Gravimetric Analyses of Enzymatic Glucan Hydrolysis and Phosphorolysis on a 27MHz Quartz-Crystal Microbalance

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  • Mori Toshiaki
    Department of Biomolecular Engineering and Frontier Collaborative Research Center, Tokyo Institute of Technology
  • Okahata Yoshio
    Department of Biomolecular Engineering and Frontier Collaborative Research Center, Tokyo Institute of Technology

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  • ２７ＭＨｚ水晶発振子マイクロバランス上でのグルカンの加水分解・加リン酸分解反応を重さで測る
  • 27MHz スイショウ ハッシンシ マイクロバランス ジョウ デ ノ グルカン ノ カスイ ブンカイ カリンサン ブンカイ ハンノウ オ オモサ デ ハカル ガン エイブン

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Catalytic cleavage reactions by glucoamylase or phosphorylase b were monitored directly on an amylopectin-immobilized 27MHz quartz-crystal microbalance (QCM). As a first example, we could follow kinetically the enzyme binding to the substrate binding (kon) and dissociation rate constants (koff) and intramolecular hydrolysis rate constant (kcat) of glucan hydrolysis by glucoamylase by detecting directly the formation and decomposition of the enzyme-substrate (ES) complex as mass changes. Secondly, glucan phosphorolysis by phosphorylase b was directly observed by two methods through reactions on a QCM. All kinetic parameters for the enzyme binding to the substrate kon and koff, and dissociation constant, Kd, the AMP binding to the enzyme as activator (KAMP), and the catalytic rate constant (kcat) were obtained from curve fittings of time-courses of frequency (mass) changes. The obtained kinetic parameters were compared with those from Michaelis-Menten kinetics in the bulk solution.

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