| Author |
Zhang D, Tu T, Wang Y, Li Y, Luo X, Zheng F, Wang X, Bai Y, Huang H, Su X, Yao B, Zhang T, Luo H.
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| Abstract |
A novel α-amylase, Amy13A, that consists of these domains was identified in Talaromyces leycettanus JCM12802: catalytic TIM-barrel fold, domain B, domain C, Thr/Ser-rich linker region, and C-terminal CBM20 domain. The wild type and three mutant enzymes were then expressed in Pichia pastoris GS115 to identify the roles of linker length (Amy13A21 and Amy13A33) and CBM20 (Amy13A-CBM) in catalysis. All enzymes had similar enzymatic properties, exhibiting optimal activities at pH 4.5-5.0 and 55-60 °C, but varied in catalytic performance. When using soluble starch as the substrate, Amy13A21 and Amy13A33 showed specific activities (926.3 and 537.8 units/mg, respectively, vs 252.1 units/mg) and catalytic efficiencies (kcat/Km, 25.7 and 22.0 mL s-1 mg-1, respectively, vs 15.4 mL s-1 mg-1) higher than those of the wild type, while Amy13A-CBM performed worse during catalysis. This study reveals the key roles of the CBM and linker length in the catalysis of GH13 α-amylase.
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