Xylulose, formation

Although our co-immobilized enzyme approach is able to sustain the necessary pH difference between isomerization and fermentation steps in SIF [35], the overall production rate of ethanol in SIF will still be limited by the total concentration of xylulose available to the yeast [9]. Under normal equilibrium conditions, the xylulose concentration is usually at best one fifth of the xylose concentration. Hence, other avenues of shifting the equilibrium towards higher xylulose formation will further increase the rate of ethanol production. 

Unaltered Pellets As an initial control experiment, the isomerization of xylose to xylulose was studied using Sweetzyme pellets, as received, before co-immobilization with urease. The time course of xylose consumption and xylulose formation was monitored for an initial xylose concentration of 60 g/1 with 0.13 g pellets at 34 °C. The isomerization mixture was buffered at pH 7.5, which is the optimal pH for XI activity. As seen in Fig. 2, curve A, the concentration of xylulose steadily increased and reached an equilibrium value of about 9 g/1, suggesting an equilibrium xylose/xylulose ratio of nearly 6 1 under these conditions. When the same experiment was repeated at a reduced pH of 4.5, no xylulose was detected in the reaction mixture, even after 40 h (data not shown). At a pH of 4.5, XI is 3 pH units below its optimum and displays essentially no activity. 

Xl/Urease Co-immobilized Pellets We next modified the SweetzymeTM pellets by adsorbing urease onto the pellets. The co-immobilized enzyme pellets (0.13 g) were added to reaction media containing 60 gA xylose buffered to pH 4.5. As with the unaltered pellets, no xylulose formation was observed under these conditions even after 48 h (see Fig. 2, curve C). Next, 0.01 M urea was added to the bulk solution buffered to a pH of 4.5. Formation of xylulose was observed in the presence of urea, and the concentration of xylulose in the reaction medium gradually increased to reach a value of about 5 g/1 by 48 h (see Fig. 2, curve B). 

Borate is able to complex, via the above mechanism, more readily with the open-chain structure of xylulose as compared to the cyclic hemiacetal form of xylose [44]. This binding preference leads to a shift in the xylose/xylulose isomerization equilibrium in favor of xylulose formation. 

Fig. 7 Efifect of initial urea concentration and mass of pellets on xylulose production for the co-immobilized enzyme pellets. All pellets were from the same co-immobilization batch and have the same urease and XI activities per g pellet at pH 7.5. The percentage conversion to xylulose is given above the bars for each experiment. The time indicated on the x-axis represents the time of apparent equilibrium for all experiments. With 0.01 M urea (A, B, and C), increasing the pellet mass results in a significant increase in xylulose formation. With 0.1 M urea D and E), the final xylulose production is only minimally increased by increasing the mass of pellets...

This portion of the pathway begins with an isomerization and an epimeriza-tion, and it leads to the formation of either D-ribose-5-phosphate or D-xylulose-5-phosphate. These intermediates can then be converted into glycolytic intermediates or directed to biosynthetic processes. 

Sprenger, G.A. et al.. Identification of a thiamin-dependent synthase in Escherichia coli required for the formation of the 1-deoxy-D-xylulose 5-phosphate precursor to isoprenoids, thiamin, and pyridoxol, Proc. Natl. Acad Sci. USA 94, 12857, 1997. Lange, B.M. et al., A family of transketolases that directs isoprenoid biosynthesis via a mevalonate-independent pathway, Proc. Natl. Acad Sci. USA 95, 2100, 1998. Lois, L.M. et al., Cloning and characterization of a gene from Escherichia coli encoding a transketolase-like enzyme that catalyzes the synthesis of D-1- deoxyxylulose 5-phosphate, a common precursor for isoprenoid, thiamin, and pyridoxol biosynthesis, Proc. Natl. Acad. Sci. USA 95, 2105, 1998. 

TAKAHASHI, S., KUZUYAMA, T WATANABE H., SETO, H., A 1-deoxy-D-xylulose 5-phosphate reductoisomerase catalyzing the formation of 2-C-methyl-D-erythritol 4-phosphate in an alternative nonmevalonate pathway, Proc. Natl. Acad. Sci. USA, 1998, 95, 9879-9884. 

It has been established" that the leaves of Liriodendron tulipifera convert 1-deoxy-D-xylulose (285) into 2-C-methyl-D-erythritol (286) via a skeletal rearrangement (see Scheme 90) reminiscent of the formation of terpene precursors from 1-deoxy-D-xylulose 4-phosphate. An esterase-catalysed regioselective 6-deacylation of... 

Xylitol is as sweet as sucrose and has been used as a food additive. Because it does not induce formation of dental plaque, it is used as a replacement for sucrose in chewing gum. It appeared to be an ideal sugar substitute for diabetics. However, despite the fact that it is already naturally present in the body, ingestion of large amounts of xylitol causes bladder tumors as well as oxalate stones in rats and mice. Its use has, therefore, been largely discontinued. A possible source of the problem may lie in the conversion by fructokinase of some of the xylitol to D-xylulose 1 -P, which can be cleaved by the xylulose 1-P aldolase to dihydroxy acetone P and glycolaldehyde. 

GA Sprenger, U Schorken, T Wiegert, S Grolle, AA de Graaf, SV Taylor, TP Begley, S Bringer-Meyer, H Sahm. Identification of a thiamin-dependent synthase in Escherichia coli required for the formation of the 1-deoxy-D-xylulose 5-phos-phate precursor to isoprenoids, thiamin, and pyridoxol. Proc Natl Acad Sci (USA) 94 12857-12862, 1997. 

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