Cloning, sequence, and phenotypic expression of katA, which encodes the catalase of Lactobacillus sake LTH677.
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.
Knauf
H J
HJ
Institut für Lebensmitteltechnologie, Universität Hohenheim, Stuttgart, Germany.
Vogel
R F
RF
Hammes
W P
WP
D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D002374:Catalase / Q000235:genetics*; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D007778:Lactobacillus / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010641:Phenotype; D010957:Plasmids; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid
D004269:DNA, Bacterial; D002374:Catalase