Gene cloning, expression, and crystallization of a thermostable exo-inulinase from Geobacillus stearothermophilus KP1289.
The gene ( inuA) encoding exo-inulinase (EC 3.2.1.80) was cloned from the thermophilic Geobacillus stearothermophilus ( Bacillus stearothermophilus) KP 1289 growing at between 41 degrees C and 69 degrees C. The inuA gene consisted of 1,482 bp encoding a protein of 493 amino acids. The deduced polypeptide of molecular mass ( M) 56,744 Da showed strong sequence similarity to Pseudomonas mucidolens exo-inulinase, Bacillus subtilis levanase, Paenibacillus polymyxa ( Bacillus polymyxa) fructosyltransferase, and so on, indicating that the enzyme belonged to glycosyl hydrolase family 32. The M of the purified exo-inulinase, expressed in Escherichia coli HB101, was estimated as approximately 54,000 Da by both SDS-PAGE and gel filtration. These results suggested that the active form of the enzyme is a monomer. The enzyme was active between 30 and 75 degrees C with an optimum at 60 degrees C. The properties were identical to those of the native enzyme. Additionally, for the first time for a prokaryotic GH32 protein, crystals of the recombinant enzyme were obtained.
Tsujimoto
Y
Y
Department of Applied Biochemistry, Kyoto Prefectural University, Shimogamo, Sakyo, 606-8522, Kyoto, Japan. yoshi_t@kpu.ac.jp
Watanabe
A
A
Nakano
K
K
Watanabe
K
K
Matsui
H
H
Tsuji
K
K
Tsukihara
T
T
Suzuki
Y
Y
D000595:Amino Acid Sequence; D001406:Bacillaceae / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D003460:Crystallization; D015342:DNA Probes; D004269:DNA, Bacterial / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D006602:Hexosyltransferases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D006358:Hot Temperature; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D015342:DNA Probes; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D006602:Hexosyltransferases; C021450:inulin fructotransferase (depolymerizing)