Molecular Biology Group, Faculty of Engineering, Toyama University, 3190 Gofuku, Toyama 930-8555, Japan.
Gene. 2012 Mar 10;495(2):154-62. doi: 10.1016/j.gene.2011.12.004. Epub 2011 Dec 16.
Though some genetic features of lactobacillar fructan hydrolases were elucidated, information about their enzymology or mutational analyses were scarce. Lactobacillus casei IAM1045 exhibits extracellular activity degrading inulin. After partial purification of the inulin-degrading protein from the spent culture medium, several fragments were obtained by protease digestion. Based on their partial amino-acid sequences, oligonucleotide primers were designed, and its structural gene (levH1) was determined using the gene library constructed in the E. coli system. The levH1 gene encoded a protein (designated as LevH1), of which calculated molecular mass and pI were 138.8-kDa and 4.66, respectively. LevH1 (1296 amino-acids long) was predicted to have a four-domain structure, containing (i) an N-terminal secretion signal of 40 amino-acids, (ii) variable domain of about 140 residues whose function is unclear, (iii) a catalytic domain of about 630 residues with glycoside-hydrolase activity consisting of two modules, a five-blade β-propeller module linked to a β-sandwich module, (iv) a C-terminal domain of about 490 residues comprising five nearly perfect repeat sequences of 80 residues homologous to equivalents of other hypothetical cell surface proteins, followed by 37-residues rich in Ser/Thr/Pro/Gly, a pentad LPQAG (the LPXTG homologue). When overproduced in E. coli, the putative variable-catalytic domain region of about 770 residues exhibited exo-inulinase activity. Deletion analyses demonstrated that the variable-catalytic domain region containing two modules is important for enzymatic activity. Presence of eight conserved motifs (I-VIII) was suggested in the catalytic domain by comparative analysis, among which motif VIII was newly identified in the β-sandwich module in this study. Site-directed mutagenesis of conserved amino-acids in these motifs revealed that D198, R388, D389 and E440, were crucial for inulinase activity. Moreover, mutations of D502A and D683A in motif VI and VIII respectively caused significant decrease in the activity. These results suggested that the variable domain and β-sandwich module, besides the β-propeller module, are important for inulin-degrading activity of LevH1.
尽管已经阐明了一些乳杆菌果聚糖水解酶的遗传特征，但关于它们的酶学或突变分析的信息却很少。干酪乳杆菌 IAM1045 表现出降解菊粉的细胞外活性。在从耗尽的培养基中部分纯化菊粉降解蛋白后，通过蛋白酶消化获得了几个片段。基于它们的部分氨基酸序列，设计了寡核苷酸引物，并使用在大肠杆菌系统中构建的基因文库确定了其结构基因（levH1）。levH1 基因编码一种蛋白质（命名为 LevH1），其计算的分子质量和等电点分别为 138.8 kDa 和 4.66。LevH1（长 1296 个氨基酸）预测具有四结构域结构，包含（i）40 个氨基酸的 N 端分泌信号，（ii）约 140 个残基的可变域，其功能尚不清楚，（iii）约 630 个残基的催化域，具有糖苷水解酶活性，由两个模块组成，一个五叶β-桨叶模块连接到一个β-三明治模块，（iv）约 490 个残基的 C 端结构域，包含 5 个几乎完美的重复序列，每个序列由 80 个残基组成，与其他假想的细胞表面蛋白的对应物同源，后面是富含 Ser/Thr/Pro/Gly 的 37 个残基，五肽 LPQAG（LPXTG 同源物）。在大肠杆菌中过表达时，约 770 个残基的假定可变-催化结构域区域表现出外切菊粉酶活性。缺失分析表明，包含两个模块的可变-催化结构域区域对于酶活性很重要。通过比较分析，在催化结构域中提出了 8 个保守基序（I-VIII），其中基序 VIII 在本研究中是在β-三明治模块中新发现的。对这些基序中保守氨基酸的定点突变表明，D198、R388、D389 和 E440 对菊粉酶活性至关重要。此外，在基序 VI 和 VIII 中分别突变 D502A 和 D683A 导致活性显著降低。这些结果表明，可变结构域和β-三明治模块，除了β-桨叶模块外，对 LevH1 的菊粉降解活性很重要。
