Fujiwara S, Kakihara H, Woo K B, Lejeune A, Kanemoto M, Sakaguchi K, Imanaka T
Department of Biotechnology, Faculty of Engineering, Osaka University, Japan.
Appl Environ Microbiol. 1992 Dec;58(12):4016-25. doi: 10.1128/aem.58.12.4016-4025.1992.
Cyclodextrin glucanotransferase (CGTase; EC 2.4.1.19) is produced mainly by Bacillus strains. CGTase from Bacillus macerans IFO3490 produces alpha-cyclodextrin as the major hydrolysis product from starch, whereas thermostable CGTase from Bacillus stearothermophilus NO2 produces alpha- and beta-cyclodextrins. To analyze the cyclization characteristics of CGTase, we cloned different types of CGTase genes and constructed chimeric genes. CGTase genes from these two strains were cloned in Bacillus subtilis NA-1 by using pTB523 as a vector plasmid, and their nucleotide sequences were determined. Three CGTase genes (cgt-1, cgt-5, and cgt-232) were isolated from B. stearothermophilus NO2. Nucleotide sequence analysis revealed that the three CGTase genes have different nucleotide sequences encoding the same amino acid sequence. Base substitutions were found at the third letter of five codons among the three genes. Each open reading frame was composed of 2,133 bases, encoding 711 amino acids containing 31 amino acids as a signal sequence. The molecular weight of the mature enzyme was estimated to be 75,374. The CGTase gene (cgtM) of B. macerans IFO3490 was composed of 2,142 bases, encoding 714 amino acids containing 27 residues as a signal sequence. The molecular weight of the mature enzyme was estimated to be 74,008. The sequence determined in this work was quite different from that reported previously by other workers. From data on the three-dimensional structure of a CGTase, seven kinds of chimeric CGTase genes were constructed by using cgt-1 from B. stearothermophilus NO2 and cgtM from B. macerans IFO3490. We examined the characteristics of these chimeric enzymes on cyclodextrin production and thermostability. It was found that the cyclization reaction was conferred by the NH2-terminal region of CGTase and that the thermostability of some chimeric enzymes was lower than that of the parental CGTases.
环糊精葡萄糖基转移酶（CGTase；EC 2.4.1.19）主要由芽孢杆菌属菌株产生。来自浸麻芽孢杆菌IFO3490的CGTase以α-环糊精作为淀粉的主要水解产物，而来自嗜热脂肪芽孢杆菌NO2的耐热CGTase则产生α-和β-环糊精。为了分析CGTase的环化特性，我们克隆了不同类型的CGTase基因并构建了嵌合基因。使用pTB523作为载体质粒，将这两种菌株的CGTase基因克隆到枯草芽孢杆菌NA-1中，并测定了它们的核苷酸序列。从嗜热脂肪芽孢杆菌NO2中分离出三个CGTase基因（cgt-1、cgt-5和cgt-232）。核苷酸序列分析表明，这三个CGTase基因具有不同的核苷酸序列，但编码相同的氨基酸序列。在这三个基因的五个密码子的第三个字母处发现了碱基替换。每个开放阅读框由2133个碱基组成，编码711个氨基酸，其中包含31个氨基酸作为信号序列。成熟酶的分子量估计为75374。浸麻芽孢杆菌IFO3490的CGTase基因（cgtM）由2142个碱基组成，编码714个氨基酸，其中包含27个残基作为信号序列。成熟酶的分子量估计为74008。本研究中测定的序列与其他研究者先前报道的序列有很大不同。根据CGTase的三维结构数据，使用来自嗜热脂肪芽孢杆菌NO2的cgt-1和来自浸麻芽孢杆菌IFO3490的cgtM构建了七种嵌合CGTase基因。我们研究了这些嵌合酶在环糊精产生和热稳定性方面的特性。结果发现，环化反应由CGTase的NH2末端区域赋予，并且一些嵌合酶的热稳定性低于亲本CGTase。
