Production of glucosyl glycerol by immobilized sucrose phosphorylase: Options for enzyme fixation on a solid support and application in microscale flow format.
2-O-(α-d-Glucopyranosyl)-sn-glycerol (αGG) is a natural osmolyte. αGG is produced industrially for application as an active cosmetic ingredient. The biocatalytic process involves a selective transglucosylation from sucrose to glycerol catalyzed by sucrose phosphorylase (SPase). Here we examined immobilization of SPase (from Leuconostoc mesenteroides) on solid support with the aim of enabling continuous production of αGG. By fusing SPase to the polycationic binding module Zbasic2we demonstrated single-step noncovalent immobilization of the enzyme chimera to different porous supports offering an anionic surface. We showed that immobilization facilitated by Zbasic2was similarly efficient as immobilization by multipoint covalent attachment on epoxy-activated supports in terms of production of αGG. Enzyme loadings of up to 90mg enzyme g-1support were obtained and the immobilized SPase was about half as effective as the enzyme in solution. The high regio- and chemo-selectivity of soluble SPase in αGG synthesis was retained in the immobilized enzyme and product yields of >85% were obtained at titers of ∼800mM. The Zbasic2-SPase immobilizates were fully recyclable: besides reuse of the enzyme activity, easy recovery of the solid support for fresh immobilizations was facilitated by the reversible nature of the enzyme attachment. Application of immobilized Zbasic2-SPase for continuous production of αGG in a microstructured flow reactor was demonstrated. Space-time yields of 500mmol αGG L-1h-1were obtained at product titers of ∼200mM. The continuous microreactor was operated for 16days and an operational half-life of about 10days was determined.
Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, NAWI Graz, Petersgasse 12, 8010 Graz, Austria.
Juan M
JM
Bolivar
Austrian Center of Industrial Biotechnology (acib), Petersgasse 14, 8010 Graz, Austria.
Christiane
C
Luley-Goedl
Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, NAWI Graz, Petersgasse 12, 8010 Graz, Austria.
Ernestine
E
Leitner
Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, NAWI Graz, Petersgasse 12, 8010 Graz, Austria.
Thornthan
T
Sawangwan
Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, NAWI Graz, Petersgasse 12, 8010 Graz, Austria; Austrian Center of Industrial Biotechnology (acib), Petersgasse 14, 8010 Graz, Austria. Electronic address: bernd.nidetzky@tugraz.at.
Bernd
B
Nidetzky
D019149:Bioreactors; D001709:Biotechnology; D057075:Enzyme Assays; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism*; D004852:Epoxy Compounds / Q000737:chemistry; D005898:Glass / Q000737:chemistry; D005960:Glucosides / Q000378:metabolism*; D005964:Glucosyltransferases / Q000737:chemistry* / Q000378:metabolism*; D005990:Glycerol / Q000378:metabolism; D006207:Half-Life; D007103:Immobilization / Q000379:methods*; D011485:Protein Binding; D012822:Silicon Dioxide / Q000737:chemistry; D013395:Sucrose / Q000378:metabolism; D013997:Time Factors
D004800:Enzymes, Immobilized; D004852:Epoxy Compounds; D005960:Glucosides; C034727:glucosylglycerol; D013395:Sucrose; D012822:Silicon Dioxide; D005964:Glucosyltransferases; C025901:sucrose phosphorylase; D005990:Glycerol
2-O-(α-d-Glucopyranosyl)-sn-glycerol; Carbohydrate biotechnology; Enzyme microreactors; Fusion protein; Oriented immobilization; Phosphorylase; Transglycosylation; Z(basic2) binding module