Rapid detection and isolation of known and putative alpha-L-arabinofuranosidase genes using degenerate PCR primers.
alpha-L-Arabinofuranosidases (AFases; EC 3.2.1.55) are exo-type enzymes, which hydrolyze terminal nonreducing arabinose residues from various polysaccharides such as arabinan and arabinoxylan. Genome-wide BLAST search showed that various bacterial strains possess the putative AFase genes with well-conserved motif sequences at the nucleotide and amino acid sequence levels. In this study, two sets of degenerate PCR primers were designed and tested to detect putative AFase genes, based on their three highly conserved amino acid blocks (PGGNFV, GNEMDG, and DEWNVW). Among 20 Bacillus-associated species, 13 species were revealed to have putative AFase genes in their genome and they share over 67% of amino acid identities with each other. Based on the partial sequence obtained from an isolate, an AFase from Geobacillus sp. was cloned and expressed in E. coli. Enzymatic characterization has verified that the resulting enzyme corresponds to a typical AFase. Accordingly, degenerate PCR primers developed in this work can be used for fast, easy, and specific detection and isolation of putative AFase genes from bacterial cells.
Department of Food Science and Technology, School of Applied Life Science and Environment, Chungbuk National University, Cheongju 361-763, Korea.
Jung-Mi
JM
Park
Nam Soo
NS
Han
Tae-Jip
TJ
Kim
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007218:Industrial Microbiology / Q000379:methods*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013997:Time Factors
D017931:DNA Primers; D006026:Glycoside Hydrolases; C019713:alpha-N-arabinofuranosidase