LYSOZYME-CATALYZED REACTION IN CONTINUOUS-FLOW SYSTEM

The lysozyme-catalyzed reaction of chitooligosaccharide was carried out in a continuous flow system in which the solution of substrate, chitooligosaccharide [(GlcNAc)n], flowed into the lysozyme solution in an ultrafiltration apparatus and the products were filtered off. The filtrate was continuously collected in test tubes with the aid of a fraction collector. The product distribution in each fraction was analyzed by high performance gel filtration. Using (GlcNAc)5 as the substrate, the concentrations of products, (GlcNAc)1-4, increased gradually and came to the steady state when the volume of the outflow amounted to sixfold of the inside volume. Before reaching the steady state, the product distribution was quite different from that observed in the closed reaction system, in which the reaction species are not exchangeable through the boundary of the system. The outflows of (GlcNAc)3-5 were delayed in comparison with those of GlcNAc and (GlcNAc)2. The delay period increased with the decrease in substrate concentration, and was shortened by using the [Asp 101 or Trp 62]-modified lysozyme instead of the native lysozyme. These results suggest that the delay in the (GlcNAc)3-5 outflows is caused by the nonproductive binding of the oligosaccharide to the lysozyme molecule. The profile of the flow reaction yields information not only on the catalytic efficiency but also on the substrate binding efficiency of the lysozyme.