No 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.

The LacLM β-galactosidase of Lactobacillus fermentum K4 is encoded by 2 consecutive genes, lacL (large subunit) and lacM (small subunit), that share 17 overlapping nucleotides. Phylogenetic analysis revealed that this enzyme was closely related to other Lactobacillus β-galactosidases and provided significant insight into its common and distinct characteristics. We cloned both the lacL and lacM genes of L. fermentum K4 and heterologously expressed each in Escherichia coli, although the recombinant enzyme was only functional when both were expressed on the same plasmid. We evaluated the enzymatic properties of this species-specific LacLM β-galactosidase and discovered that it acts as both a hydrolase, bioconverting lactose into glucose and galactose, and a transgalactosylase, generating prebiotic galacto-oligosaccharides (GOS). The recombinant β-galactosidase showed a broad pH optimum and stability around neutral pH. The optimal temperature and Michaelis constant (K(m)) for the substrates o-nitrophenyl-β-D-galactopyranoside and lactose were, respectively, 40°C and 45 to 50°C and 1.31 mM and 27 mM. The enzyme activity was stimulated by some cations such as Na⁺, K⁺, and Mg²⁺. In addition, activity was also enhanced by ethanol (15%, wt/vol). The transgalactosylation activity of L. fermentum K4 β-galactosidase effectively and rapidly generated GOS, up to 37% of the total sugars from the reaction. Collectively, our results suggested that the β-galactosidase from L. fermentum K4 could be exploited for the formation of GOS.