Identification of a novel DFA I-producing inulin fructotransferase from Streptomyces davawensis

Abstract

In this work, a novel gene encoding DFA I-forming inulin fructotransferase (IFTase) from Streptomyces davawensis SK39.001 was cloned and expressed in Escherichia coli. The enzyme was purified, identified, and characterized. The results showed that this IFTase (DFA I-forming) is a trimer (molecular weight of 125KDa) consisting of three identical subunits (the molecular weight as assayed by SDS-PAGE was approximately 40KDa). At pH 5.5 and 40°C, the maximum specific activity (approximately 100Umg⿿1) was achieved. Moreover, the enzyme was stable up to 70°C. Km and Vmax were 2.89±0.2mM and 1.94±0.9mMmin⿿1, respectively. For exploring putative active sites and probable catalytic mechanisms, homology modelling and molecular docking methods after site-directed mutagenesis were applied to IFTase (DFA I-forming). The results revealed that D183 and E194 were potential catalytic residues of the purified enzyme.