期刊
CANADIAN JOURNAL OF MICROBIOLOGY
卷 57, 期 12, 页码 1032-1041出版社
CANADIAN SCIENCE PUBLISHING, NRC RESEARCH PRESS
DOI: 10.1139/W11-106
关键词
Pseudomonas sp strain KS-408; alginate lyase; alginate oligosaccharides; polyM-specific lyase
资金
- New and Renewable Energy Center of the Korean Institute of Energy Technology Evaluation and Planning (KETEP) [20093020090020]
- Ministry of Knowledge Economy (MKE), Korean Government
- Ministry of Knowledge Economy (MKE)
- Korea Industrial Technology Foundation (KOTEF)
- Korea Evaluation Institute of Industrial Technology (KEIT) [20093020090020] Funding Source: Korea Institute of Science & Technology Information (KISTI), National Science & Technology Information Service (NTIS)
An alginate lyase gene of a newly isolated Pseudomonas sp. strain KS-408 was cloned by using PCR with the specific primers designed from homologous nucleotide sequences. A partial protein sequence of KS-408 alginate lyase was homology-modeled on the basis of the crystal structure of A1-III alginate lyase from Sphingomonas sp. strain Al. The proposed 3-D structure of KS-408 alginate lyase shows that Asn-198, His-199, Arg-246, and Tyr-253 residues are conserved for the catalytic active site. The recombinant KS-408-1F (with signal peptide) and KS-408-2F (without signal peptide) alginate lyases with the (His)(6) tag consist of 393 (44.5 kDa) and 372 (42.4 kDa) amino acids with isoelectric points of 8.64 and 8.46, respectively. The purified recombinant KS-408 alginate lyase was very stable when it was incubated at 40 degrees C for 30 min. Alginate oligosaccharides produced by the KS-408-2F alginate lyase were purified on a Bio-Gel P2 column and analyzed by thin-layer chromatography, fast-protein liquid chromatography, and electrospray ionization mass spectrometry. H-1-1 NMR data showed that the KS-408-2F alginate lyase cleaved the glycosidic linkages between two mannuronates (mannuronate-beta(1-4)-mannuronate) or mannuronate and guluronate (mannuronate-beta(1-4)-guluronate), indicating that the KS-408 alginate lyase is a polyM-specific lyase.
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