Characterization of an indican-hydrolyzing enzyme from Sinorhizobium meliloti

Abstract

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, K M and V max , 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.