Cloning, expression and characterization of a new aspartate aminotransferase from Bacillus subtilis B3

Authors

  • Hui-Jun Wu,

    1. Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, Key Laboratory of Monitoring and Management of Crop Diseases and Pest Insects, Ministry of Agriculture, China
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    • These authors contributed equally to this work

  • Yang Yang,

    1. Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, Key Laboratory of Monitoring and Management of Crop Diseases and Pest Insects, Ministry of Agriculture, China
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    • These authors contributed equally to this work

  • Shuai Wang,

    1. Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, China
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    • These authors contributed equally to this work

  • Jun-Qing Qiao,

    1. Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, Key Laboratory of Monitoring and Management of Crop Diseases and Pest Insects, Ministry of Agriculture, China
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  • Yan-Fei Xia,

    1. Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, Key Laboratory of Monitoring and Management of Crop Diseases and Pest Insects, Ministry of Agriculture, China
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  • Yu Wang,

    1. Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, Key Laboratory of Monitoring and Management of Crop Diseases and Pest Insects, Ministry of Agriculture, China
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  • Wei-Duo Wang,

    1. Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, Key Laboratory of Monitoring and Management of Crop Diseases and Pest Insects, Ministry of Agriculture, China
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  • Sheng-Feng Gao,

    1. Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, Key Laboratory of Monitoring and Management of Crop Diseases and Pest Insects, Ministry of Agriculture, China
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  • Jun Liu,

    1. Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, Key Laboratory of Monitoring and Management of Crop Diseases and Pest Insects, Ministry of Agriculture, China
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  • Peng-Qi Xue,

    1. Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, Key Laboratory of Monitoring and Management of Crop Diseases and Pest Insects, Ministry of Agriculture, China
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  • Xue-Wen Gao

    1. Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, Key Laboratory of Monitoring and Management of Crop Diseases and Pest Insects, Ministry of Agriculture, China
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X.-W. Gao, Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, Key Laboratory of Monitoring and Management of Crop Diseases and Pest Insects, Ministry of Agriculture, Nanjing 210095, China
Fax: +86 25 84395268
Tel: +86 25 84395268
E-mail: gaoxw@njau.edu.cn

Abstract

In the present study, we report the identification of a new gene from the Bacillus subtilis B3 strain (aatB3), which comprises 1308 bp encoding a 436 amino acid protein with a monomer molecular weight of 49.1 kDa. Phylogenetic analyses suggested that this enzyme is a member of the Ib subgroup of aspartate aminotransferases (AATs; EC 2.6.1.1), although it also has conserved active residues and thermostability characteristic of Ia-type AATs. The Asp232, Lys270 and Arg403 residues of AATB3 play a key role in transamination. The enzyme showed maximal activity at pH 8.0 and 45 °C, had relatively high activity over an alkaline pH range (pH 7.0–9.0) and was stable up to 50 °C. AATB3 catalyzed the transamination of five amino acids, with l-aspartate being the optimal substrate. The Km values were determined to be 6.7 mm for l-aspartate, 0.3 mm for α-ketoglutarate, 8.0 mm for l-glutamate and 0.6 mm for oxaloacetate. A 32-residue N-terminal amino acid sequence of this enzyme has 53% identity with that of Bacillus circulans AAT, although it is absent in all other AATs from different organisms. Further studies on AATB3 may confirm that it is potentially beneficial in basic research as well as various industrial applications.

Database
The nucleotide sequence data have been deposited in the GenBank database under accession Numbers AY040867.1

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