Levan Formation Research Articles

Fermentpolymerisation VIII

The temperature dependance of the reaction of levansucrase with sucrose was studied, and the variation of the equilibrium constants of the Michaelis and acceptor reaction, and the maximum velocities for all three parallel reactions (levan formation, inversion and oligosaccharide formation) have been determined. The activation energies of these three reactions were evaluated. The following values were found: oligosaccharide formation 22, inversion 8, levan formation below 24°C 7 and above this temperature 0.7 kcal/Mol. The abnormal behaviour of the levan formation could be interpreted by assuming that different subreactions proceed simultanously, and the one which ends last determines the temperature dependance. More detailed ideas of the nature of these subreactions are presented. This new reaction mechanism must be distinguished from the consecutive reaction mechanism in which different subreactions follow each other. Together with the reaction mechanism as evaluated in an earlier paper, a hypothetical model of the course of the total reaction at the active site of the enzyme has been proposed.

Fermentpolymerisation VII.

The action of levan sucrase of aerobacter levanicum on sucrose was investigated at different substrate concentrations. Furthermore the influence of certain acceptors (glucose and glycerol) on this reactions was studied with respect to variations of the rates of the three reactions (levan formation, inversion and oligosaccharide formation). The kinetic data give evidence that all subreactions proceed over the same enzyme substrate complex. It is assumed that the polymerisation reaction, building up the levan chains, follows an insertion mechanism. A reaction mechanism is proposed for the simple reaction. Equations for the rates of the three reactions are developed. In order to be able to perform these calculations it was assumed that the reaction of the complexes are rate determining in the overall reaction, and the Michaelis and acceptor equilibria are undisturbed during the reaction. The constants in the equations of these reaction rates (Michaelis constant, acceptor constant and the maximum rates of the three reactions) have been determined from experimental data. It is shown that the equations are in good agreement with the experimental data. If acceptors are added to the reaction, the reaction mechanism has to be extended. The equations for the reaction rates and the additional acceptor constants were determined in the same manner as above. Again, good agreement with the experimental data was obtained. Adding glycerol to the reaction a new product was detected and identified as dioxypropylfructoside. The rate equation of its formation was evaluated from the reaction mechanism.

Fermentpolymerisation VI

The action of levan sucrase from aerobacter levanicum on sucrose can be divided into three reactions: levan formation, inversion and oligosaccharide formation. As in dextran synthesis, levan has extremely high molecular weights which are formed even at very small conversions. The oligosaccharides consist mainly of 1β-fructosyl sucrose. From kinetic data of reactions with enzyme preparations which have been prepared on sucrose containing cultivation media, we could conclude that a levanase was present in our enzyme preparations. By application of fructose instead of sucrose in our cultivation media we could get enzyme preparations completely free of levanase and levan. Temperature- and pH-stability measurements and kinetic data lead to the conclusion that all three reactions are catalysed by the same enzyme. This was confirmed by investigations in the ultracentrifuge; the molecular weight of the enzyme as measured by sedimentation analysis was found to be 22 000.

FORMATION OF SEROLOGICALLY REACTIVE DEXTRANS BY STREPTOCOCCI FROM SUBACUTE BACTERIAL ENDOCARDITIS

Serologically reactive dextrans were synthesized from sucrose by 22 of 48 strains of streptococci isolated from the blood of patients with subacute bacterial endocarditis. One strain formed an appreciable amount of levan as well as dextran. Nine strains of group H streptococci from throats formed dextran serologically similar to that formed by the described endocarditis streptococci. Six strains of S. salivarius from throats and feces, which were included for comparison, formed levan which agreed in serological properties with the purified levan previously described for one strain of that species. The experimental method comprised a series of serological tests that were controlled and supplemented by physical and chemical tests, and furnished a relatively simple but entirely adequate way of recognizing and distinguishing dextrans and levans in bacterial cultures without isolation of the purified polysaccharides. The possible use of dextran and levan formation in the differential description of non-hemolytic streptococci is suggested.

Enzymatic Synthesis of Levan

STUDY of the formation of micro-organisms of polysaccharides from sucrose has been hampered by the absence of a satisfactory technique for the cell-free isolation of the enzyme catalysts concerned1. This obstacle has now been removed. The separation of a dextran-synthesizing enzyme from Leuconostoc mesenteroides has been reported recently by Hehre2. It is the purpose of the present note to report the isolation from Bacillus subtilis and Aerobacter of an enzyme or enzymes which act on sucrose with formation of levan.

STUDIES ON REACTIONS RELATING TO CARBOHYDRATES AND POLYSACCHARIDES: XLI. STRUCTURE OF RAFFINOSE LEVAN

Raffinose levan has been synthesized by the action of B. subtilis on raffinose. The product is identical in structure with sucrose levan previously synthesized by the action of B. subtilis and B. mesentericus respectively on sucrose, and thus represents a polymerized 2:6-anhydro-fructofuranose. The work serves to confirm the theory put forward previously to the effect that levan formation takes place only with sugars containing a terminal fructofuranose grouping. B. subtilis and B. mesentericus thus exert quite a specific action with respect to the synthesis of levan.