A microtiter plate assay for the determination of the synthetic activity of protease.

Proteases have several industrial applications due to their hydrolytic activity [1] and have attracted considerable interest due to their ability to synthesize peptides in organic media [2,3]. Protease-catalyzed peptide synthesis occurs either by a thermodynamically controlled process (condensation of the acid and amine groups) or by a kinetically controlled process (aminolysis of an ester). In the kinetically controlled process the rapidly formed covalent acyl enzyme can transfer the acyl moiety either to the nucleophilic amino component, resulting in the formation of the desired peptide, or to water, resulting in hydrolysis [3]. The hydrolysis step can be suppressed by performing the reaction at subzero temperature [4-6]. Serine proteases have been employed in the synthesis of many biological active oligopeptides [2], including kyotorphin, which is an analgesic dipeptide (Tyr-Arg). This dipeptide has been synthesized using L-Tyr-OEt or Bz-L-Tyr-OEt and H-Arg-OH or L-Arg-OEt as the nucleophile in organic solvent [6,7] and analyzed using high-performance liquid chromatography (HPLC). The presence of water-miscible organic solvents in enzyme-catalyzed reaction increases the solubility of hydrophobic substrates and shifts the thermodynamic equilibrium of hydrolytic reaction to synthetic reaction [8,9]. In addition water-miscible organic solvents such as dimethylformamide (DMF) when added to a subtilisin BPN-catalyzed reaction have shown preference toward aminolysis as His on the active site flips in and a strong hydrogen bond observed between His and Asp in water is disrupted [10]. However, increasing solvent concentration has led to enzyme inactivation [11,12]. Hence, techniques such as site-directed mutagenesis and directed evolution have been employed to improve the stability of enzymes in organic solvents. Directed evolution has been used as an elegant approach to generate and identify new enzyme variants [13]. A range of methods for generation of enzyme libraries is available [14], but the major problem is the lack of a rapid and reliable assay system for the screening of improved enzyme variants out of a pool of 10-10 mutants. To overcome such difficulty, hydrolase variants were identified using chromogenic esters to identify an active esterase [15], to increase the stereoselectivity of a lipase [16], and to identify active esterases using pH indicators [17]. Due to the high number of clones generated during directed evolution techniques, the use of HPLC or gas chromatography for screening is often a cumbersome and time-consuming method. It can, however, be used as the tool to finally confirm the results. In the present study, a microtiter plate (MTP) assay based on p-nitroanilide (pNA) release has been developed for the first time for rapid analysis of synthetic activity of protease.