Cloning of the thermostable phytase gene (phy) from Bacillus sp. DS11 and its overexpression in Escherichia coli.
Phytase hydrolyzes phytate to release inorganic phosphate, which would decrease the addition of phosphorus to feedstuffs for monogastric animals and thus reduce environmental pollution. The gene encoding phytase from Bacillus sp. DS11 was cloned in Escherichia coli and its sequence determined. A 560-bp DNA fragment was used as a probe to screen the genomic library. It was obtained through PCR of Bacillus sp. DS11 chromosomal DNA and two oligonucleotide primers based on N-terminal amino acid sequences of the purified protein and the cyanogen bromide-cleaved 21-kDa fragment. The phy cloned was encoded by a 2.2-kb fragment. This gene comprises 1152 nucleotides and encodes a polypeptide of 383 amino acids with a deduced molecular mass of 41,808 Da. Phytase was produced to 20% content of total soluble proteins in E. coli BL21 (DE3) using the pET22b(+) vector with the inducible T7 promoter. This is the first nucleic sequence report on phytase from a bacterial strain.
Kim
Y O
YO
Microbial Enzyme RU, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Taejon, South Korea.
Lee
J K
JK
Kim
H K
HK
Yu
J H
JH
Oh
T K
TK
D010832:6-Phytase / Q000737:chemistry / Q000235:genetics*; D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial / Q000235:genetics; D006358:Hot Temperature; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry; D015183:Restriction Mapping; D017421:Sequence Analysis; D017422:Sequence Analysis, DNA
D011993:Recombinant Fusion Proteins; D010832:6-Phytase