Catalytic cleavage reactions of phosphorylase b were monitored directly on an amylopectin-immobilized 27 MHz quartz-crystal microbalance (QCM). When the inactivated phosphorylase b was injected into a phosphate buffer solution of amylopectin-immobilized QCM (method A), the binding of the enzyme to amylopectin was observed as a frequency decrease (mass increase). Then, when AMP (adenosine monophosphate) was added to activate the enzyme, the frequency gradually increased (mass decreased) due to the phosphorolysis of amylopectin in the presence of phosphates as buffers. When the AMP-activated phosphorylase b was employed (method B), the continuous reaction was observed which includes both the mass increase due to the enzyme binding to amylopectin at first and then the following mass decrease due to the phosphorolysis by the AMP-activated enzyme. All kinetic parameters for the enzyme binding to the substrate (binding and dissociation rate constants, k(on) and k(off), and dissociation constant, K(d)), the AMP binding to the enzyme as activator (K(AMP)), the catalytic rate constant (k(cat)) were obtained from curve fittings of time-courses of frequency (mass) changes. The obtained kinetic parameters were compared with those from Michaelis-Menten kinetics.
All kinetic parameters such as the enzyme binding process and the catalytic processes (phosphorolysis and polymerization) by phosphorylase from Potato could be obtained using an amylopectin-immobilized 27-MHz quartz-crystal microbalance (QCM).
A novel alkyl spacer-conjugated derivative of P(k) trisaccharide (P(k)), one of the active receptors of Shiga toxins (Stxs; Stx1 and Stx2) produced by pathogenic Escherichia coli (STEC), was designed and synthesized by a combination of cellulase-mediated condensation from Trichoderma reesei and α1,4-galactosyltransferase (LgtC) from Neisseria gonorrhoeae. The specific activity of N. gonorrhoeae LgtC was 66U/mg, which was 13-fold higher than that from N. meningitidis expressed in E. coli. 5-trifluoroacetamidopentyl-β-P(k) (TFAP-P(k)) was synthesized (yield of 86%, based on the amount of TFAP-lactose added) and its binding to Stx1a-B and Stx2a-B was evaluated. The dissociation constants (KDs) of Stx1a-B and Stx2a-B to the spacer-linked P(k), immobilized on a CM5 sensor chip, were 6.8×10(-6) M (kon=4.1×10(1)M(-1)S(-1), koff=2.8×10(-4)S(-1)) and 2.2×10(-5)M (kon=3.9×10(2)M(-1)S(-1), koff=8.6×10(-3)S(-1)), respectively. This result suggests that the monovalent P(k)-derivative, conjugated to a pentylamino group, represents a promising Stx-neutralizing agent. This cellulase-mediated condensation using cellulase and glycosyltransferase is a valuable tool for the synthesis of spacer-linked oligosaccharide.
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