Fructose dehydrogenase (FDH) catalyzes oxidation of D-fructose into 2-keto-D-fructose and is one of the enzymes allowing a direct electron transfer (DET)-type bioelectrocatalysis. FDH is a heterotrimeric membrane-bound enzyme (subunit I, II, and III) and subunit II has a C terminal hydrophobic region (CHR), which was expected to play a role in anchoring to membranes from the amino acid sequence. We have constructed a mutated FDH lacking of CHR (DchrFDH). Contrary to the expected function of CHR, DchrFDH is expressed in the membrane fraction, and subunit I/III subcomplex (DcFDH) is also expressed in a similar activity level but in the soluble fraction. In addition, the enzyme activity of the purified DchrFDH is about one twentieth of the native FDH. These results indicate that CHR is concerned with the binding between subunit I(/III) and subunit II and then with the enzyme activity. DchrFDH has clear DET activity that is larger than that expected from the solution activity, and the characteristics of the catalytic wave of DchrFDH are very similar to those of FDH. The deletion of CHR seems to increase the amounts of the enzyme with the proper orientation for the DET reaction at electrode surfaces. Gel filtration chromatography coupled with urea treatment shows that the binding in DchrFDH is stronger than that in FDH. It can be considered that the rigid binding between subunit I(/III) and II without CHR results in a conformation different from the native one, which leads to the decrease in the enzyme activity in solution. (C) 2015 Elsevier Ltd. All rights reserved.