Site-directed mutagenesis of the calcium-binding site of α-amylase of Bacillus licheniformis

Abstract

Amylases that are active under acidic conditions (pH <6), at higher temperatures (>70 degrees C) and have less reliance on Ca(2+) are required for starch hydrolysis. The alpha-amylase gene of Bacillus licheniformis MTCC 6598 was cloned and expressed in Escherichia coli BL21. The calcium-binding site spanning amino acid residues from 104 to 200 in the loop regions of domain B and D430 in domain C of amylase were changed by site-directed mutagenesis and the resultant mutant amylases were analyzed. Calcium-binding residues, N104, D161, D183, D200 and D430, were replaced with D104 and N161, N183, N200 and N430, respectively. Mutant amylase with N104D had a slightly decreased activity at 30 degrees C but a significantly improved specific activity at pH 5 and 70 degrees C, which is desirable character for a food enzyme. The amylase mutants with D183N or D200N lost all activity while the mutant amylase with D161N retained its activity at 30 degrees C but had significantly less activity at 70 degrees C. On the other hand, the activity of the mutant amylase with D430N was not changed at 30 degrees C but had an improved activity at 70 degrees C.

Significance of Tyr302, His235 and Asp194 in the α-amylase from Bacillus licheniformis

“…Priyadharshini and Gunasekaran (2007) reported that the metal-binding residues Asp161, Asp183, Asp200, and Asp430 of amylase were subject to site-directed mutagenesis, among which, D183N and D200N lose their amylase activity; only the activity of mutant amylase with D430N showed improved activity at 70°C. In the present study, three mutant sites were chosen.…”

Mutational analysis of the proposed calcium-binding aspartates of a truncated α-amylase from Bacillus sp. strain TS-23

“…As shown in 193,217,228,234,236, and 238 do not abolish the amylolytic activity but significantly affect the catalytic function of the enzyme. It has been reported that replacements of Asp183 and Asp200 in BLA with asparagine will cause a complete loss of the amylolytic activity (Priyadharshini and Gunasekaran 2007). However, substitutions of the corresponding residues, Asp217 and Asp234, with asparagine did not abolish BACΔNC activity.…”

“…Most of the mutations that change the thermostability are concentrated in domain B and its interface with domain A, where the triadic metal and the substrate-binding site are located (Declerck et al 2002). The recent studies further demonstrated that the mutations of the calcium-binding residues are highly deleterious to the thermostability of BLA and BAA (Liu et al 2010;Priyadharshini and Gunasekaran 2007). However, the mutational effects of the calcium-binding residues on BACΔNC properties have not been studied.…”

“…The third calcium-binding site (CaIII), which acts as a bridging ion, is located at the interface between domains A and C of α-amylases. The importance of the calcium-binding residues (Asp161, Asp183, Asp200, and Asp430) of BLA has been explored, and their Asn replacements exhibited a significant reduction of the enzymatic activity and thermostability (Priyadharshini and Gunasekaran 2007). The role of the calcium-binding residues of B. amyloliquefaciens α-amylase (BAA) have also been investigated, and the results demonstrated that residues Asp231, Asp233, and Asp438 are critical for the amylase activity as well as thermostability (Lee et al 2006;Liu et al 2010).…”

Quantitative digital image analysis of chromogenic assays for high throughput screening of α-amylase mutant libraries

“…E. coli BL21(DE3) (pBLA1), harbouring an a-amylase gene (amyL) (Priyadharshini and Gunasekaran 2007) was used. Eighty colonies were separately grown in a 96-well microplate containing 100 ll LB medium supplemented with 0.1 g ampicillin/l in each well.…”