Profiles of xylose reductase, xylitol dehydrogenase and xylitol production under different oxygen transfer volumetric coefficient values

BACKGROUND: Xylitol is a sugar alcohol (polyalcohol) with many interesting properties for pharmaceutical and food products. It is currently produced by a chemical process, which has some disadvantages such as high energy requirement. Therefore microbiological production of xylitol has been studied as an alternative, but its viability is dependent on optimisation of the fermentation variables. Among these, aeration is fundamental, because xylitol is produced only under adequate oxygen availability. In most experiments with xylitol-producing yeasts, low oxygen transfer volumetric coefficient (K(L)a) values are used to maintain microaerobic conditions. However, in the present study the use of relatively high K(L)a values resulted in high xylitol production. The effect of aeration was also evaluated via the profiles of xylose reductase (XR) and xylitol clehydrogenase (XD) activities during the experiments. RESULTS: The highest XR specific activity (1.45 +/- 0.21 U mg(protein)(-1)) was achieved during the experiment with the lowest K(L)a value (12 h(-1)), while the highest XD specific activity (0.19 +/- 0.03 U mg(protein)(-1)) was observed with a K(L)a value of 25 h(-1). Xylitol production was enhanced when K(L)a was increased from 12 to 50 h(-1), which resulted in the best condition observed, corresponding to a xylitol volumetric productivity of 1.50 +/- 0.08 g(xylitol) L(-1) h(-1) and an efficiency of 71 +/- 6.0%. CONCLUSION: The results showed that the enzyme activities during xylitol bioproduction depend greatly on the initial KLa value (oxygen availability). This finding supplies important information for further studies in molecular biology and genetic engineering aimed at improving xylitol bioproduction. (C) 2008 Society of Chemical Industry

CTAB, Triton X-100 and freezing-thawing treatments of Candida guilliermondii: Effects on permeability and accessibility of the glucose-6-phosphate dehydrogenase, xylose reductase and xylitol dehydrogenase enzymes

Cells of Candida guilliermondii (ATCC 201935) were permeabilised with surfactant treatment (CTAB or Triton X-100) or a freezing-thawing procedure. Treatments were monitored by in situ activities of the key enzymes involved in xylose metabolism, that is, glucose-6-phosphate dehydrogenase (G6PD), xylose reductase (XR) and xylitol dehydrogenase (XD). The permeabilising ability of the surfactants was dependent on its concentration and incubation time. The optimum operation conditions for the permeabilisation of C. guilliermondii with surfactants were 0.41 mM (CTAB) or 2.78 mM (Triton X-100), 30 degrees C, and pH 7 at 200 rpm for 50 min. The maximum permeabilisation measured in terms of the in situ G6PD activity observed was, in order, as follows: CTAB (122.4 +/- 15.7 U/g(cells)) > freezing-thawing, , (54.3 +/- 1.9 U/g(cells)) > Triton X-100 (23.5 +/- 0.0 U/g(cells)). These results suggest that CTAB surfactant is more effective in the permeabilisation of C. guilliermondii cells in comparison to the freezing-thawing and Triton X-100 treatments. Nevertheless, freezing-thawing was the only treatment that allowed measurable in situ XR activity. Therefore, freezing-thawing permeabilised yeast cells could be used as a source of xylose reductase for analytical purposes or for use in biotransformation process such as xylitol preparation from xylose. The level of in situ xylose reductase was found to be 13.2 +/- 0.1 U/g(cells).

Evaluation of hexose and pentose in pre-cultivation of Candida guilliermondii on the key enzymes for xylitol production in sugarcane hemicellulosic hydrolysate

The evaluation of hexose and pentose in pre-cultivation of Candida guilliermondii FTI 20037 yeast on xylose reductase (XR) and xylitol dehydrogenase (XDH) enzymes activities was performed during fermentation in sugarcane bagasse hemicellulosic hydrolysate. The xylitol production was evaluated by using cells previously growth in 30.0 gl(-1) xylose, 30.0 gl(-1) glucose and in both sugars mixture (30.0 gl(-1) xylose and 2.0 gl(-1) glucose). The vacuum evaporated hydrolysate (80 gl(-1)) was detoxificated by ion exchange resin (A-860S; A500PS and C-150-Purolite(A (R))). The total phenolic compounds and acetic acid were 93.0 and 64.9%, respectively, removed by the resin hydrolysate treatment. All experiments were carried out in Erlenmeyer flasks at 200 rpm, 30A degrees C. The maximum XR (0.618 Umg (Prot) (-1) ) and XDH (0.783 Umg (Prot) (-1) ) enzymes activities was obtained using inoculum previously growth in both sugars mixture. The highest cell concentration (10.6 gl(-1)) was obtained with inoculum pre-cultivated in the glucose. However, the xylitol yield and xylitol volumetric productivity were favored using the xylose as carbon source. In this case, it was observed maximum xylose (81%) and acetic acid (100%) consumption. It is very important to point out that maximum enzymatic activities were obtained when the mixture of sugars was used as carbon source of inoculum, while the highest fermentative parameters were obtained when xylose was used.

Fermentation kinetics for xylitol production by a Pichia stipitis D-Xylulokinase mutant previously grown in spent sulfite liquor

Spent sulfite pulping liquor (SSL) contains lignin, which is present as lignosulfonate, and hemicelluloses that are present as hydrolyzed carbohydrates. To reduce the biological oxygen demand of SSL associated with dissolved sugars, we studied the capacity of Pichia stipitis FPL-YS30 (xyl3 Delta) to convert these sugars into useful products. FPL-YS30 produces a negligible amount of ethanol while converting xylose into xylitol. This work describes the xylose fermentation kinetics of yeast strain P.stipitis FPL-YS30. Yeast was grown in rich medium supplemented with different carbon sources: glucose, xylose, or ammonia-base SSL. The SSL and glucose-acclimatized cells showed similar maximum specific growth rates (0.146 h(-1)). The highest xylose consumption at the beginning of the fermentation process occurred using cells precultivated in xylose, which showed relatively high specific activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49). However, the maximum specific rates of xylose consumption (0.19 g(xylose)/g(cel) h) and xylitol production (0.059 g(xylitol)/g(cel) h) were obtained with cells acclimatized in glucose, in which the ratio between xylose reductase (EC 1.1.1.21) and xylitol dehydrogenase (EC 1.1.1.9) was kept at higher level (0.82). In this case, xylitol production (31.6 g/l) was 19 and 8% higher than in SSL and xylose-acclimatized cells, respectively. Maximum glycerol (6.26 g/l) and arabitol (0.206 g/l) production were obtained using SSL and xylose-acclimatized cells, respectively. The medium composition used for the yeast precultivation directly reflected their xylose fermentation performance. The SSL could be used as a carbon source for cell production. However, the inoculum condition to obtain a high cell concentration in SSL needs to be optimized.

The behavior of key enzymes of xylose metabolism on the xylitol production by Candida guilliermondii grown in hemicellulosic hydrolysate

A variety of raw materials have been used in fermentation process. This study shows the use of rice straw hemicellulosic hydrolysate, as the only source of nutrient, to produce high added-value products. In the present work, the activity of the enzymes xylose reductase (XR); xylitol dehydrogenase (XD); and glucose-6-phosphate dehydrogenase (G6PD) during cultivation of Candida guilliermondii on rice straw hemicellulosic hydrolysate was measured and correlated with xylitol production under different pH values (around 4.5 and 7.5) and initial xylose concentration (around 30 and 70 g l(-1)). Independent of the pH value and xylose concentration evaluated, the title of XD remained constant. On the other hand, the volumetric activity of G6PD increased whereas the level of XR decreased when the initial xylose concentration was increased from 30 to 70 g l(-1). The highest values of xylitol productivity (Q (P) a parts per thousand 0.40 g l(-1)) and yield factor (Y (P/S) a parts per thousand 0.60 g g(-1)) were reached at highest G6PD/XR ratio and lowest XR/XD ratio. These results suggest that NADPH concentrations influence the formation of xylitol more than the activity ratios of the enzymes XR and XD. Thus, an optimal rate between G6PD and XR must be reached in order to optimize the xylitol production.

A study of cell disruption of Candida mogii by glass bead mill for the recovery of xylose reductase

The release of xylose reductase (XR) from Candida mogii by cell disruption in a glass beads mill was studied using an experimental design. Statistical analysis of the results indicated that XR volumetric activity increases by using lower glass beads diameter and cell concentration, and by increasing the number of agitation pulses. Based on results attained in experimental design, assays were carried out aiming at the maximization of XR release. Under optimized conditions (300 mu m glass beads, 45 g/l of cell concentration and 50 pulses), the XR volumetric activity reach 0.683 U/ml. Disruption with glass beads showed to be the most efficient method for XR release when compared to sonication process. The highest specific activity (0.175 U/mg of protein) was found in extracts obtained by suspension freezing and thawing, which suggests that this method can be used as a selective process of cell disruption for XR release. (c) 2008 Elsevier B.V. All rights reserved.

A novel use for sugarcane bagasse hemicellulosic fraction: Xylitol enzymatic production

The excess of sugarcane bagasse (SCB) from the sugar-alcohol industry is considered a by-product with great potential for many bioproducts production. This work had as objective to verify the performance of sugarcane bagasse hemicellulosic hydrolysate (SCBHH) as source of sugars for enzymatic or in vitro xylitol production. For this purpose, xylitol enzymatic production was evaluated using different concentrations of treated SCBHH in the commercial reaction media. The weak acid hydrolysis of SCB provided a hydrolysate with 18 g L(-1) and 6 g L(-1) of xylose and glucose, respectively. Considering the reactions, changes at xylose xylitol conversion efficiency and volumetric productivity in xylitol were not observed for the control experiment and using 20 and 40% v.v (1) of SCBHH in the reaction media. The conversion efficiency achieved 100% in all the experiments tested. The results showed that treated SCBHH is suitable as xylose and glucose source for the enzymatic xylitol production and that this process has potential as an alternative for traditional xylitol production ways. (C) 2011 Published by Elsevier Ltd.

Xylitol production from DEO hydrolysate of corn stover by Pichia stipitis YS-30

Corn stover that had been treated with vapor-phase diethyl oxalate released a mixture of mono- and oligosaccharides consisting mainly of xylose and glucose. Following overliming and neutralization, a d-xylulokinase mutant of Pichia stipitis, FPL-YS30 (xyl3-a dagger 1), converted the stover hydrolysate into xylitol. This research examined the effects of phosphoric or gluconic acids used for neutralization and urea or ammonium sulfate used as nitrogen sources. Phosphoric acid improved color and removal of phenolic compounds. d-Gluconic acid enhanced cell growth. Ammonium sulfate increased cell yield and maximum specific cell growth rate independently of the acid used for neutralization. The highest xylitol yield (0.61 g(xylitol)/g(xylose)) and volumetric productivity (0.18 g(xylitol)/g(xylose) l) were obtained in hydrolysate neutralized with phosphoric acid. However, when urea was the nitrogen source the cell yield was less than half of that obtained with ammonium sulfate.

Contribution of Tris Buffer on Xylitol Enzymatic Production

Xylitol enzymatic production can be an alternative to chemical and microbial processes, because of advantages like higher conversion efficiency. However, for an adequate conversion, it is necessary to investigate the effect of many parameters, such as buffer initial concentration, pH, temperature, agitation, etc. In this context, the objective of this work was to evaluate xylitol enzymatic production under different Tris buffer initial concentrations in order to determine the best condition for this parameter to begin the reaction. The best results were obtained when Tris buffer initial concentration was 0.22 M, reaching 0.31 g L(-1) h(-1) xylitol volumetric productivity with 99% xylose-xylitol conversion efficiency. Although the increase in buffer concentration allowed better pH maintenance, it hindered the catalysis. The results demonstrate that this bioreaction is greatly influenced by involved ions concentrations.

Use of chewing gum containing 15% of xylitol and reduction in mutans streptococci salivary levels

Frequent use of Xylitol may decrease the S. mutans levels. However, very little is known about whether this effect on the levels of cariogenic bacteria is maintained after the interruption of short-term usage of xylitol. This study aimed at evaluating changes in mutans streptococci (MS) salivary levels after using a chewing gum containing xylitol. Twelve volunteers harboring > 10(5) CFU MS/ml saliva levels were asked to chew Happydent-xylit® for 5 minutes, 5 X/day, for 30 days. Saliva samples were collected at baseline, at 30 days after xylitol usage began, and at 30 days beyond its interruption. MS salivary levels were estimated. The average salivary levels of MS in the ten subjects who completed the study were 13.17 (NL-CFU) at baseline (A). After the 30 days experimental period (B), this average decreased to 9.45 (NL-CFU). Nine of ten subjects studied showed a reduction in MS salivary levels in relation to baseline, whereas salivary levels were maintained in the remaining subject. At thirty days beyond the interruption of xylitol usage (C), the average levels of MS were still reduced to 10.31 (NL-CFU). Multiple sample comparison using the Bonferroni test revealed that the decrease in MS levels observed from baseline (A) to the time immediately after 30 days of xylitol usage (B) was statistically significant (p < 0.05), and those levels were still decreased between baseline and 30 days beyond the interruption of xylitol usage (C). So, the use of xylitol induced a reduction in MS salivary levels after a short period of usage which persisted beyond its interruption.

Alcohol dehydrogenase activities and ethanol tolerance in Anastrepha (Diptera, Tephritidae) fruit-fly species and their hybrids

The ADH (alcohol dehydrogenase) system is one of the earliest known models of molecular evolution, and is still the most studied in Drosophila. Herein, we studied this model in the genus Anastrepha (Diptera, Tephritidae). Due to the remarkable advantages it presents, it is possible to cross species with different Adh genotypes and with different phenotype traits related to ethanol tolerance. The two species studied here each have a different number of Adh gene copies, whereby crosses generate polymorphisms in gene number and in composition of the genetic background. We measured certain traits related to ethanol metabolism and tolerance. ADH specific enzyme activity presented gene by environment interactions, and the larval protein content showed an additive pattern of inheritance, whilst ADH enzyme activity per larva presented a complex behavior that may be explained by epistatic effects. Regression models suggest that there are heritable factors acting on ethanol tolerance, which may be related to enzymatic activity of the ADHs and to larval mass, although a pronounced environmental effect on ethanol tolerance was also observed. By using these data, we speculated on the mechanisms of ethanol tolerance and its inheritance as well as of associated traits.

A new bianthron glycoside as inhibitor of Trypanosoma cruzi glyceraldehyde 3-phosphate dehydrogenase activity

A phytochemical investigation of the ethanolic extract of stalks of Senna martiana Benth. (Leguminoseae), native specie of northeast Brazil, resulted in the isolation and spectroscopic characterization of a new bianthrone glycoside, martianine 1 (10,10'-il-chrysophanol-10-oxi10,10'-bi-glucosyl). Its identification was established by HRMS, IR and 2D NMR experiments. The evaluation of martianine trypanocidal activity was carried out against gliceraldehyde 3-phosphate dehydrogenase enzyme from Trypanosoma cruzi. Its inhibitory constant (Ki) is in the low micromolar concentration and it was determined by isothermal titration calorimetry to be 27.3 ± 2.47 µmol L-1. The non-competitive mechanism is asserted to be putative of the mode of action martianine displays against T. cruzi GAPDH. Results show that martianine has a great potential to become new lead molecule by inhibiting this key enzyme and for the development of new drugs against Chagas disease.

Screening of Trypanosoma cruzi glycosomal glyceraldehyde-3-phosphate dehydrogenase enzyme inhibitors

The inhibitory activity of crude extracts of Meliaceae and Rutaceae plants on glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) enzyme from Trypanosoma cruzi was evaluated at 100 μg/mL. Forty-six extracts were tested and fifteen of them showed significant inhibitory activity (IA % > 50). The majority of the assayed extracts of Meliaceae plants (Cedrela fissilis, Cipadessa fruticosa and Trichilia ramalhoi) showed high ability to inhibit the enzymatic activity. The fractionation of the hexane extract from branches of C. fruticosa led to the isolation of three flavonoids: flavone, 7-methoxyflavone and 3',4',5',5,7-pentamethoxyflavone. The two last compounds showed high ability to inhibit the gGAPDH activity. Therefore, the assayed Meliaceae species could be considered as a promising source of lead compounds against Chagas' disease.

Defects in vesicle core induced by Escherichia coli dihydroorotate dehydrogenase

Dihydroorotate dehydrogenase (DHODH) catalyzes the oxidation of dihydroorotate to orotate during the fourth step of the de novo pyrimidine synthesis pathway. In rapidly proliferating mammalian cells, pyrimidine salvage pathway is insufficient to overcome deficiencies in that pathway for nucleotide synthesis. Moreover, as certain parasites lack salvage enzymes, relying solely on the de novo pathway, DHODH inhibition has turned out as an efficient way to block pyrimidine biosynthesis. Escherichia coli DHODH (EcDHODH) is a class 2 DHODH, found associated to cytosolic membranes through an N-terminal extension. We used electronic spin resonance (ESR) to study the interaction of EcDHODH with vesicles of 1,2-dioleoyl-sn-glycero-phosphatidylcholine/detergent. Changes in vesicle dynamic structure induced by the enzyme were monitored via spin labels located at different positions of phospholipid derivatives. Two-component ESR spectra are obtained for labels 5- and 1 0-phosphatidylcholine in presence of EcDHODH, whereas other probes show a single-component spectrum. The appearance of an additional spectral component with features related to fast-motion regime of the probe is attributed to the formation of a defect-like structure in the membrane hydrophobic region. This is probably the mechanism used by the protein to capture quinones used as electron acceptors during catalysis. The use of specific spectral simulation routines allows us to characterize the ESR spectra in terms of changes in polarity and mobility around the spin-labeled phospholipids. We believe this is the first report of direct evidences concerning the binding of class 2 DHODH to membrane systems.

The development of an immobilized enzyme reactor containing glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi: the effect of species' specific differences on the immobilization

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays an important role in the life cycle of the Trypanosoma cruzi, and an immobilized enzyme reactor (IMER) has been developed for use in the on-line screening for GAPDH inhibitors. An IMER containing human GAPDH has been previously reported; however, these conditions produced a T. cruzi GAPDH-IMER with poor activity and stability. The factors affecting the stability of the human and T. cruzi GAPDHs in the immobilization process and the influence of pH and buffer type on the stability and activity of the IMERs have been investigated. The resulting T. cruzi GAPDH-IMER was coupled to an analytical octyl column, which was used to achieve chromatographic separation of NAD+ from NADH. The production of NADH stimulated by D-glyceraldehyde-3-phosphate was used to investigate the activity and kinetic parameters of the immobilized T. cruzi GAPDH. The Michaelis-Menten constant (K-m) values determined for D-glyceraldehyde-3-phosphate and NAD(+) were K-m = 0.5 +/- 0.05 mM and 0.648 +/- 0.08 mM, respectively, which were consistent with the values obtained using the non-immobilized enzyme.