Characterization of two novel heat-active α-galactosidases from thermophilic bacteria.
Two genes (agal1 and agal2) encoding α-galactosidases were identified by sequence-based screening approaches. The gene agal1 was identified from a data set of a sequenced hot spring metagenome, and the deduced amino-acid sequence exhibited 99% identity to an α-galactosidase from the thermophilic bacterium Dictyoglomus thermophilum. The gene agal2 was identified from the whole genome sequence of the thermophile Meiothermus ruber. The amino-acid sequences exhibited structural motifs typical for glycoside hydrolase (GH) family 36 members and were also differentiated into different subgroups of this family. Recombinant production of the heat-active GH36b enzyme Agal1 (87 kDa) and GH36bt enzyme Agal2 (57 kDa) was carried out in E. coli. Agal1 exhibited a specific activity of 1502.3 U/mg at 80 °C, pH 6.5, and Agal2 225.4 U/mg at 60-70 °C, pH 6.5. Half-lives of 14 h (Agal1) and 39 h (Agal2) were obtained at 50 °C, and Agal1 showed half-lives of 4 and 2 h at 70 and 80 °C, respectively. In addition to the natural substrates melibiose, raffinose, and stachyose, 4NP α-D-galactopyranoside was hydrolyzed. Galactose was also liberated from locust bean gum. Both heat-active enzymes are attractive candidates for application in food and feed industry for high-temperature processes for the degradation of raffinose family oligosaccharides.
Institute of Technical Microbiology, Hamburg University of Technology, 21073, Hamburg, Germany.
Carola
C
Schröder
Institute of Technical Microbiology, Hamburg University of Technology, 21073, Hamburg, Germany.
Viktoria-Astrid
VA
Janzer
Clariant Produkte (Deutschland) GmbH, Group Biotechnology, 81477, Munich, Germany.
Georg
G
Schirrmacher
Clariant Produkte (Deutschland) GmbH, Group Biotechnology, 81477, Munich, Germany.
Jörg
J
Claren
Institute of Technical Microbiology, Hamburg University of Technology, 21073, Hamburg, Germany. antranikian@tuhh.de.
Garabed
G
Antranikian
D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D006090:Gram-Negative Bacteria / Q000201:enzymology* / Q000235:genetics; D006358:Hot Temperature; D013379:Substrate Specificity; D000519:alpha-Galactosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D000519:alpha-Galactosidase
Dictyoglomus thermophilum; Raffinose family oligosaccharides; Thermostable α-galactosidase