Characterization of an indican-hydrolyzing enzyme from Sinorhizobium meliloti |
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| Affiliation: | 1. Department of Biological Sciences, College of Natural Sciences, Chonnam National University, Yong-Bong Dong, Buk-Gu, Gwangju 500-757, Republic of Korea;2. Department of Clothing and Textiles, Chonnam National University, Gwangju 500-757, Republic of Korea;1. Laboratório Nacional de Ciência e Tecnologia do Bioetanol (CTBE), Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Rua Giuseppe Máximo Scolfaro, nº 10000, 13083-970 Campinas, SP, Brazil;2. Laboratório de Genômica e Expressão (LGE), Departamento de Genética, Evolução e Bioagentes da Universidade Estadual de Campinas (UNICAMP), Campinas, Brazil;3. Centro de Hematologia e Hemoterapia, Universidade Estadual de Campinas (UNICAMP), Campinas, Brazil;4. Departamento de Biologia, Instituto de Biociências, Universidade Estadual Paulista (UNESP), Rio Claro, Brazil;5. Departamento de Física, Instituto de Biociências, Letras e Ciências Exatas (IBILCE), Universidade Estadual Paulista (UNESP), São José do Rio Preto, SP, Brazil;1. Plant Biotechnology and Metabolic Engineering, Technische Universität Darmstadt, Schnittspahnstraße 4, 64287 Darmstadt, Germany;2. Lehrstuhl für Pharmazeutische Biologie, Julius-von-Sachs-Institut der Universität Würzburg, Julius-von-Sachs-Platz 2, 97082 Würzburg, Germany;3. Clemens-Schöpf-Institut für Organische Chemie und Biochemie, Technische Universität Darmstadt, Alarich-Weiss-Str. 4, 64287 Darmstadt, Germany |
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| Abstract: | A novel β-glucosidase capable of hydrolyzing indican to indigo was mined and isolated from Sinorhizobium meliloti using a systematic approach. The corresponding gene was amplified by PCR and overexpressed in the soluble fraction as an MBP fusion protein. The resulting enzyme easily purified to apparent homogeneity via a consecutive step in the affinity column. The recombinant enzyme was determined to be a monomer with a calculated molecular mass of 52 kDa and showed the maximum activity for indican at pH 7.0 and 45 °C. The kinetic parameters for indican, KM and Vmax, were determined to be 0.97 mM and 355.6 μM/min/mg protein, respectively, at pH 7.0 and 35 °C. Additionally, this enzyme hydrolyzed both the β-(1-4)- and β-(1-6)-glucosidic bonds and revealed a minor activity against α-d-glucosides. Furthermore, the enzyme was severely inhibited by DTT, indicating a possibility that the oxidation of amino acids could play a crucial role in the activity of the enzyme. |
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