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Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration
Published on: January 22, 2014
Gene cloning, expression, and crystallization of a thermostable exo-inulinase from Geobacillus stearothermophilus
Y Tsujimoto1, A Watanabe, K Nakano
1Department of Applied Biochemistry, Kyoto Prefectural University, Shimogamo, Sakyo, 606-8522, Kyoto, Japan. yoshi_t@kpu.ac.jp
Abstract:
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.
