The DNA polymerase gene from the archaeon Pyrococcus sp. strain KOD1 (KOD DNA polymerase) contains a long open reading frame of 5,013 bases that encodes 1,671 amino acid residues (GenBank accession no. D29671). Similarity analysis revealed that the DNA polymerase contained a putative 3-5 exonuclease activity and two in-frame intervening sequences of 1,080 bp (360 amino acids; KOD pol intein-1) and 1,611 bp (537 amino acids; KOD pol intein-2), which are located in the middle of regions conserved among eukaryotic and archaeal ␣-like DNA polymerases. The mature form of the DNA polymerase gene was expressed in Escherichia coli, and the recombinant enzyme was purified and characterized. 3-5 exonuclease activity was confirmed, and although KOD DNA polymerase's optimum temperature (75°C) and mutation frequency (3.5 ؋ 10 ؊3) were similar to those of a DNA polymerase from Pyrococcus furiosus (Pfu DNA polymerase), the KOD DNA polymerase exhibited an extension rate (100 to 130 nucleotides/s) 5 times higher and a processivity (persistence of sequential nucleotide polymerization) 10 to 15 times higher than those of Pfu DNA polymerase. These characteristics enabled the KOD DNA polymerase to perform a more accurate PCR in a shorter reaction time.
a-L-Arabinofuranosidase (a-L-arafase) was purified from fruit of Japanese pear (Pyrus pyrifolia). The enzyme solubilized from the cell wall by NaCl and Triton X-100 had the homogeneity of a single 62-kD polypeptide on SDS-PAGE after purification through the steps of hydroxyapatite, anion-exchange chromatography, and size-exclusion chromatography. A related cDNA clone was isolated (PpARF2). The transcript and related protein were detected solely in the ripening fruit corresponding to the increase of a-L-arafase activity. Transcripts of PpARF2 were not detected in buds, leaves, roots, or shoots of the Japanese pear. The deduced amino acid sequences of PpARF2 had low identity with those of other plants or bacteria. This a-L-arafase belonged to glycoside hydrolase family 3, which includes some b-xylosidases. The purified enzyme hydrolyzed mainly p-nitrophenyl a-L-arabinofuranoside and also reacted bifunctionally with p-nitrophenyl b-D-xylopyranoside. However, it released only arabinose from native cell wall polysaccharides prepared from Japanese pear and from sugar beet arabinan. The enzyme did not release xylose from arabinoxylan and xylan. The only activity of the a-L-arafase presented here was hydrolyzing the arabinosyl residue from native polysaccharides, whereas it showed bifunctional activity against artificial substrates. According to the expression pattern and properties of the enzyme, it is a new member of the glycoside hydrolase family 3 isolated from fruit, and it may be responsible for modification of the cell wall architecture during fruit softening.The modification of cell wall architecture is involved in plant growth, development, and formation of shape. The cooperative biosynthesis and degradation of several cell wall components are necessary, and numerous cell wall-related enzymes are implicated in these processes. Fruit softening or textural changes are important factors that decide fruit quality, and they are caused by modification of cell wall polysaccharide architecture during fruit ripening. Several cell wallmetabolizing enzymes contribute to the changes in cell wall architecture (Fischer and Bennett, 1991). During fruit ripening, pectic and some hemicellulosic polysaccharides become increasingly soluble and depolymerize with the release of neutral sugar residues from side chains of matrix polysaccharides (Huber and O'
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.