To biotransform rutin into isoquercitrin. A alpha-l-rhamnosidase from Bifidobacterium breve was produced by using Escherichia coli BL21 for biotransformation of rutin to isoquercitrin. The enzyme was purified by Ni2+-NTA chromatography to yield a soluble protein with a specific activity of 56 U protein mg(-1). The maximum enzyme activities were at pH 6.5, 55 A degrees C, 20 mM rutin, and 1.2 U enzyme ml(-1). Under optimal conditions, the half-life of the enzyme was 96 h. The K (m) and V (max) values were 2.2 mM, 56.4 mu mol mg(-1) min(-1) and 2.1 mM, 57.5 mu mol mg(-1) min(-1) using pNP-Rha and rutin as substrates, respectively. The kinetic behavior indicated that the recombinant alpha-l-rhamnosidase has good catalytic performance for producing isoquercitrin. 20 mM rutin was biotransformed into 18.25 and 19.87 mM isoquercitrin after 60 and 240 min. The specific biotransformation of rutin to isoquercitrin using recombinant alpha-l-rhamnosidase from B. breve is a feasible method for use in industrial processes.