title: Bioengineering of bacterial polymer inclusions catalyzing the synthesis of N-acetylneuraminic acid.
abstract: N-Acetylneuraminic acid is produced by alkaline epimerization of N-acetylglucosamine to N-acetylmannosamine and then subsequent condensation with pyruvate catalyzed by free N-acetylneuraminic acid aldolase. The high-alkaline conditions of this process result in the degradation of reactants and products, while the purification of free enzymes to be used for the synthesis reaction is a costly process. The use of N-acetylglucosamine 2-epimerase has been seen as an alternative to the alkaline epimerization process. In this study, these two enzymes involved in N-acetylneuraminic acid production were immobilized to biopolyester beads in vivo in a one-step, cost-efficient process of production and isolation. Beads with epimerase-only, aldolase-only, and combined epimerase/aldolase activity were recombinantly produced in Escherichia coli. The enzymatic activities were 32 U, 590 U, and 2.2 U/420 U per gram dry bead weight, respectively. Individual beads could convert 18% and 77% of initial GlcNAc and ManNAc, respectively, at high substrate concentrations and near-neutral pH, demonstrating the application of this biobead technology to fine-chemical synthesis. Beads establishing the entire N-acetylneuraminic acid synthesis pathway were able to convert up to 22% of the initial N-acetylglucosamine after a 50-h reaction time into N-acetylneuraminic acid.
authors: Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand.
authors: David O
authors: DO
authors: Hooks
authors: Paul A
authors: PA
authors: Blatchford
authors: Bernd H A
authors: BH
authors: Rehm
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D057005:Bioengineering; D002238:Carbohydrate Epimerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D006595:Hexosamines / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008863:Microspheres; D019158:N-Acetylneuraminic Acid / Q000378:metabolism*; D007652:Oxo-Acid-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054813:Polyhydroxyalkanoates / Q000737:chemistry*; D011108:Polymers / Q000737:chemistry*; D019289:Pyruvic Acid / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D046939:Synechocystis
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D006595:Hexosamines; D054813:Polyhydroxyalkanoates; D011108:Polymers; D011993:Recombinant Fusion Proteins; D019289:Pyruvic Acid; D007652:Oxo-Acid-Lyases; C018728:N-acetylneuraminate lyase; D002238:Carbohydrate Epimerases; C024519:N-acyl-D-glucosamine 2-epimerase; D019158:N-Acetylneuraminic Acid; C002022:N-acetylmannosamine