title: Protein engineering of selected residues from conserved sequence regions of a novel Anoxybacillus α-amylase.
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 (t1/2 43 h) and L189I (t1/2 36 h) mutants had a lower thermostability at 65°C than the native ASKA (t1/2 48 h), the mutants F113V and A161D exhibited an improved t1/2 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.
authors: Faculty of Biosciences and Medical Engineering, Universiti Teknologi Malaysia, Skudai, 81310 Johor, Malaysia.
authors: Velayudhan
authors: V
authors: Ranjani
authors: 1] Laboratory of Protein Evolution, Institute of Molecular Biology, Slovak Academy of Sciences, SK-84551 Bratislava, Slovakia [2] Department of Biology, Faculty of Natural Sciences, University of SS. Cyril and Methodius, SK-91701 Trnava, Slovakia.
authors: Stefan
authors: S
authors: Janeček
authors: Faculty of Biosciences and Medical Engineering, Universiti Teknologi Malaysia, Skudai, 81310 Johor, Malaysia.
authors: Kian Piaw
authors: KP
authors: Chai
authors: Faculty of Biosciences and Medical Engineering, Universiti Teknologi Malaysia, Skudai, 81310 Johor, Malaysia.
authors: Shafinaz
authors: S
authors: Shahir
authors: Enzyme and Microbial Technology Research Centre, Faculty of Biotechnology and Biomolecular Science, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.
authors: Raja Noor Zaliha Raja
authors: RN
authors: Abdul Rahman
authors: Division of Genetics and Molecular Biology, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia.
authors: Kok-Gan
authors: KG
authors: Chan
authors: Faculty of Biosciences and Medical Engineering, Universiti Teknologi Malaysia, Skudai, 81310 Johor, Malaysia.
authors: Kian Mau
authors: KM
authors: Goh
mesh_terms: D000595:Amino Acid Sequence; D058677:Anoxybacillus / Q000737:chemistry* / Q000201:enzymology; D001407:Bacillus / Q000737:chemistry / Q000201:enzymology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017124:Conserved Sequence; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D056506:Geobacillus / Q000737:chemistry / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008320:Maltose / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013213:Starch / Q000378:metabolism; D000516:alpha-Amylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D008320:Maltose; D013213:Starch; D000516:alpha-Amylases