Xylose immobilization

Since these early discoveries, xylose isomerases have been isolated from many bacterial species, and these enzymes have been intense investigated, especially those of the genera Streptomyces, Lactobacillus, and Bacillus. The characteristics of substrate specificity (xylose glucose > ribose), divalent metal cation activation (Mg, Mn or Co ), and activity at alkaline pH are properties that most of the enzymes share to a certain extent, but significant variations exist. Some of these em es have been immobilized and patented for commercial use. There are many good reviews in the literature that describe the enzymatic characteristics of the xylose isomerases 9,28,29). 

Krishnan, M.S., Blanco, M., Shattuck, G.K., Nghiem, N.P and Davison, B.H., Ethanol production from glucose and xylose by immobilized Zymomonas mobtlis GP4(pZB5), Appl. Biochem. Biotech., 84 (2000) 525-542. 

Scheme 5 Xylose can be dehydrated to produce furfural. The reaction has been reported using several different catalysts including zeolites, sulfonic acid functionalized MCM-41 and immobilized heteropolyacids. The best selectivity towards furfural was achieved using zeolite H-mordenite, although at low conversion of xylose.Overall, the best yield of furfural was obtained using sulfonic acid functionalized MCM-41.

We finish this Section with enzymic conversions that are difficult to classify elsewhere Takasweet, a commercial variety of immobilized glucose-iso-merase, converts 6-O-methyl-D-fructose and 6-deoxy-D-fructose into the gluco isomers in not very satisfactory yield.34 A mixture of catalase (75 U/mmol) and glucose oxidase (80 U/mmol) oxidizes xylitol to L-xylose in 50% yield, on the 100- mol scale.106 The enzyme cyclodextrin a-(l—>4)-glucosyltransferase (1000 U immobilized on silica gel-glutaraldehyde) allows107 preparation of cyclomaltohexaose (0.3 g), cyclomaltoheptaose... 

An immobilized enzyme preparation of pyranose-2-oxidase catalase and 2-ulose dehydratase effects the conversion of D-xylose and 6-deoxy-D-glucose into 5-hydroxy-2,3-dioxopentanal (266) and D-glycero-5-hydroxy-2,3-dioxohexanal (267), respectively 492... 

The key to the successful scale-up was the immobilization of the enzyme, which increased stability and reduced the enzyme costs to an acceptable level. The isomerization step is typically carried out in a parallel series of packed-bed reactors, where the enzyme is immobilized on silica or inert cellulose carriers (Figure 5.22). A ton of immobilized xylose isomerase can catalyze the production of 5000 tons of HFCS [35]. Current research is concentrating on developing a more thermostable enzyme variant which would reach the 5 5 45 ratio directly in the reactor column. 

Batch fermentations with immobilized cells were carried out both in 500-mL Erlenmeyer flasks and in the 2-L Biostat B from BBInternational. All results are the mean of two duplicate experiments. For the shake-flask experiments, 100 mL of the culture medium supplemented with glucose and xylose or aspen hydrolysates was used. pH corrections were done discontinuously every 10 h. 

The fermentative performance of P. stipitis immobilized in Ca-algi-nate beads was first investigated using concentrated solutions of xylose. The higher ethanol yield and productivity obtained from the fermentation of 60 g/L of xylose working with a cell concentration of 2.34 x 109 cells/L were 0.46 ge,hanoi/gmitiaisugars (ge/gs) and 0.40 g/(L-h), respectively. Only traces of secondary metabolites were detected. 

Fig. 2. Sequential fermentation of model solutions of glucose (60 g/L) and xylose (20 g/L) in shake flasks containing same amount of suspended S. cerevisiae (3 g/L) and different gel fractions (percentages in parenthesis) of immobilized P. stipitis. The initial cell concentrations of P. stipitis were ( ), 7.02 x 109 ( ), 1.17 x 1010 (A), 4.10 x 1010 cells/L.

Fig. 3. Sequential fermentation of model solutions of glucose and xylose in shake flasks. Systems A, B, and C contained suspended S. cerevisiae and immobilized P. stipitis with a loading ratio of P. stipitis/S. cerevisiae of 1.4 g/g dry wt. The gel fractions were as follows system A, 0.36 system B, 0.18 and system C, 0.09 g/g. The concentrations of P. stipitis cells were as follows system A, 2.83 x 1012 system B, 1.41 x 1012 and system C 7.31 X 10u cells/L.

Fig. 7. Cofermentation of model solutions of glucose and xylose with P. st ip it is and S. cerevisiae separately immobilized (system G) and coimmobilized (system H) in Ca-alginate beads. The gel fraction in system G was made of 0.20 g/g of beads containing P. stipitis and 0.05 g/g of beads containing S. cerevisiae. The initial concentrations of P. stipitis and S. cerevisiae cells were 5.64 x 1012 and 1.89 x 10u cells/L, respectively. The gel fraction in system H was made of 0.25 g/g of beads containing P. stipitis and S. cerevisiae coimmobilized with a loading ratio of P. stipitis/S. cerevisiae of 4 g/g of dry cells. The total cells concentration was 6.01 x 1012 cells/L.

Microbacteruim arborescens, Streptomyces rubiginosus var., or Streptomyces murinus) Produced as an off-white to tan, brown, or pink, amorphous powder, granules, or a liquid by controlled fermentation using any of the above-named organisms. The products may be soluble in water but practically insoluble in alcohol, in chloroform, and in ether, or if immobilized, may be insoluble in water and partially soluble in alcohol, in chloroform, and in ether. Major active principle glucose (or xylose) isomerase. Typical applications manufacture of high-fructose corn syrup and other fructose starch syrups. 

Fumaric acid can also be produced from xylose. The rate of xylose fermentation is much slower than with glucose with a specific productivity of only about 0.075 g fumaric acid/h/g biomass. Kautola and Linko [73] used immobilized R. arrhizus with polyurethane foam to ferment xylose. A specific productivity of 0.087 g/l/h was obtained when the initial xylose concentration was 100 g/1 and the resident time was 10.25 days. 

The reactor was loaded with 75 ml granular carrier material [14], and finally, the entire reactor system, including tubing and recirculation reservoir, was autoclaved at 120°C for 30 min. Before use, the reactor system was gassed for 15 min with N2/CO2 (4 1) to ensure anaerobic conditions and filled with BA medium with an initial xylose concentration of 10 g/1. The reactor was started up in batch mode by inoculation with 80 ml of cell suspension with an optical density (OD578) of 0.9-1. The batch mode of operation was maintained for 24 h to allow cells to attach and to immobilize on the carrier matrix. After the batch run, the system was switched to continuous mode, applying a hydraulic retention time (HRT the volume of the reactor divided by the influent flowrate) of 8 h and up-flow velocity of 1 m/h. To achieve operational stability, the reactor was run for 7 days under... 

To overcome the disparity in the optimal pH s for the isomerization and fermentation, our group [29, 35, 36] proposed a novel scheme of isomerization that incoiporates urease co-immobilized with xylose isomerase. This technique uses XI immobilized in a porous pellet for isomerization and the immobilized urease enzyme for pH control (Fig. 1). These co-immobilized enzyme pellets are dispersed in a fermentation broth, which contains urea in addition to the other necessary ingredients for fermentation. Theoretically, it is possible to sustain a significant pH gradient between the bulk liquid and the core region of the pellet... 

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. 

---