Abstract
High oxidative stability and catalytic efficiency are required for the alkaline α-amylases to keep the enzymatic performance under the harsh conditions in detergent industries. In this work, we attempted to significantly improve both the oxidative stability and catalytic efficiency of an alkaline α-amylase from Alkalimonas amylolytica by engineering the five oxidation-prone methionine residues around the catalytic domain via a systematic approach. Specifically, based on the tertiary structure analysis, five methionines (Met 145, Met 214, Met 229, Met 247 and Met 317) were individually substituted with oxidation-resistant threonine, isoleucine and alaline, respectively. Among the created 15 mutants, 7 mutants M145A, M145I, M214A, M229A, M229T, M247T and M317I showed significantly enhanced oxidative stability or catalytic efficiency. In previous work, we found that the replacement of M247 with leucine could significantly improve the oxidative stability. Thus, these 8 positive mutants (M145A, M145I, M214A, M229A, M229T, M247T, M247L and M317I) were used to conduct the second round of combinational mutations. Among the constructed 85 mutants (25 two-point mutants, 36 three-point mutants, 16 four-point mutants and 8 five-point mutants), the mutant M145I-214A-229T-247T-317I showed a 5.4-fold increase in oxidative stability and a 3.0-fold increase in catalytic efficiency. Interestingly, the specific activity, alkaline stability and thermal stability of this mutant were also increased. The increase of salt bridge and hydrogen bonds around the catalytic domain contributed to the significantly improved catalytic efficiency and stability, as revealed by the three-dimensional structure model of wild-type alkaline α-amylase and its mutant M145I-214A-229T-247T-317I. With the significantly improved oxidative stability and catalytic efficiency, the mutant M145I-214A-229T-247T-317I has a great potential as a detergent additive, and this structure-guided systems engineering strategy may be useful for the protein engineering of the other microbial enzymes to fulfill industrial requirements.
Highlights
The a-amylases (1, 4-a-D-glucan glucanohydrolase; EC 3.2.1.1) responsible for starch-hydrolyzing are being widely utilized in the food, textile and pharmaceutical industries [1]
We have shown that the oxidative stability can be enhanced to a certain extent by replacing these five methionine residues with serine or leucine, while the catalytic efficiency of most mutants decreased [10,11]
These might be the main reason why the enzymes were more compatible with solid washing powder than liquid detergents. This observation agreed well with other studies where the activity of AmyUS100DIG/M197A from Geobacillus stearothermophilus was increased by 10,20% after incubation in the presence of Lav+ and Nadhif [7]. We significantly improved both the oxidative stability and catalytic efficiency by a systems protein engineering strategy based on the protein structure information
Summary
The a-amylases (1, 4-a-D-glucan glucanohydrolase; EC 3.2.1.1) responsible for starch-hydrolyzing are being widely utilized in the food, textile and pharmaceutical industries [1]. Alkaline a-amylases are of particular importance due to the high catalytic capability under alkaline conditions, and find wide applications in detergent and textile industries [2,3,4]. Oxidative stability is one of the most important parameters for alkaline a-amylase, especially for its application in detergents and textile industries where the washing environment is oxidative [5]. The methionine and cysteine residues of a-amylase are oxidized to sulfoxide under the oxidative conditions [6]. TS-23 a-amylase with leucine enhanced the oxidative stability by 2.4-fold [8]. In most cases the enhanced oxidative stability is at the cost of decreased catalytic efficiency or specific activity. The specific activity of a-amylase from Geobacillus stearothermophilus US100 decreased from 1, 000 to
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