Laminaribiose (3-β-D-glucosyl-D-glucose) is of interest for the food industry and also for the pharmaceutical industry. It can be extracted from laminarin, occurring in brown algae, however it is an expensive process.
In this project laminaribiose production is studied by establishing an immobilized trienzymatic system as suggested already by Kitaoka (1993). Sucrose (Suc) as substrate is transformed with sucrose phosphorylase (SP), glucose isomerase (GI) and laminaribiose phosphorylase (LP) to produce laminaribiose (Lam) in a trienzymatic process.
As a first step laminaribiose had to be produced since it is not commercially available. The optimum enzyme production could be realized under the heterotrophic cultivation of Euglena gracilis at 33°C and pH 4.2 to reduce the lag phase. Harvesting the cells at the beginning of the stationary phase give the highest enzyme yields. Ultrasonic disintegration was found the best method to obtain the enzyme in high quantity (0.52 g/l culture volume) and quality. Ammonium sulfate precipitation with 30%-60% saturation and dialysis delivered more purified laminaribiosephosphorylase.
All three enzymes were characterized and an integrated kinetical model established. SP produces glucose-1-phosphate, with fructose und glucose as side products. Optimal conditions for SP are found between 40-50 °C at pH 7. Double-substrate kinetics with inhibition describes the mechanism. GI was integrated (Topt.: 60°C, pHopt.: 6.5) with a simple first order equilibrium kinetics (production of glucose from the side product fructose). LP uses glucose-1-phosphate and glucose to produce laminaribiose at temperatures from 40-45 °C at pH 6.5. Double-substrate kinetics was successfully applied.
Immobilization of the three enzymes was achieved by using a chitosan-matrix for entrapment with GI already fixed within glutaraldehyde cross-linked cells. Different experimental set-ups were studied to establish the combined system and will be shown.
Kitaoka et al (1993): Purification and Properties of Laminaribiose Phosphorylase (EC 2.4.1.31) from Euglena gracilis Z. Arch Biochem Biophys 508–514.
