Proteins sequence analysis of glycohydrolases and application to cloning enzymes from Thai plants
1998
Ketudat-Cairns, James R., Suranaree Univ. of Technology, Nakhon Ratchasima (Thailand). Inst. of Science
Glycohydrolase enzymes have many present and potential applications. In order to identify novel glycohydrolases from Thai plants, protein sequence-based approach was undertaken. Known protein sequences of alpha-mannosidases, beta-galactosidases, and B-glucosidases were retrieved from on-line databases, and grouped into sequence families as previously described by Henrisatt (1991, Biochem. J. 280:309-316). Multiple sequence alignments were performed and phylogenetic trees constructed with the computer programs of Feng and Doolittle (1996, Meth. Enzymol. 266:368-382). Sequences likely to be conserved in a broad range of plant species were identified from the alignment and phylogenetic relationships. These sequences were used to design degenerate primers, which were used for PCR screening of plant DNA. Initial characterization of primer conditions were carried out with rice and bean DNA as monocot and dicot models, respectively, and using cloned beta-glucosidase cDNA in a plasmid. Several products of the size expected form protein sequence alignments have been identified by gel electrophoresis.
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