A mutant α-amylase with only part of the catalytic domain and its structural implication

Abstract

A truncated mutant alpha-amylase, Xa-S2, was obtained from Xanthomonas campestris wild type alpha-amylases (Xa-WT) through random mutagenesis that contained 167 amino acid residues (approx 65% shorter than that of Xa-WT). Secondary structure prediction implied that Xa-S2, would be unable to form the whole (beta/alpha)(8)-barrel catalytic domain and did not have the three conserved catalytic residues of wild type alpha-amylase, but it still displays the starch-hydrolyzing activity. Xa-S2 was prepared, characterized and compared to the recombinant wild-type enzymes. The K (m) for starch was 32 mg/ml; activity was optimal at pH 6.2 and 30 degrees C. In contrast, the K (m) for starch of Xa-WT was 8 mg/ml and optimal enzyme activity was at pH 6.0-6.2 and 45-50 degrees C. Our results suggested that Xa-S2 is a new amylase with a minimal catalytic domain for hydrolyzing substrates with of alpha-1,4-glucosidic bonds.

Thermal stability and activity improvements of a Ca‐independent α‐amylase from Bacillus subtilisCN7 by C‐terminal truncation and hexahistidine‐tag fusion

“…A major type of these engineered α‐amylases is usually in their truncated forms, which is produced by naturally occurring spontaneous truncation or by man‐made protein truncation manipulation. All the existing truncated α‐amylases can be classified into three main categories: first, the original amylolytic activity was unchanged but the thermostability somehow affected ; second, the thermostability was increased or unchanged but the activity declined ; and third, both activity and thermostability were unchanged or declined . So far, there has been no report on simultaneous improvements of the thermostability and the activity of α‐amylase by applying only the truncation strategy.…”

A new group of glycoside hydrolase family 13 α-amylases with an aberrant catalytic triad

“…The absence of any one of the catalytic residues normally causes partial or complete loss of hydrolysis activity27. Remarkably, the mutant α-amylase from Xanthomonas campestris truncated from the C-terminal part of domain B and thus lacking any of the three conserved catalytic residues, still exhibited starch-hydrolyzing activity28, but that observation has never been supported by other examples.…”