Rashid M H, Mori M, Sekiguchi J
Department of Applied Biology, Faculty of Textile Science & Technology, Shinshu University, Nagano, Japan.
Microbiology (Reading). 1995 Oct;141 ( Pt 10):2391-404. doi: 10.1099/13500872-141-10-2391.
The 90 kDa glucosaminidase protein was purified to apparent homogeneity from vegetative cells of Bacillus subtilis AC327, and then the corresponding gene was cloned into Escherichia coli in two inactive forms by standard procedures. Nucleotide sequencing of the glucosaminidase region revealed a monocistronic operon, (designated lytD = cwIG) encoding a 95.6 kDa protein, comprising 880 amino acid residues, which has a typical signal peptide. Moreover, another monocistronic operon (designated pmi = orfX), encoding a 35.4 kDa protein, was found upstream of the glucosaminidase gene. Expression of a lytD-lacZ fusion gene, driven by lytD regulatory sequences, was observed during the exponential growth phase. The introduction of a sigD null mutation greatly reduced (by about 95%) the expression of the fusion. Amino acid sequence analysis of the glucosaminidase showed two types of direct repeats, each type being present twice, in the N-terminal-to-central region of the glucosaminidase: these repeats probably represent the cell-wall-binding domain. Zymographic analysis revealed that the 90 kDa glucosaminidase is partly processed to several smaller proteins (35-39 kDa), retaining lytic activity. Processing of these proteins occurred between the N-terminal cell-wall-binding and C-terminal catalytic domains of the glucosaminidase, the site being located between the 569th and 606th codons of the glucosaminidase. Serial deletions from the N-terminus of the glucosaminidase revealed that the loss of more than one repeating unit drastically reduces its lytic activity toward cell walls. The lytD gene product, in either an intact or a truncated form, was found to be lethal for E. coli, and the N-terminally truncated glucosaminidase proteins, produced in E. coli, were very unstable. The partially purified glucosaminidase from B. subtilis was found to be very unstable at low ionic strength at 37 degrees C, but this instability was overcome by the addition of either SDS-purified cell wall or protease inhibitor (PMSF) to the enzyme or after purification of the glucosaminidase to apparent homogeneity.
从枯草芽孢杆菌AC327的营养细胞中纯化出了90 kDa的氨基葡萄糖苷酶蛋白，使其达到表观均一性，然后通过标准程序将相应基因以两种无活性形式克隆到大肠杆菌中。氨基葡萄糖苷酶区域的核苷酸测序揭示了一个单顺反子操纵子（命名为lytD = cwIG），它编码一种95.6 kDa的蛋白质，由880个氨基酸残基组成，具有典型的信号肽。此外，在氨基葡萄糖苷酶基因上游发现了另一个单顺反子操纵子（命名为pmi = orfX），它编码一种35.4 kDa的蛋白质。在指数生长期观察到由lytD调控序列驱动的lytD-lacZ融合基因的表达。sigD缺失突变的引入极大地降低了（约95%）融合基因的表达。氨基葡萄糖苷酶的氨基酸序列分析表明，在氨基葡萄糖苷酶的N端到中央区域存在两种类型的直接重复序列，每种类型出现两次：这些重复序列可能代表细胞壁结合结构域。酶谱分析表明，90 kDa的氨基葡萄糖苷酶部分加工成几种较小的蛋白质（35 - 39 kDa），并保留了裂解活性。这些蛋白质的加工发生在氨基葡萄糖苷酶的N端细胞壁结合结构域和C端催化结构域之间，该位点位于氨基葡萄糖苷酶第569和606个密码子之间。从氨基葡萄糖苷酶的N端进行系列缺失表明，失去一个以上的重复单元会显著降低其对细胞壁的裂解活性。发现lytD基因产物无论是完整形式还是截短形式对大肠杆菌都是致死的，并且在大肠杆菌中产生的N端截短的氨基葡萄糖苷酶蛋白非常不稳定。从枯草芽孢杆菌中部分纯化的氨基葡萄糖苷酶在37℃低离子强度下非常不稳定，但通过向酶中添加SDS纯化的细胞壁或蛋白酶抑制剂（PMSF），或者在将氨基葡萄糖苷酶纯化至表观均一性后，这种不稳定性得以克服。
