Cloning of the maltose phosphorylase gene from Bacillus sp. strain RK-1 and efficient production of the cloned gene and the trehalose phosphorylase gene from Bacillus stearothermophilus SK-1 in Bacillus subtilis

• Published in: Bioscience, biotechnology, and biochemistry Vol. 66; no. 12; pp. 2594 - 2599
• Main Authors: Inoue, Y. (Showa Sangyo Co. Ltd., Tsukuba, Ibaraki (Japan)), Yasutake, N, Oshima, Y, Yamamoto, Y, Tomita, T, Miyoshi, S, Yatake, T
• Format: Journal Article
• Language: English
• Published: Tokyo Japan Society for Bioscience Biotechnology and Agrochemistry 01.12.2002
• Subjects: Amino Acid Sequence; BACILLUS; Bacillus - classification; Bacillus - enzymology; Bacillus - genetics; Bacillus amyloliquefaciens; BACILLUS STEAROTHERMOPHILUS; BACILLUS SUBTILIS; Bacteriology; Base Sequence; Biological and medical sciences; Biology of microorganisms of confirmed or potential industrial interest; Biotechnology; Cloning, Molecular; Fundamental and applied biological sciences. Psychology; GENE EXPRESSION; GENES; Genetics; Glucosyltransferases - biosynthesis; Glucosyltransferases - chemistry; Glucosyltransferases - genetics; MALTOSE; Maltose - metabolism; Microbiology; Mission oriented research; MOLECULAR CLONING; Molecular Sequence Data; PHOSPHORYLASE; TREHALOSE; Trehalose - metabolism; trehalose phosphorylase; Maltose phosphorylase; Enzyme; Transcription promoter; Bacillus subtilis; Transferases; trehalose phosphorylase; Glycosyltransferases; Bacillaceae; Gene expression; Regulation(control); Bacillales; α,α-Trehalose phosphorylase; Bacillus stearothermophilus; Bacteria; Hexosyltransferases; Molecular cloning; intracellular expression
• ISSN: 0916-8451; 1347-6947
• DOI: 10.1271/bbb.66.2594

The maltose phosphorylase (MPase) gene of Bacillus sp. strain RK-1 was cloned by PCR with oligonucleotide primers designed on the basis of a partial N-terminal amino acid sequence of the purified enzyme. The MPase gene consisted of 2,655 bp encoding a theoretical protein with a Mr of 88,460, and had no secretion signal sequence, although most of the MPase activity was detected in the culture supernatant of RK-1. This cloned MPase gene and the trehalose phosphorylase (TPase) gene from Bacillus stearothermophilus SK-1 were efficiently expressed intracellularly under the control of the Bacillus amyloliquefaciens alpha-amylase promoter in Bacillus subtilis. The production yields were estimated to be more than 2 g of enzyme per liter of medium, about 250 times the production of the original strains, in a simple shake flask. About 60% of maltose was converted into trehalose by the simultaneous action of both enzymes produced in B. subtilis.