Screening, Gene Cloning, and Characterizations of an Acid-Stable ��-Amylase

Abstract

Based on its α-amylase activity at pH 5.0 and optimal pH of the crude enzyme, a strain (named B-5) with acid α-amylase production was screened. The B-5 strain was identified as Bacillus amyloliquefaciens through morphological, physiological, and biochemical characteristics analysis, as well as 16S rDNA phylogenetic analysis. Its α-amylase gene of GenBank Accession No. GU318401 was cloned and expressed in Escherichia coli. The purified recombinant α-amylase AMY-Ba showed the optimal pH of 5.0, and was stable at a pH range of 4.0-6.0. When hydrolyzing soluble starch, amylose, and amylopectin, AMY-Ba released glucose and maltose as major end products. The α-amylase AMY-Ba in this work was a different type from the well-investigated J01542 (GenBank Accession No.)-type α-amylase from the same species. AMY-Ba exhibited notable adsorption and hydrolysis ability towards various raw starches. Structure analysis of AMY-Ba suggested the presence of a new starch-binding domain at its C-terminal region.

Effect of differential processing of the native and recombinant α-amylase from Bacillus amyloliquefaciens JJC33M on specificity and enzyme properties

“…4a). In general, native and recombinant a-amylases from Bacillus show optimum activity at neutral or slightly acidic pH, as has been reported for AmyJ33 (optimum pH 5.0) (Herna ´ndez-Heredia and del Moral 2016) AmyQ A and B (optimum pH 7 and 7.5, respectively) (Liu et al 2015); AmyQ (optimum pH 5-7) (Sun et al 2010), AMY-Ba (optimum pH 5) (Liu et al 2015). It is also known that the activity in the most of native a-amylase decreases rapidly at alkaline pH values (Bessler et al 2003;Goyal et al 2005), which seems to apply for some recombinant enzymes (Yang et al 2012) but not for AmyJ33r.…”

“…TS 23 a-amylase lost the N-terminal when it was expressed in E. coli (Lin et al 1997); in further research, the authors found that the N-terminal is essential for translocation of the enzyme to the culture medium in E. coli (Lo et al 2001). In contrast, the B. amyloliquefaciens a-amylase, AMY-Ba, with 95% of identity with AmyJ33r, was cloned without the signal peptide and expressed in E. coli BL21; the protein of 66 kDa produced was located in the intracellular extract (Liu et al 2015). In the same way, the recombinant a-amylase from B. aquimaris (BaqA, without signal peptide) expressed in the same system was located in the intracellular extract (Puspasari et al 2012).…”

“…AmyJ33 enzymatic activity was reduced 20% to the highest concentration used of EDTA (10 mM); instead, AmyJ33r was not affected in neither of the EDTA concentrations tested. Some a-amylases lost their activity on 1 mM EDTA and others require EDTA concentrations higher (10 mM) and long incubation times (60 h) (Aygan et al 2008;Gangadharan et al 2009;Liu et al 2015). EDTA a-amylases sensitivity varies according to the calcium sites located in the enzyme (Gupta et al 2003).…”

“…Native a-amylases have been isolated from different strains of B. amyloliquefaciens and their substrate specificity towards raw starch or soluble starch has been evaluated (Gangadharan et al 2009; Quintero Moreno and Gutie ´rrez Sa ´nchez 2010; Deb et al 2013). Recombinant aamylases have been produced in Escherichia coli and also characterized (Demirkan et al 2005;Liu et al 2015). Some studies have been conducted to understand the role of some residues in calcium-binding and thermal stability (Sun et al 2010;Zonouzi et al 2013).…”

Overexpression of an endogenous raw starch digesting mesophilic α‐amylase gene in Bacillus amyloliquefaciens Z3 by in vitro methylation protocol

“…Similar properties of temperature and pH were found in the previous study of Amy1 from B. amyloliquefacien JH‐06 . However, after pre‐incubation at 60 °C, AmyZ3 showed better thermostability compared to AMY‐Ba from B. amyloliquefacien B‐5 . α ‐Amylases find a broad range of applications because of their versatilely pH and temperature characteristics .…”