (CNP) b-maltooligosides with degree of polymerisation (DP) 4-10 as model substrates. Action patterns, cleavage frequencies and subsite binding energies were determined at 50 °C, 80 °C and 100 °C. Subsite map at 80 °C indicates more favourable bindings compared to the hydrolysis at 50 °C. Hydrolysis at 100 °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-G<sub>1</sub> was never formed. In the preferred binding mode of shorter oligomers, CNP-G<sub>2</sub> serves as the leaving group (79%, 50%, 59% and 62% from CNP-G<sub>4</sub>, CNP-G<sub>5</sub>, CNP-G<sub>6</sub> and CNP-G<sub>7</sub>, respectively), while CNP-G<sub>3</sub> is the dominant hydrolysis product from CNP-G<sub>8</sub>, CNP-G<sub>9</sub>, and CNP-G<sub>10</sub> (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-G<sub>2</sub>. Subsite mapping at 80 °C and 100 °C confirms that there are no further binding sites despite the presence of longer products.
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