Galactomannanases Man2 and Man5 from Thermotogaspecies: Growth physiology on galactomannans, gene sequence analysis, and biochemical properties of recombinant enzymes

Abstract

The enzymatic hydrolysis of mannan-based hemicelluloses is technologically important for applications ranging from pulp and paper processing to food processing to gas and oil well stimulation. In many cases, thermostability and 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 maritima were isolated, cloned, and expressed in Escherichia coli. The amino acid sequences for the mannosidases from these organisms were 77% identical and corresponded to proteins with an M(r) of approximately 92 kDa. The translated nucleotide sequences for the beta-mannanase genes (man5) encoded polypeptides with an M(r) of 76 kDa that exhibited 84% amino acid sequence identity. The recombinant versions of Man2 and Man5 had similar respective biochemical and biophysical properties, which were also comparable to those determined for the native versions of these enzymes in T. neapolitana. The optimal temperature and pH for the recombinant Man2 and Man5 from both organisms were approximately 90 degrees C 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-mannanase and beta-mannosidase activities were significantly stimulated when T. neapolitana was grown on guar or carob galactomannan. Maximum cell densities increased by at least tenfold when either guar or carob galactomannan was added to the growth medium. For T. neapolitana grown on guar at 83 degrees C, Man5 was secreted into the culture media, whereas Man2 was intracellular. These localizations were consistent with the presence and lack of signal peptides for Man5 and Man2, respectively. The identification of the galactomannan-degrading enzymes in these Thermotoga species adds to the list of biotechnologically important hemicellulases produced by members of this hyperthermophilic genera.

Cloning and Characterization of a β-1,4-Mannanase 5C Possessing a Family 27 Carbohydrate-Binding Module from a Marine Bacterium,Vibriosp. Strain MA-138

“…Biochemical characterization of Man5C revealed that the optimal temperature of the enzyme was 50 C, and that it was stable up to 45 C for 20 min, whereas the enzyme showed high similarities to thermostablemannanases derived from thermophilic bacteria, such as G. stearothermophilus (optimal temperature: 70 C), 35) T. maritima MSB8 (90 C), 36) and T. neapolitana (92 C) 36) (Table 1). However, the optimal temperature of Man5C was 10 C higher than that of ManA from strain MA-138, which has also been classified in GH5.…”

Substrate Specificities and Expression Patterns Reflect the Evolutionary Divergence of Maltose ABC Transporters in Thermotoga maritima

“…The high level of expression of MalE1 in guar gum-grown cells and the ability of MalE1 to bind mannooligosaccharides suggest that the role of MalE1 is to transport oligosaccharides generated from galactomannan hydrolysis. Both endo-1-4-␤-mannanase and ␤-galactosidase are extracellular in T. maritima, so ␤-1-4-linked mannooligosaccharides would be available for transport into cells (24). MalE1 binds only ␤-(134)-mannotetraose and not ␤-(134)-mannobiose and ␤-(134)-mannotriose, showing that it has affinity only for larger mannooligosaccharides.…”

Molecular Characterization of aRhodotorula-lytic Enzyme fromPaecilomyces lilacinusHaving β-1,3-Mannanase Activity

“…The results from BLAST program showed homology only in their Nterminal half (amino acid residues 43-201) with -1,4-mannanases (E.C.3.2.1.78), which is derived from Thermotoga neapolitana (27% identity), 9) Arabidopsis thaliana (30%, accession no. CAB82763), Thermotoga maritima (26%), 9) and Aspergillus aculeatus (24%).…”