Enzymic synthesis of levan and fructo-oligosaccharides by Bacillus circulans and improvement of levansucrase stability by carbohydrate coupling

Abstract

Bacillus circulans was able to produce extracellular levansucrase using sucrose as carbon source optimally at 35°C. The enzymic synthesis of levan and fructo-oligosaccharides was studied using a 50% ethanol fraction of crude extract. The molecular weight of the synthesized levan was markedly affected by sucrose concentration, the molecular weight of levan decreased with increased sucrose concentration up to 32% whereby fructooligosaccharides were isolated. Temperature and the reaction time clearly affected the conversion of fructose to levan with molecular weight values ranging from 10 to 38 kDa. Identification of levan indicated that fructose was the building unit of the levan obtained. Thermal and pH stabilities of B. circulans levansucrase could be improved by enzyme glycosylation using sodium metaperiodate treatment. Chemical modification provides additional points of attachment of the enzyme to the support which offered the modified enzyme greater stabilization than did the free enzyme. The modified enzyme exhibited thermal tolerance up to 50°C, where it retained 88.25% of its activity, while the free enzyme only retained 64.55% of its original activity. The half-life significantly increased from 130 min for the free enzyme to 347 min for the modified enzyme at 50°C, however, it increased from 103 min for the free enzyme to 210 min for the modified enzyme at 60°C. Other properties i.e., the response to some metal ions as well as the ability to convert higher substrate levels and tolerance to an extension of the reaction periods were also improved upon modification. Obviously, the results obtained outlined the conditions leading to the formation of important high or low molecular weight or levan and fructo-oligosaccharides suitable for different industrial applications.

Immobilization and stabilization of levansucrase biocatalyst of high interest for the production of fructooligosaccharides and levan

“…The thermal stability of LS from Bacillus circulans was improved through cross‐linking the enzyme with oxidized dextrans. The modified LS had a stability factor of 2.65 at 50 °C and 2.22 at 55 °C compared with the free enzyme . When LS from Z. mobilis was immobilized onto titanium‐activated magnetite, it maintained almost complete activity after heating at 45 °C for 15 min, while the free enzyme only retained 41% of its initial activity …”

Exopolymeric substances (EPS) from Bacillus subtilis : polymers and genes encoding their synthesis

“…Levan type I consists of β‐2,6‐linked d ‐fructose units, whereas type II is a fructose polymer with a glucose residue linked to the terminal fructose by α‐glycoside bond. Levan can be synthesized outside the cell following the extrusion of the extracellular enzyme levansucrase (Abdel‐Fattah et al , 2005; El‐Refai et al , 2009). Further details on levansucrase extrusion and induction are included in the section describing active EPS.…”

Properties of Geobacillus stearothermophilus levansucrase as potential biocatalyst for the synthesis of levan and fructooligosaccharides

“…The E a being close for both hydrolytic and transfructosylation activities of LS suggests that their limiting catalytic step is similar. In fact, the activation energy of the total activity LS from Bacillus circulans and B. subtilis has been reported to vary from 6.62 to 53.9 kJ mol −1 . Contrary to pH, the temperature variation had no significant effect on the selectivity of G. stearothermophilus LS‐catalyzed reaction.…”