Characterization of trehalose phosphorylase from Bacillus stearothermophilus SK-1 and nucleotide sequence of the corresponding gene

• Published in: Bioscience, biotechnology, and biochemistry biotechnology, and biochemistry, 2002-09, Vol.66 (9), p.1835-1843
• Main Authors: "Inoue, Y. (Showa Sangyo Co. Ltd., Tsukuba, Ibaraki (Japan)), Ishii, K, Tomita, T, Yatake, T, Fukui, F.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; AMINO ACID SEQUENCES; BACILLUS STEAROTHERMOPHILUS; Base Sequence; Biological and medical sciences; Biology of microorganisms of confirmed or potential industrial interest; Biotechnology; ENZYME ACTIVITY; Escherichia coli; Fundamental and applied biological sciences. Psychology; GENES; Genetics; Geobacillus stearothermophilus - enzymology; Geobacillus stearothermophilus - genetics; Glucosyltransferases - chemistry; Glucosyltransferases - genetics; Glucosyltransferases - metabolism; Hydrogen-Ion Concentration; Isoelectric Point; Kinetics; Mission oriented research; MOLECULAR CLONING; Molecular Sequence Data; Molecular Weight; NUCLEOTIDE SEQUENCE; PHOSPHORYLASE; Sequence Alignment; Sequence Homology, Amino Acid; Substrate Specificity; Temperature; TRANSGENICS; trehalose phosphorylase; trehalose phosphorylase gene
• ISSN: 0916-8451; 1347-6947
• DOI: 10.1271/bbb.66.1835

"A bacterial trehalose phosphorylase (TPase; EC 2.4.1.64) was purified from the culture supernatant of Bacillus stearothermophilus SK-1 to apparent homogeneity, and some properties were investigated. Furthermore, a gene from SK-1 responsible for the TPase was cloned by Southern hybridization with a degenerate oligonucleotide probe synthesized on the basis of the N-terminal sequence of the purified enzyme. The Mr of the enzyme was estimated to be 150,000 by gel filtration and 83,000 by SDS-PAGE, so the enzyme is likely to be a homodimer. The enzyme had optimum activity at pH 7.0-8.0 or nearby and the optimum temperature was about 75degC. The deduced amino acid sequence of the SK-1 TPase encodes a theoretical protein with a Mr of 87,950. Alignment of amino acid sequences with a maltose phosphorylase from Lactobacillus brevis the crystal structure and active site of which had been analyzed suggested that these two phosphorylases evolved from a common ancestor. The Escherichia coli cells harboring the plasmid containing the cloned TPase gene had about 100 times the activity of SK-1."