EXTRACELLULAR ENZYME-ACTIVITY IN ANAEROBIC BACTERIAL CULTURES - EVIDENCE OF PULLULANASE ACTIVITY AMONG MESOPHILIC MARINE-BACTERIA

The extracellular enzymatic activity of a mixed culture of anaerobic m arine bacteria enriched on pullulan [alpha(1,6)-linked maltotriose uni ts] was directly assessed with a combination of gel permeation chromat ography (GPC) and nuclear magnetic resonance spectroscopy (NMR). Hydro lysis products of pullulan were separated by GPC into three fractions with molecular weights of greater than or equal to 10,000, similar to 5,000, and less than or equal to 1,200. NMR spectra of these fractions demonstrated that pullulan was rapidly and specifically hydrolyzed at alpha(1,6) linkages by pullulanase enzymes, most likely type II pullu lanase. Although isolated pullulanase enzymes have been shown to hydro lyze pullulan completely to maltotriose (S. H. Brown, H. R. Costantino , and R. M. Kelly, Appl. Environ. Microbiol. 56:1985-1991, 1990; M. Kl ingeberg, H. Hippe, and G. Antranikian, FEMS Microbiol. Lett. 69:145-1 52, 1990; R. Koch, P. Zablowski, A. Spreinat, and G. Antranikian, FEMS Microbiol. Lett. 71:21-26, 1990), the smallest carbohydrate detected in the bacterial cultures consisted of two maltotriose units linked th rough one alpha(1,6) linkage. Either the final hydrolysis step was clo sely linked to substrate uptake, or specialized porins similar to malt oporin might permit direct transport of large oligosaccharides into th e bacterial cell. This is the first report of pullulanase activity amo ng mesophilic marine bacteria. The combination of GPC and NMR could ea sily be used to assess other types of extracellular enzyme activity in bacterial cultures.