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Protein engineering of selected residues from conserved sequence regions of a novel Anoxybacillus α-amylase

Goh, Kian Mau and Shahir, Shafinaz and Velayudhan, Ranjani and Chai, Kian Piaw and Stefan, Janecek and Raja Abdul Rahman, Raja Noor Zaliha and Kok, Gan Chan (2014) Protein engineering of selected residues from conserved sequence regions of a novel Anoxybacillus α-amylase. Scientific Reports, 4 . ISSN 2045-2322

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Official URL: http://dx.doi.org/10.1038/srep05850

Abstract

The α-amylases from Anoxybacillus species (ASKA and ADTA), Bacillus aquimaris (BaqA) and Geobacillus thermoleovorans (GTA, Pizzo and GtamyII) were proposed as a novel group of the α-amylase family GH13. An ASKA yielding a high percentage of maltose upon its reaction on starch was chosen as a model to study the residues responsible for the biochemical properties. Four residues from conserved sequence regions (CSRs) were thus selected, and the mutants F113V (CSR-I), Y187F and L189I (CSR-II) and A161D (CSR-V) were characterised. Few changes in the optimum reaction temperature and pH were observed for all mutants. Whereas the Y187F (t 1/2 43 €...h) and L189I (t 1/2 36 €...h) mutants had a lower thermostability at 65°C than the native ASKA (t 1/2 48 €...h), the mutants F113V and A161D exhibited an improved t 1/2 of 51 €...h and 53 €...h, respectively. Among the mutants, only the A161D had a specific activity, k cat and k cat /K m higher (1.23-, 1.17- and 2.88-times, respectively) than the values determined for the ASKA. The replacement of the Ala-161 in the CSR-V with an aspartic acid also caused a significant reduction in the ratio of maltose formed. This finding suggests the Ala-161 may contribute to the high maltose production of the ASKA.

Item Type:Article
Uncontrolled Keywords:amylase, bacterial protein
Subjects:Q Science > QH Natural history
Divisions:Biosciences and Medical Engineering
ID Code:62358
Deposited By: Widya Wahid
Deposited On:06 Jun 2017 14:30
Last Modified:06 Jun 2017 14:30

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