3 NEOCALLIMASTIX-PATRICIARUM ESTERASES ASSOCIATED WITH THE DEGRADATION OF COMPLEX POLYSACCHARIDES ARE MEMBERS OF A NEW FAMILY OF HYDROLASES

Acetylesterase and cinnamoyl ester hydrolase activities were demonstra ted in culture supernatant of the anaerobic ruminal fungus Neocallimas tix patriciarum. A cDNA expression library from N. patriciarum was scr eened for esterases using beta-naphthyl acetate and a model cinnamoyl ester compound. cDNA clones representing four different esterase genes (bnaA-D) were isolated. None of the enzymes had cinnamoyl ester hydro lase activity, but two of the enzymes (BnaA and BnaC) had acetylxylan esterase activity. bnaA, bnaB and bnaC encode proteins with several di stinct domains. Carboxy-terminal repeats in BnaA and BnaC are homologo us to protein-docking domains in other enzymes from Neocallimastix spe cies and another anaerobic fungus, a Piromyces sp. The catalytic domai ns of BnaB and BnaC are members of a recently described family of Ser/ His active site hydrolases [Upton, C. & Buckley, J.T. (1995). Trends B iochem Sci 20, 178-179]. BnaB exhibits 40% amino acid identity to a do main of unknown function in the CelE cellulase from Clostridium thermo cellum and BnaC exhibits 52% amino acid identity to a domain of unknow n function in the XynB xylanase from Ruminococcus flavefaciens. BnaA, whilst exhibiting less than 10% overall amino acid identity to BnaB or BnaC, or to any other known protein, appears to be a member of the sa me family of hydrolases, having the three universally conserved amino acid sequence motifs. Several other previously described esterases are also shown to be members of this family, including a rhamnogalacturon an acetylesterase from Aspergillus aculeatus. However, non of the othe r previously described enzymes with acetylxylan esterase activity are members of this family of hydrolases.