Engineering of Bacillus amyloliquefaciens α‐Amylase with Improved Calcium Independence and Catalytic Efficiency by Error‐Prone PCR

Abstract

Bacillus amyloliquefaciens α‐amylase (BAA) is one of the most important amylases and presents a wide range of applications in many processes involving starch‐liquefaction. However, its activity and thermostability largely depend on the existence of calcium ions, which is a major obstacle for its wide‐scale industrial applications. In the present study, to enhance the calcium independence of a mesophilic α‐amylase from B. amyloliquefaciens CICIM B2125, a novel calcium‐independent mutant Q264S is evolved by directed evolution using error‐prone polymerase chain reaction (PCR). Enzymatic properties and kinetic parameters of mutant Q264S were subsequently analyzed. Although the thermostability at 60°C and optimum temperature of Q264S are slightly decreased, its calcium independence and catalytic efficiency are significantly improved, with no apparent changes of specific activity and optimum pH compared with those of the wild‐type enzyme. Furthermore, three‐dimensional structure analysis showed that the improved calcium independence and catalytic efficiency of Q264S are most likely to be a subtle balance among the hydrophobic interactions, electrostatic forces, and hydrogen bonds around calcium‐binding sites and active‐site regions. To the best of our knowledge, this is the first report on increasing calcium independence of BAA by error‐prone PCR. These results could aid the understanding of mechanistic bases of calcium independence of BAA and thus facilitate the design and engineering of more efficient and robust calcium‐independent BAA variants.

Novel Thermotolerant Amylase from Bacillus licheniformis Strain LB04: Purification, Characterization and Agar-Agarose

“…The study of the calcium ions’ influence over α-amylase activity, demonstrated how enzymatic activity is partially reduced by EDTA additions. These results agree with previous studies on the Ca 2+ -independent α-amylase produced by native B. licheniformis species [ 38 , 39 ], indicating a partial independence from calcium ions due to a continuous starch hydrolysis despite a lower efficiency [ 40 , 41 ]. As pH increased, the activity dropped; an effect that could be attributed to the enzyme’s structure configuration, more specifically due to irregularities in segments belonging to the loop regions that participate in the metal-binding zones 177–199 [ 41 ].…”

“…These results agree with previous studies on the Ca 2+ -independent α-amylase produced by native B. licheniformis species [ 38 , 39 ], indicating a partial independence from calcium ions due to a continuous starch hydrolysis despite a lower efficiency [ 40 , 41 ]. As pH increased, the activity dropped; an effect that could be attributed to the enzyme’s structure configuration, more specifically due to irregularities in segments belonging to the loop regions that participate in the metal-binding zones 177–199 [ 41 ]. Amino acids in the domain B region were reported to play a fundamental role in thermostability [ 42 ].…”

Engineering ofBacillus amyloliquefaciensα‐Amylase for Improved Catalytic Efficiency by Error‐Prone PCR

“…The specific activity of BAA (103.75 U mg −1 ) was similar to that of the wild‐type BAA (112.5 U mg −1 ) of B. amyloliquefaciens reported by Wu et al. [ 16 ] Compared with the wild type, the specific activities of BAA28 and BAA294 increased by 40% and 62%, respectively ( Table 1 ). [ 8 ] A similar study was also reported by Wu et al.…”

Progress in Ameliorating Beneficial Characteristics of Microbial Cellulases by Genetic Engineering Approaches for Cellulose Saccharification

“…The resulting mutants are further screened for improved characteristics (Nannemann et al, 2011). This method has become a valuable tool for ameliorating various enzymes such as xylanases (Xu et al, 2016;Acevedo et al, 2017), laccases (Mateljak et al, 2019), phytases (Shivange et al, 2016;Körfer et al, 2018), amylases (Wu et al, 2018;Huang et al, 2019) and cellulases (Liu et al, 2014;Larue et al, 2016;Lin et al, 2016;Goedegebuur et al, 2017;Yang et al, 2017;Cao et al, 2018). Cao et al (2018) modified β-glucosidase (Ks5A7) for improved thermostability, catalytic efficiency and resistance to glucose inhibition.…”