title: Evaluation of four microbial Class II fructose 1,6-bisphosphate aldolase enzymes for use as biocatalysts.
abstract: Fructose 1,6-bisphosphate (FBP) aldolase has been used as biocatalyst in the synthesis of several pharmaceutical compounds such as monosaccharides and analogs. Is has been suggested that microbial metal-dependant Class II aldolases could be better industrial catalysts than mammalian Class I enzyme because of their greater stability. The Class II aldolases from four microbes were subcloned into the Escherichia coli vector pT7-7, expressed and purified to near homogeneity. The kinetic parameters, temperature stability, pH profile, and tolerance to organic solvents of the Class II enzymes were determined, and compared with the properties of the Class I aldolase from rabbit muscle. Contrary to results obtained previously with the E. coli Class II aldolase, which was reported to be more stable than the mammalian enzyme, other recombinant Class II aldolases were found to be generally less stable than the Class I enzyme, especially in the presence of organic solvents. Class II aldolase from Bacillus cereus showed higher temperature stability than the other enzymes tested, but only the Mycobacterium tuberculosis Class II aldolase had a stability comparable to the Class I mammalian enzyme under assay conditions. The turnover number of the recombinant M. tuberculosis and Magnaporthe grisea Class II type A aldolases was comparable or higher than that of the Class I enzyme. The recombinant B. cereus and Pseudomonas aeruginosa Class II type B aldolases had very low turnover numbers and low metal content, indicating that the E. coli overexpression system may not be suitable for the Class II type B aldolases from these microorganisms.
authors.affiliation: Department of Chemistry, University of Waterloo, 200 University Ave. W, Waterloo, ON N2L3G1, Canada.
authors.forename: Geneviève
authors.initials: G
authors.lastname: Labbé
authors.forename: Sarah
authors.initials: S
authors.lastname: de Groot
authors.forename: Timothy
authors.initials: T
authors.lastname: Rasmusson
authors.forename: Gorica
authors.initials: G
authors.lastname: Milojevic
authors.forename: Gary I
authors.initials: GI
authors.lastname: Dmitrienko
authors.forename: J Guy
authors.initials: JG
authors.lastname: Guillemette
mesh_terms: D000818:Animals; D001409:Bacillus cereus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004789:Enzyme Activation; D057075:Enzyme Assays; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005634:Fructose-Bisphosphate Aldolase / Q000302:isolation & purification / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D005798:Genes, Bacterial; D005993:Glycerolphosphate Dehydrogenase / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D020082:Magnaporthe / Q000201:enzymology* / Q000235:genetics; D013058:Mass Spectrometry; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D055550:Protein Stability; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012997:Solvents / Q000378:metabolism; D013696:Temperature; D014305:Triose-Phosphate Isomerase / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D005656:Fungal Proteins; D011994:Recombinant Proteins; D012997:Solvents; D005993:Glycerolphosphate Dehydrogenase; D005634:Fructose-Bisphosphate Aldolase; D014305:Triose-Phosphate Isomerase