Gutshall K R, Trimbur D E, Kasmir J J, Brenchley J E
Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, USA.
J Bacteriol. 1995 Apr;177(8):1981-8. doi: 10.1128/jb.177.8.1981-1988.1995.
We have characterized a new psychrotrophic Arthrobacter isolate which produces beta-galactosidase isozymes. When DNA from this isolate was transformed into an Escherichia coli host, we obtained three different fragments, designated 12, 14, and 15, each encoding a different beta-galactosidase isozyme. The beta-galactosidase produced from fragment 12 was of special interest because the protein subunit was smaller (about 71 versus 116 kDa) than those typically encoded by the lacZ family. The isozyme encoded by fragment 12 was purified, and its activity and thermostability were examined. Although the enzyme is highly specific towards beta-D-galactoside substrates, its levels in the isolate do not increase in cells grown with lactose. Nucleotide sequence determination showed that the gene encoding isozyme 12 is not similar to the other members of the lacZ family but has regions similar to beta-galactosidase isozymes from Bacillus stearothermophilus and B. circulans. Addition of the isozyme 12 sequence to the database made it possible to examine these enzymes as possible members of a new, separate family. Our analysis of this new family showed some conserved amino acids corresponding to the lacZ acid-base catalytic region but no homology with the nucleophilic region. On the basis of these comparisons, we designated this a new lacG family.
我们已鉴定出一种新的嗜冷节杆菌分离株，它能产生β-半乳糖苷酶同工酶。当将该分离株的DNA转化到大肠杆菌宿主中时，我们获得了三个不同的片段，分别命名为12、14和15，每个片段编码一种不同的β-半乳糖苷酶同工酶。片段12产生的β-半乳糖苷酶特别引人关注，因为其蛋白质亚基比通常由lacZ家族编码的亚基小（约71 kDa对116 kDa）。对片段12编码的同工酶进行了纯化，并检测了其活性和热稳定性。尽管该酶对β-D-半乳糖苷底物具有高度特异性，但在以乳糖培养的细胞中，其在分离株中的水平并未增加。核苷酸序列测定表明，编码同工酶12的基因与lacZ家族的其他成员不同，但具有与嗜热脂肪芽孢杆菌和环状芽孢杆菌的β-半乳糖苷酶同工酶相似的区域。将同工酶12序列添加到数据库中，使得可以将这些酶作为一个新的独立家族的可能成员进行研究。我们对这个新家族的分析表明，有一些与lacZ酸碱催化区域相对应的保守氨基酸，但与亲核区域没有同源性。基于这些比较，我们将其命名为一个新的lacG家族。
