Characterization of two novel heat-active α-galactosidases from thermophilic bacteria

• Published in: Extremophiles : life under extreme conditions 2017, Vol.21 (1), p.85-94
• Main Authors: Schröder, Carola, Janzer, Viktoria-Astrid, Schirrmacher, Georg, Claren, Jörg, Antranikian, Garabed
• Format: Article
• Language: English
• Subjects: 11th International Congress on Extremophiles; alpha-Galactosidase - chemistry; alpha-Galactosidase - genetics; alpha-Galactosidase - metabolism; Amino Acid Motifs; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biochemistry; Biomedical and Life Sciences; Biotechnology; Enzyme Stability; Escherichia coli; Gram-Negative Bacteria - enzymology; Gram-Negative Bacteria - genetics; Hot Temperature; Life Sciences; Meiothermus; Microbial Ecology; Microbiology; Space life sciences; Special Feature: Original Paper; Substrate Specificity
• ISSN: 1431-0651; 1433-4909
• DOI: 10.1007/s00792-016-0885-z

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.