Rhamnogalacturonan [alpha]-L-Rhamnopyranohydrolase (A Novel Enzyme Specific for the Terminal Nonreducing Rhamnosyl Unit in Rhamnogalacturonan Regions of Pectin)

Abstract

Two [alpha]-L-rhamnohydrolases with different substrate specificities were isolated from a commercial preparation produced by Aspergillus aculeatus. The first rhamnohydrolase was active toward p-nitrophenyl-[alpha]-L-rhamnopyranoside, naringin, and hesperidin and was termed p-nitrophenyl-[alpha]-L-rhamnopyranohydrolase (pnp-rhamnohydrolase). From the data collected, the enzyme seemed specific for the [alpha]-1,2- or [alpha]-1,6-linkage to [beta]-D-glucose. The pnp-rhamnohydrolase had a molecular mass of 87 kD (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), a pH optimum of 5.5 to 6, a temperature optimum of 60[deg]C, and a specific activity toward pnp-[alpha]-L-rhamnopyranoside (pnp-Rha) of 13 units mg-1 protein. The second rhamnohydrolase, on the contrary, was active toward rhamnogalacturonan (RG) fragments, releasing Rha, and was therefore termed RG-rhamnohydrolase. The RG-rhamnohydrolase had a molecular mass of 84 kD, a pH optimum of 4, a temperature optimum of 60[deg]C, and a specific activity toward RG oligomers of 60 units mg-1 protein. The RG-rhamnohydrolase liberated Rha from the nonreducing end of the RG chain and appeared specific for the [alpha]-1,4-linkage to [alpha]-D-galacturonic acid. The enzyme was hindered when this terminal Rha residue was substituted at the 4-position by a [beta]-D-galactose. The results so far obtained did not indicate particular preference of the enzyme for low or high molecular mass RG fragments. From the results it can be concluded that a new enzyme, an RG [beta]-L-rhamnopyranohydrolase, has been isolated with high specificity toward RG regions of pectin.