Improving the Product Specificity of Maltotetraose-Forming Amylase from Pseudomonas saccharophila STB07 by Removing the Carbohydrate-Binding Module

Maltotetraose (G4) is composed of four glucose units linked by the α-1,4-glycosidic bond, which has excellent adaptability in food processing and specific physiological functions. Maltotetraose-forming amylases (MFAses) are used in the industry as a promising tool for G4 production. The MFAse from Pseudomonas saccharophila STB07 (MFAPS), which belongs to the GH13, can preferentially hydrolyze substrates to G4. MFAPS contains a carbohydrate-binding module (CBM). In this study, we removed the CBM to obtain the mutant MFAPS-ΔCBM. We explored the aspects affecting the catalytic performance of enzymes through structural simulations and molecular docking. Results showed that when the CBM was removed, the thermal stability of MFAPS was slightly reduced, and its catalytic ability for long-chain substrates, such as corn starch, was significantly reduced. However, the catalytic ability and product specificity of the substrates with shorter chain length, such as maltodextrin (DE 7-9), were improved. The G1-G7 (glucose (G1), maltose (G2), maltotriose (G3), maltotetraose (G4), maltopentaose (G5), maltohexaose (G6), and maltoheptaose (G7)) contents and G4 proportion of the mutant MFAPS-ΔCBM reaction at 24 h were 11.1 and 11.6% higher than those of MFAPS, respectively. The results also showed that the forces of MFAPS on the substrate near the -4, -1, +1, and +3 subsites were critical for its product specificity.