Thompson J, Nguyen N Y, Sackett D L, Donkersloot J A
Laboratory of Microbial Ecology, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1991 Aug 5;266(22):14573-9.
Sucrose-6-phosphate hydrolase from Lactococcus lactis subsp. lactis K1-23 (formerly Streptococcus lactis K1-23) has been purified 600-fold to electrophoretic homogeneity. Purification of the enzyme was achieved by DEAE-Sephacel, phosphocellulose P-11, and gel exclusion (Ultrogel AcA 54) chromatography. The purified enzyme (specific activity 31 units/mg) catalyzed the hydrolysis of both 6-O-phosphoryl-alpha-D-glucopyranosyl-1,2-beta-D-fructofuranoside (sucrose 6-phosphate) and sucrose (Km = 0.1 and 100 mM, respectively). Ultracentrifugal analysis of sucrose-6-phosphate hydrolase indicated an Mr = 52,200. The purified enzyme migrated as a single protein during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr = 52,000). However, four distinct polypeptides were detected by analytical electrofocusing, and all four species hydrolyzed sucrose and sucrose 6-phosphate. The amino acid composition of sucrose-6-phosphate hydrolase, and the sequence of the first 12 amino acids from the NH2 terminus, have been determined. Hybridization studies with oligonucleotide probes show that the genes for sucrose-6-phosphate hydrolase (scrB), Enzyme IIScr of the phosphoenolypyruvate-dependent sucrose:phosphotransferase system (scrA), and N5-(carboxyethyl)ornithine synthase (ceo) are encoded by the same approximately 20-kilobase EcoRI fragment. This fragment is part of a large transposon Tn5306 that also encodes the nisin precursor gene, spaN, and IS904. In L. lactis ATCC 11454, spaN, IS904, scrA, and scrB (but not ceo) are encoded on a related transposon, Tn5307.
来自乳酸乳球菌乳酸亚种K1-23（原乳酸链球菌K1-23）的蔗糖-6-磷酸水解酶已被纯化600倍，达到电泳纯。通过DEAE-葡聚糖凝胶、磷酸纤维素P-11和凝胶排阻（Ultrogel AcA 54）色谱法实现了该酶的纯化。纯化后的酶（比活性为31单位/毫克）催化6-O-磷酸-α-D-吡喃葡萄糖基-1,2-β-D-呋喃果糖苷（蔗糖6-磷酸）和蔗糖的水解（Km分别为0.1和100 mM）。对蔗糖-6-磷酸水解酶的超速离心分析表明其Mr = 52,200。纯化后的酶在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳中迁移为单一蛋白（Mr = 52,000）。然而，通过分析性电聚焦检测到四种不同的多肽，且所有四种多肽都能水解蔗糖和蔗糖6-磷酸。已确定了蔗糖-6-磷酸水解酶的氨基酸组成以及从NH2末端起的前12个氨基酸的序列。用寡核苷酸探针进行的杂交研究表明，蔗糖-6-磷酸水解酶（scrB）、磷酸烯醇丙酮酸依赖性蔗糖：磷酸转移酶系统的酶IIScr（scrA）和N5-（羧乙基）鸟氨酸合酶（ceo）的基因由同一个约20千碱基的EcoRI片段编码。该片段是一个大转座子Tn5306的一部分，该转座子还编码乳链菌肽前体基因spaN和IS904。在乳酸乳球菌ATCC 11454中，spaN、IS904、scrA和scrB（但不包括ceo）由一个相关的转座子Tn5307编码。
