Heterologous expression and characterization of Bacillus coagulans L-arabinose isomerase.
Bacillus coagulans has been of great commercial interest over the past decade owing to its strong ability of producing optical pure L: -lactic acid from both hexose and pentose sugars including L: -arabinose with high yield, titer and productivity under thermophilic conditions. The L: -arabinose isomerase (L-AI) from Bacillus coagulans was heterologously over-expressed in Escherichia coli. The open reading frame of the L-AI has 1,422 nucleotides encoding a protein with 474 amino acid residues. The recombinant L-AI was purified to homogeneity by one-step His-tag affinity chromatography. The molecular mass of the enzyme was estimated to be 56 kDa by SDS-PAGE. The enzyme was most active at 70°C and pH 7.0. The metal ion Mn(2+) was shown to be the best activator for enzymatic activity and thermostability. The enzyme showed higher activity at acidic pH than at alkaline pH. The kinetic studies showed that the K (m), V (max) and k (cat)/K (m) for the conversion of L: -arabinose were 106 mM, 84 U/mg and 34.5 mM(-1)min(-1), respectively. The equilibrium ratio of L: -arabinose to L: -ribulose was 78:22 under optimal conditions. L: -ribulose (97 g/L) was obtained from 500 g/l of L: -arabinose catalyzed by the enzyme (8.3 U/mL) under the optimal conditions within 1.5 h, giving at a substrate conversion of 19.4% and a production rate of 65 g L(-1) h(-1).
Institute of Chemical and Engineering Sciences, Agency for Science, Technology and Research (A STAR), 1 Pesek Road, Jurong Island 627833, Singapore.
Xingding
X
Zhou
Jin Chuan
JC
Wu
D019747:Aldose-Ketose Isomerases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001089:Arabinose / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D020536:Enzyme Activators / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008345:Manganese / Q000378:metabolism; D008970:Molecular Weight; D016366:Open Reading Frames; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature
D020536:Enzyme Activators; D011994:Recombinant Proteins; D008345:Manganese; D001089:Arabinose; D019747:Aldose-Ketose Isomerases; C019715:L-arabinose isomerase