Expression, crystallization and preliminary X-ray crystallographic studies of Klebsiella pneumoniae maltohexaose-producing alpha-amylase.
A recombinant form of Klebsiella pneumoniae maltohexaose-producing alpha-amylase has been overexpressed in Escherichia coli and purified to homogeneity. Crystals were obtained at 293 K by the microbatch technique using 80 mM sodium/potassium phosphate buffer pH 6.2 containing 8% polyethylene glycol 3000, 4% polyethylene glycol 3350 and 40 mM sodium thiocyanate. Crystals of the overexpressed recombinant enzyme diffracted to better than 2.5 A resolution at 95 K using a synchrotron-radiation source. The crystals belong to the primitive monoclinic space group P2(1), with unit-cell parameters a = 74.8, b = 107.6, c = 82.2 A, beta = 96.2 degrees. Assuming the presence of two molecules per asymmetric unit, the V(M) value for the crystal was 2.3 A(3) Da(-1), indicating a solvent content of 47%.
Momma
Mitsuru
M
Department of Biochemistry, National Institute of Agrobiological Sciences, Kan-nondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan. momma@nias.affrc.go.jp
Fujimoto
Zui
Z
D002021:Buffers; D003460:Crystallization; D018360:Crystallography, X-Ray / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007711:Klebsiella pneumoniae / Q000201:enzymology*; D009154:Mutation; D009844:Oligosaccharides / Q000737:chemistry*; D010710:Phosphates / Q000737:chemistry / Q000494:pharmacology; D011092:Polyethylene Glycols / Q000737:chemistry; D017680:Potassium Compounds / Q000737:chemistry; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D017356:Synchrotrons; D013696:Temperature; D013861:Thiocyanates / Q000494:pharmacology; D014961:X-Ray Diffraction; D000516:alpha-Amylases / Q000737:chemistry*
D002021:Buffers; D009844:Oligosaccharides; D010710:Phosphates; D017680:Potassium Compounds; D011994:Recombinant Proteins; D013861:Thiocyanates; C016549:maltohexaose; D011092:Polyethylene Glycols; C024553:sodium thiocyanate; C013216:potassium phosphate; D000516:alpha-Amylases; C018279:sodium phosphate