Molecular cloning of the phytase gene from Citrobacter braakii and its expression in Saccharomyces cerevisiae.
The gene, appA, encoding phytase was cloned from a size-selected genomic library of Citrobacter braakii YH-15 by Southern hybridization using a degenerate probe based on the N-terminal amino acid sequence of the phytase. The deduced amino acid sequence of appA contained the N-terminal RHGXRXP motif and the C-terminal HD motif, which are common in histidine acid phosphatases. It also had significant homology (60% identity) with phytase from Escherichia coli, while the physical mapping analysis of appA revealed that gene organization near appA in C. braakii was similar to that in Salmonella typhimurium genome. C. braakii AppA contained five putative N-glycosylation sites. The recombinant phytases, rAppAEc and rAppASc, were produced in E. coli and Saccharomyces cerevisiae, respectively, with both being fused with C-terminal His-tag. After purification, rAppASc was shown to be hyperglycosylated by Endo-H treatment. It had greater thermostability than the wild type phytase and rAppAEc.
Kim
Young-Ok
YO
Biotechnology research center, National Fisheries Research and Development Institute (NFRDI), 408-1 Sirang-ri, 619-902, Gigang-eup, Gigang-gun, Busan, Korea.
Kim
Han-Woo
HW
Lee
Jung-Ho
JH
Kim
Kyung-Kil
KK
Lee
Sang-Jun
SJ
D010832:6-Phytase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001483:Base Sequence; D002876:Chromosomes, Bacterial; D002954:Citrobacter / Q000235:genetics*; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D015870:Gene Expression; D008969:Molecular Sequence Data; D010802:Phylogeny; D020161:Physical Chromosome Mapping; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D011994:Recombinant Proteins; D010832:6-Phytase