CLONING, SEQUENCE, AND PHENOTYPIC-EXPRESSION OF KATA, WHICH ENCODES THE CATALASE OF LACTOBACILLUS-SAKE LTH677
CLONING, SEQUENCE, AND PHENOTYPIC-EXPRESSION OF KATA, WHICH ENCODES THE CATALASE OF LACTOBACILLUS-SAKE LTH677
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DOI：
10.1128/aem.58.3.832-839.1992
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发表时间：
1992-03-01
影响因子：
4.4
通讯作者：
HAMMES, WP
中科院分区：
文献类型：
--
作者：
KNAUF, HJ;VOGEL, RF;HAMMES, WP
清酒乳杆菌（Lactobacillus sake）LTH 677是从发酵香肠中分离的菌株，其形成血红素依赖性过氧化氢酶。 这种罕见的特性在香肠发酵中非常理想，因为它可以防止过氧化氢引起的酸败和变色。 构建了含有清酒乳杆菌（Lactobacillus sake）LTH 677染色体DNA的MboI片段的大肠杆菌质粒pBR 328基因库。 通过异源互补E. coli UM2。 通过质粒pHK 1000中最初克隆片段的缺失分析，将过氧化氢酶结构基因（命名为katA）分配到2.3-kb区域。 通过Southern杂交确定原始染色体排列。 蛋白质分析表明，过氧化氢酶亚基的分子大小为65，000 Da，活性过氧化氢酶具有六聚体结构。 从核苷酸序列推断的亚基的分子大小被确定为54，504 Da。65，000 Da蛋白质的N-末端氨基酸序列对应于从DNA序列推断的氨基酸序列。 katA在E.大肠杆菌-乳球菌穿梭载体pGKV 210中，该基因被成功地转移并表型表达在干酪乳杆菌中，该干酪乳杆菌天然缺乏过氧化氢酶活性。
Lactobacillus sake LTH677 is a strain, isolated from fermented sausage, which forms a heme-dependent catalase. This rare property is highly desirable in sausage fermentation, as it prevents rancidity and discoloration caused by hydrogen peroxide. A gene bank containing MboI fragments of chromosomal DNA from Lactobacillus sake LTH677 in Escherichia coli plasmid pBR328 was constructed. The catalase gene was cloned by heterologous complementation of the Kat- phenotype of E. coli UM2. The catalase structural gene, designated katA, was assigned to a 2.3-kb region by deletion analysis of the originally cloned fragment in plasmid pHK1000. The original chromosomal arrangement was determined by Southern hybridization. Protein analysis revealed that the catalase subunit has a molecular size of 65,000 Da and that the active catalase possesses a hexameric structure. The molecular size of the subunit deduced from the nucleotide sequence was determined to 54,504 Da. The N-terminal amino acid sequence of the 65,000-Da protein corresponded to the one deduced from the DNA sequence. After recloning of katA in the E. coli-Lactococcus shuttle vector pGKV210, the gene was successfully transferred and phenotypically expressed in Lactobacillus casei, which is naturally deficient in catalase activity.