Characterization of two novel heat-active α-galactosidases from thermophilic bacteria
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- 作者：Carola Schröder ; Viktoria-Astrid Janzer ; Georg Schirrmacher ; Jörg Claren…
- 关键词：Thermostable α ; galactosidase ; Raffinose family oligosaccharides ; Dictyoglomus thermophilum
- 刊名：Extremophiles
- 出版年：2017
- 出版时间：January 2017
- 年：2017
- 卷：21
- 期：1
- 页码：85-94
- 全文大小：1056KB
- 刊物类别：Biomedical and Life Sciences
- 刊物主题：Microbiology; Biotechnology; Biochemistry, general; Microbial Ecology;
- 出版者：Springer Japan
- ISSN：1433-4909
- 卷排序：21
文摘
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.