Galactomannanases man2 and man5 from Thermotoga species: Growth physiologyon galactomannans, gene sequence analysis, and biochemical properties of recombinant enzymes

The enzymatic hydrolysis of mannan-based hemicelluloses is technologically important for applications ranging from pulp and paper processing to food p rocessing to gas and oil well stimulation. In many cases, thermostability a nd activity at elevated temperatures can be advantageous, To this end, the genes encoding beta -mannosidase (man2) and beta -mannanase (man5) from the hyperthermophilic bacteria Thermotoga neapolitana 5068 and Thermotoga mari tima were isolated, cloned, and expressed in Escherichia coli. The amino ac id sequences for the mannosidases from these organisms were 77% identical a nd corresponded to proteins with an Mr of approximately 92 kDa. The transla ted nucleotide sequences for the beta -mannanase genes (man5) encoded polyp eptides with an Mr of 76 kDa that exhibited 84% amino acid sequence identit y. The recombinant versions of Man2 and Man5 had similar respective biochem ical and biophysical properties, which were also comparable to those determ ined for the native versions of these enzymes in T. neapolitana. The optima l temperature and pH for the recombinant Man2 and Man5 from both organisms were approximately 90 degreesC and 7.0, respectively. The presence of Man2 and Man5 in these two Thermotoga species indicates that galactomannan is a potential growth substrate. This was supported by the fact that beta -manna nase and beta -mannosidase activities were significantly stimulated when T. neapolitana was grown on guar or carob galactomannan. Maximum cell densiti es increased by at least tenfold when either guar or carob galactomannan wa s added to the growth medium. For T. neapolitana grown on guar at 83 degree sC, Man5 was secreted into the culture media, whereas Man2 was intracellula r. These localizations were consistent with the presence and lack of signal peptides for Man5 and Man2, respectively. The identification of the galact omannan-degrading enzymes in these Thermotoga species adds to the list of b iotechnologically important hemicellulases produced by members of this hype rthermophilic genera. (C) 2001 John Wiley & Sons, Inc.