The study of shear inactivation of dextransucrase preparation from Leuconostoc mesenteroides

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Virginia Polytechnic Institute and State University

The extracellular enzyme dextransucrase (α 1,6-glucan: D-fructose 2-glucosyltransferase EC was concentrated and purified from the fermentor culture of Leuconostoc mesenteroides (ATCC 10830) by a three-step procedure: centrifugation, membrane ultrafiltration and dialysis, and gel permeation chromatography. Two enzyme preparations which contained dextransucrase activity of 0.95 U/ml and 1.59 U/ml were used for this study. The contaminating enzyme levansucrase was determined to be less than 2%.

Shear inactivation study was performed on a batch reactor after subjecting the enzyme solution to a shear rate of 1046 second⁻¹ in the couette viscometer for several different periods of shear exposure time. The kinetic data showed dextransucrase lost part of its catalytic power due to shear inactivation.

A correlation curve of the remaining dextransucrase activity versus a dimensionless group-shear strain was generated in the absence of substrate. The data revealed there was not inactivation until shear strain reached 10⁵. Two sets of the correlation curves were generated on the same system but with the presence of substrate at two different levels of enzyme loading. Both results indicated less shear inactivation effect in the presence of substrate and this protective effect on substrate binding strongly endorses Ebert and Schenk's hypothesis that there are substrate induced conformation changes of the enzyme molecule.

In order to find any long range effect on the catalytic specificity of the enzyme molecule associated with shear inactivation, the dextransucrase solution with different shear histories was incubated with sucrose. The synthesized dextrans were then precipitated out by a 83% ethanol-water mixture. When the molecular structure of the dextran precipitates was analyzed on ¹³C NMR spectroscopy at 90°C, it was found that there was no change in the polymer structure. But one of the spectra which corresponded to the dextransucrase preparation with the longest shear history contained resonance peaks of levan. All these findings lead to the conclusion that shear induced conformational changes of the enzyme molecule can not alter their catalytic specificity, and that the sensitivity toward shear inactivation is much greater by dextransucrase than by the small quantity of levansucrase present.