Novel enzyme-membrane reactor for polysaccharide synthesis
Introduction
Some widely known and easily available polysaccharides can be used for a lot of very different applications; poly-β-(2,1)-fructan (inulin) from chicory, dahlia, or Jerusalem artichoke is one example 1, 2. However, even more interesting are special well-defined polysaccharides which can be isolated from natural sources only in very small amounts and/or with help of tedious procedures. As an alternative, there is increasing interest in developing cell-free processes with isolated biocatalysts and using, e.g., sucrose as one of the naturally growing raw materials.
The batch enzymatic synthesis of a polysaccharide from sucrose is disadvantageous due to a large loss of enzyme activity by product inhibition. In addition, the separation of enzyme and product having a similarly high molecular weight is rather difficult. Therefore, the enzyme should be immobilized. Using a porous membrane as carrier, the product molecules could be removed quickly if the enzymes are bound within the membrane pores. In such a pore “microreactor”, convective transport of both substrate and product can be expected by using pressure as driving force. A flat-sheet membrane should serve as an assembly of such microreactors (enzyme-membrane reactor, EMR). Our particular objective was to synthesize enzymatically and continuously the polymer inulin from sucrose with immobilized inulinsucrase in an EMR [3].
The enzyme for the in vitro inulin synthesis is a fusion protein of the fructosyltransferase (FTF) of Streptococcus mutans (the only known bacterial source of inulin) and the maltose binding protein of Escherichia coli [4]. The FTF catalyzed reaction involves cleavage of sucrose in glucose and fructose and sequentially coupling fructose via (β-2,1) links yielding inulin (see Scheme. 1). Compared with the relatively low molecular weight inulin from plants (MW<35000 g/mol with a rather broad distribution [5]), the inulin synthesized by bacteria has a very high MW of more than 106 g/mol [6].
In preliminary studies, two flat-sheet membrane types had been used for covalent enzyme immobilization: (i) selfmade asymmetric ultrafiltration membranes prepared by phase inversion from special polymers with reactive epoxide groups for direct covalent coupling of enzymes [7], (ii) commercial symmetric microfiltration membranes (MFM) from non-reactive polymers after a heterogenous photo-grafting functionalization of the pore surface with carboxylic or amino groups and sequential activation/coupling 7, 8, 9, 10. Enzyme binding capacities and activities, membrane permeabilities and EMR performance were evaluated. Membranes most suited for inulin synthesis seemed to be aminofunctionalized capillary pore MFM because they had the lowest blocking tendency [7]. This application is different from all the other previously reported porous enzyme-membranes where the product had either a lower or a similar MW compared to the educt (see, e.g. [11]). A technically interesting example was the synthesis of cyclodextrin from starch with a cyclodextrin glucanotransferase membrane [12].
In this paper, we describe the results for the FTF–EMR (see Scheme. 2). The influence of MFM pore size and flux through the enzyme-membrane onto FTF activity, yield as well as MW for the product inulin, and the membrane blocking /FTF deactivation tendency were analyzed. Conclusions can be drawn with respect to the applicability of the EMR concept for polysaccharide synthesis.
Section snippets
Materials
Polyethylene terephthalate (PET) nuclear track membranes with straight cylindrical capillary pores (RoTrac®), all 23 μm thick, were obtained from Oxyphen AG (Dresden, Germany). These membranes are almost isoporous with pore diameters of 0.4, 1.0 and 3.0 μm, respectively (see Table 1).
2-Aminoethyl methacrylate hydrochloride (AEMA; Polysciences), benzophenone (BP; >99%; Merck), glutardialdehyde (GDA; 50% in water; Merck), the ionic surfactant sodium dodecyl sulfate (SDS; Merck), the non-ionic
Results and discussion
Irrespective the different DG values relative to the outer surface area for the MFM with different pore sizes, the functionalization yields about the same surface coverage on every membrane (DGSA; see Table 1). With the reasonable assumption of an insignificant modification gradient through the 23 μm thick membranes (cf. 9, 10), the entire PET surface is evenly covered with a grafted layer of less than 10 nm thickness in the non-swollen state. The only minor changes of membrane permeability due
Conclusions
EMR based on surface functionalized capillary pore membranes are in principle suited for the synthesis of polysaccharides. A very interesting product, inulin with extraordinarily high MW and low polydispersity, can be obtained in vitro after covalent immobilization of FTF in membrane pores. The problems with the studied system are to a large extent caused by the biochemistry of the enzyme, namely the hypothesized FTF/inulin aggregation/blocking scenario. Obviously, the ratio of convective and
Nomenclature
ATP adenosine 5′-triphosphate DG degree of grafting EMR enzyme-membrane reactor FTF fructosyltransferase G6PD glucose-6-phosphate dehydrogenase HK hexokinase MFM microfiltration membrane NADP nicotinamide adenine dinucleotide phosphate PAEMA poly-(2-amino ethyl methacrylate) PET poly-(ethylene terephthalate) PGI phosphoglucose isomerase UFM ultrafiltration membrane
Acknowledgements
Many thanks are due to Dr. A. Oechel (GKSS) for the protein analyses, and to A. Pfeiffer (GKSS), B. Schroeer (MPI) and G. Reimer (FhG IAP) for technical assistance. The financial support of the BMBF (BEO 0311134; Germany) is gratefully acknowledged.
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