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Characterization of a Bifidobacterium longum BORI Dipeptidase Belonging to the U34 Family

Department of Agricultural Biotechnology and Center for Agricultural Biomaterials, Seoul National University, 56-1 Sillim-dong, Gwanak-gu, Seoul 151-921, Korea,¹ Department of Food and Nutrition, Seoul National University, 56-1 Sillim-dong, Gwanak-gu, Seoul 151-742, Korea,² Research Institute, Bifido Inc., Ltd., Hongchon 250-804, Korea,³ Department of Hotel Culinary Art, Anyang Technical College, Anyang 430-749, Korea⁴

Received 20 March 2007/ Accepted 23 June 2007

A dipeptidase was purified from a cell extract of Bifidobacterium longum BORI by ammonium sulfate precipitation and chromatography on DEAE-cellulose and Q-Sepharose columns. The purified dipeptidase had a molecular mass of about 49 kDa and was optimally active at pH 8.0 and 50°C. The enzyme was a strict dipeptidase, being capable of hydrolyzing a range of dipeptides but not tri- and tetrapeptides, p-nitroanilide derivatives of amino acids, or N- or C-terminus-blocked dipeptides. A search of the amino acid sequence of an internal tryptic fragment against protein sequences deduced from the total genome sequence of B. longum NCC2705 revealed that it was identical to an internal sequence of the dipeptidase gene (pepD), which comprised 1,602 nucleotides encoding 533 amino acids with a molecular mass of 60 kDa, and thereby differed considerably from the 49-kDa mass of the purified dipeptidase. To understand this discrepancy, pepD was cloned into an Escherichia coli expression vector (pBAD-TOPO derivative) to generate the recombinant plasmids pBAD-pepD and pBAD-pepD-His (note that His in the plasmid designation stands for a polyhistidine coding region). Both plasmids were successfully expressed in E. coli, and the recombinant protein PepD-His was purified using nickel-chelating affinity chromatography and reconfirmed by internal amino acid sequencing. The PepD sequence was highly homologous to those of the U34 family of peptidases, suggesting that the B. longum BORI dipeptidase is a type of cysteine-type N-terminal nucleophile hydrolase and has a ß-hairpin motif similar to that of penicillin V acylase, which is activated by autoproteolytic processing.

Published ahead of print on 29 June 2007.