A chimeric α-amylase engineered from Bacillus acidicola and Geobacillus thermoleovorans with improved thermostability and catalytic efficiency

Abstract

The α-amylase (Ba-amy) of Bacillus acidicola was fused with DNA fragments encoding partial N- and C-terminal region of thermostable α-amylase gene of Geobacillus thermoleovorans (Gt-amy). The chimeric enzyme (Ba-Gt-amy) expressed in Escherichia coli displays marked increase in catalytic efficiency [K cat: 4 × 104 s−1 and K cat/K m: 5 × 104 mL−1 mg−1 s−1] and higher thermostability than Ba-amy. The melting temperature (T m) of Ba-Gt-amy (73.8 °C) is also higher than Ba-amy (62 °C), and the CD spectrum analysis revealed the stability of the former, despite minor alteration in secondary structure. Langmuir–Hinshelwood kinetic analysis suggests that the adsorption of Ba-Gt-amy onto raw starch is more favourable than Ba-amy. Ba-Gt-amy is thus a suitable biocatalyst for raw starch saccharification at sub-gelatinization temperatures because of its acid stability, thermostability and Ca2+ independence, and better than the other known bacterial acidic α-amylases.

Production of Chimeric Acidic α-Amylase by the Recombinant Pichia pastoris and Its Applications

“…P. pastoris X-33 (genotype: wild type) (Invitrogen, Carlsbad, CA, USA) was used as the expression host. E. coli clone harboring chimeric α-amylase gene (Ba-Gt-amy) generated earlier (Parashar and Satyanarayana, 2016b) was procured from the laboratory culture collection. pPICZαA vector (Invitrogen) was used as a cloning and expression vector.…”

“…The activity of α-amylase was determined according to Parashar and Satyanarayana (2016b). The reaction mixture consisted of 0.5 mL of 0.5% potato starch (Sigma) prepared in 100 mM sodium acetate buffer (pH 4.0) and 0.5 mL appropriately diluted enzyme (0.21 U), incubated for 15 min at 60°C.…”

Raw starch degrading α-amylases: an unsolved riddle

“…This chimeric enzyme Ba-Gt-amy was expressed in E. coli and tested on 25% raw wheat and corn starch slurries and hydrolysis of 29.5% and 32.3% was achieved, respectively. For comparison, Bacillus acidicola α-amylase Ba-amy had 12% and 8% hydrolysis yield, respectively, under same conditions [62].…”

“…Fusion can improve catalytic efficiency, stability, activity, expression, secretion, and solubility of enzymes [82]. In one of such investigations, a chimera was engineered by fusing the truncated acidic α-amylase gene of Bacillus acidicola Ba-amy with partial N-and C-terminal regions of the thermostable α-amylase gene Gt-amy of G. thermoleovorans [62] (Table 1). This chimeric enzyme Ba-Gt-amy was expressed in E. coli and tested on 25% raw wheat and corn starch slurries and hydrolysis of 29.5% and 32.3% was achieved, respectively.…”

“…There are not many α-amylases that are able to perform effectively on high concentration of raw starch (>25%) [28, [60][61][62][63][64][65] (Table 1), due to substrate or product inhibition [66] and because of the need for higher enzyme concentrations in raw starch digestion, which have to be obtained through the optimized upstream and downstream processing to obtain high quantity of biocatalyst. It is likely that during amylase attack on starch granule, enzymestarch physical, irreversible or non-catalytic binding may be both, productive and non-productive, which is why high enzyme loadings give rise to the formation of productive interactions [8].…”