Okamoto T, Yamano S, Ikeaga H, Nakamura K
Central Laboratories for Key Technology Kirin Brewery Co., Yokohama-shi, Japan.
Appl Microbiol Biotechnol. 1994 Dec;42(4):563-8. doi: 10.1007/BF00173921.
A DNA fragment corresponding to carboxymethylcellulase activity of Acetobacter xylinum IFO 3288 was isolated and cloned in Escherichia coli, and the DNA sequence was determined. The DNA fragment sequenced had an open-reading frame of 654 base pairs that encoded a protein of 218 amino acid residues with a deduced molecular mass of 23,996 Da. The protein encoded in the DNA fragment expressed in E. coli hydrolyzed a carboxymethylcellulose. This gene was subcloned into the shuttle vector [pZA22; Misawa et al. (1986) Agric Biol Chem 50:3201-3203] between Zymomonas mobilis and E. coli. The recombinant plasmid pZAAC21 was introduced into Z. mobilis IFO 13756 by electroporation. The carboxymethylcellulase gene was efficiently expressed in both bacteria, although the level of expression in Z. mobilis was ten times greater than that in E. coli. Approximately 75% of the total carboxymethylcellulase activity detected in Z. mobilis was located in the periplasmic space (outside of the cytoplasmic space). Enzyme activity was not detected in the periplasmic space, but in the cytoplasm of E. coli.
分离出与木醋杆菌IFO 3288的羧甲基纤维素酶活性相对应的DNA片段，并将其克隆到大肠杆菌中，然后测定了该DNA序列。测序的DNA片段有一个654个碱基对的开放阅读框，编码一个由218个氨基酸残基组成的蛋白质，推导分子量为23,996道尔顿。在大肠杆菌中表达的该DNA片段编码的蛋白质可水解羧甲基纤维素。该基因被亚克隆到运动发酵单胞菌和大肠杆菌之间的穿梭载体[pZA22；三泽等人（1986年），农业生物化学50：3201 - 3203]中。通过电穿孔将重组质粒pZAAC21导入运动发酵单胞菌IFO 13756。羧甲基纤维素酶基因在两种细菌中均有效表达，尽管在运动发酵单胞菌中的表达水平比在大肠杆菌中的高十倍。在运动发酵单胞菌中检测到的总羧甲基纤维素酶活性约75%位于周质空间（细胞质空间之外）。在大肠杆菌的周质空间中未检测到酶活性，而是在细胞质中检测到。
