Overexpression, purification and preliminary X-ray analysis of pullulanase from Bacillus subtilis strain 168

Journal Article · Sat Apr 01 04:00:00 EST 2006 · Acta Crystallographica. Section F

DOI:https://doi.org/10.1107/S1744309106007901· OSTI ID:22356339

Malle, Dominggus; Itoh, Takafumi ^[1]; Hashimoto, Wataru; Murata, Kousaku ^[2]; Utsumi, Shigeru; Mikami, Bunzo ^[1]

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1. Laboratory of Food Quality Design and Development, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan)
2. Laboratory of Molecular Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan)

The AmyX gene encoding pullulanase from the common spore-forming bacterium B. subtilis strain 168 has been cloned, overexpressed in Escherichia coli, purified and crystallized. The AmyX gene encoding pullulanase from the common spore-forming bacterium Bacillus subtilis strain 168 was cloned, overexpressed in Escherichia coli, purified and crystallized. The recombinant pullulanase was purified to homogeneity using ammonium sulfate precipitation, hydrophobic chromatography and anion-exchange chromatography, resulting in a specific activity of 24.10 U per milligram of protein. SDS–PAGE analysis showed that the molecular weight of the protein is approximately 81.0 kDa, which is similar to the calculated molecular weight, 81.1 kDa, from its translated cDNA sequence. The k{sub cat} and K{sub m} of the purified enzyme with pullulan as substrate were approximately 79 s{sup −1} and 1.284 mg ml{sup −1}, respectively. X-ray crystallographic analysis of the pullulanase crystal showed that the crystal belongs to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 70.568, b = 127.68, c = 189.25 Å. The crystal contains two molecules of pullulanase in the asymmetric unit, with a solvent content of 53.15%. The crystal diffracted to 2.1 Å resolution at a synchrotron and is suitable for structure determination.

• OSTI ID: 22356339
• Journal Information: Acta Crystallographica. Section F, Journal Name: Acta Crystallographica. Section F Journal Issue: Pt 4 Vol. 62; ISSN ACSFCL; ISSN 1744-3091
• Country of Publication: United Kingdom
• Language: English

Related Subjects

75 CONDENSED MATTER PHYSICS
SUPERCONDUCTIVITY AND SUPERFLUIDITY
AMMONIUM SULFATES
CRYSTALS
ESCHERICHIA COLI
MOLECULAR WEIGHT
MOLECULES
PRECIPITATION
RESOLUTION
SOLVENTS
SPACE GROUPS
STRAINS
SYNCHROTRONS