Induction of aldose reductase activity inCandida guilliermondii by pentose sugars

Summary Cell extracts ofCandida guilliermondii grown ind-xylose,l-arabinose,d-galactose,d-glucose,d-mannose and glycerol as sole carbon sources possessed NADPH-dependent aldose reductase activity, but no NADH-dependent activity was detected.d-xylose andl-arabinose were the best inducers of aldose reductase activity. The highest enzyme activity ind-xylose orl-arabinose-grown cells was observed first withl-arabinose followed byd-xylose as substrates of the enzymatic reaction. However, only low activity was found ind-glucose,d-mannose andd-galactose-grown cells, indicating that these carbon sources cause catabolite repression. Enzyme activities induced ind-xylose-grown cells were twice as high as those obtained from the cells under resting conditions. Furthermore, the level of induction of aldose reductase activity depended on the initial concentration ofd-xylose. The present study shows that aldose reductase activity may be efficiently induced by pentose sugars of hemicellulosic hydrolysates and weakly by hemicellulosic hexoses.     

Repression of galactose utilization by glucose in the citrate-producing yeastCandida guilliermondii

Summary A strain of the yeastCandida guilliermondii has been shown to produce citric acid from galactose to a similar extent, and at a similar rate, as from glucose. At an initial concentration of 36 g/l of either glucose or galactose, citric acid production exceeds 13 g/l. When galactose and glucose are present in a mixture, however, galactose utilization is delayed until most of the glucose has been utilized, providing evidence for catabolite repression.     

Kinetic behavior of Candida guilliermondii yeast during xylitol production from Brewer's spent grain hemicellulosic hydrolysate

Brewer's spent grain, the main byproduct of breweries, was hydrolyzed with dilute sulfuric acid to produce a hemicellulosic hydrolysate (containing xylose as the main sugar). The obtained hydrolysate was used as cultivation medium by Candidaguilliermondii yeast in the raw form (containing 20 g/L xylose) and after concentration (85 g/L xylose), and the kinetic behavior of the yeast during xylitol production was evaluated in both media. Assays in semisynthetic media were also performed to compare the yeast performance in media without toxic compounds. According to the results, the kinetic behavior of the yeast cultivated in raw hydrolysate was as effective as in semisynthetic medium containing 20 g/L xylose. However, in concentrated hydrolysate medium, the xylitol production efficiency was 30.6% and 42.6% lower than in raw hydrolysate and semisynthetic medium containing 85 g/L xylose, respectively. In other words, the xylose-to-xylitol bioconversion from hydrolysate medium was strongly affected when the initial xylose concentration was increased; however, similar behavior did not occur from semisynthetic media. The lowest efficiency of xylitol production from concentrated hydrolysate can be attributed to the high concentration of toxic compounds present in this medium, resulting from the hydrolysate concentration process.     

Batch xylitol production from wheat straw hemicellulosic hydrolysate using Candida guilliermondii in a stirred tank reactor

Batch production of xylitol from the hydrolysate of wheat straw hemicellulose using Candida guilliermondii was carried out in a stirred tank reactor (agitation speed of 300 rpm, aeration rate of 0.6 vvm and initial cell concentration of 0.5 g l^–1). After 54 h, xylitol production from 30.5 g xylose l^–1 reached 27.5 g l^–1, resulting in a xylose-to-xylitol bioconversion yield of 0.9 g g^–1 and a productivity of 0.5 g l^–1 h^–1.     

Improvement of xylitol production by Candida guilliermondii FTI 20037 previously adapted to rice straw hemicellulosic hydrolysate

AIMS: To evaluate a simple and economical technique to improve xylitol production using concentrated xylose solutions prepared from rice straw hemicellulosic hydrolysate. METHODS AND RESULTS: Experiments were carried out with rice straw hemicellulosic hydrolysate containing 90 g l-1 xylose, with and without the addition of nutrients, using the yeast Candida guilliermondii previously grown on the hydrolysate (adapted cells) or on semi-defined medium (unadapted cells). By this method, the yield of xylitol increased from 17 g l-1 to 50 g l-1, and xylose consumption increased from 52% to 83%, after 120 h of fermentation. The xylitol production rates were very close to that (0.42 g l-1 h-1) attained in a medium simulating hydrolysate sugars. CONCLUSION: Yeast strain adaptation to the hydrolysate showed to be a suitable method to alleviate the inhibitory effects of the toxic compounds. Adapted cells of Candida guilliermondii can efficiently produce xylitol from hydrolysate with high xylose concentrations. SIGNIFICANCE AND IMPACT OF THE STUDY: Yeast adaptation helps the bioconversion process in hydrolysate made from lignocellulosic materials. This low-cost technique provides an alternative to the detoxification methods used for removal of inhibitory compounds. In addition, the use of adapted inocula makes it possible to schedule a series of batch cultures so that the whole plant can be operated almost continuously with a concomitant reduction in the overall operation time.     

Xylitol formation by Candida guilliermondii grown in a cane bagasse hemicellulosic hydrolysate: Effect of aeration and inoculum adaptation

The xylose conversion into by Candida guilliermondii was evaluated in sugar cane bagasse hemicellulosic hydrolysate. The effect of air flow rates of 0.4, 0.6 and 0.8 vvm cn xylitol formation was studied. In addition, inoculum previously adapted to the hydrolysate was also tested in the fermentation carried out at 0.6 vvm. The results showed that xylitol production depends markedly on the aeration rate and on the previous adaptation of the yeast to the hydrolysate. When the highest productivity of xylitol was 0.39 g/l × h. However, during the fermentation carried out at an air flow rate of 0.6 vvm with adapted inoculum, the productivity increased to 0.65 g/l × h. Furthermore, the adapted cells performed quite well in the presencel of acetic concentrations of about 4.5 g/l in the medium.     

Induction of aldose reductase and xylitol dehydrogenase activities in Candida tenuis CBS 4435

In this study the ability of various sugars and sugar alcohols to induce aldose reductase (xylose reductase) and xylitol dehydrogenase (xylulose reductase) activities in the yeast Candida tenuis was investigated. Both enzyme activities were induced when the organism was grown on d-xylose or l-arabinose as well as on the structurally related sugars d-arabinose or d-lyxose. Mixtures of d-xylose with the more rapidly metabolizable sugar d-glucose resulted in a decrease in the levels of both enzymes formed. These results show that the utilization of d-xylose by C. tenuis is regulated by induction and catabolite repression. Furthermore, the different patterns of induction on distinct sugars suggest that the synthesis of both enzymes is not under coordinate control.     

Catabolite Repression and Pyruvate Metabolism in Escherichia coli

A study was made of the reactions involved in the cellular regulatory function known as catabolite repression. These studies employed the glucose-repressible, beta-galactosidase system of Escherichia coli and involved an investigation of glucose dissimilation under cultural conditions capable of permitting or preventing expression of catabolite repression. The results indicated that reactions associated with pyruvate decarboxylation are of particular importance in influencing repression. This conclusion was based on results obtained by measurement of differential rates of C(14)O(2) evolution from specifically labeled (14)C-glucose substrates, and by measurements of H(2) evolution during anaerobic growth. Catabolite repression measured in relation to steady-state growth rates indicated that the repression mechanism may in fact be a direct consequence of a cell's energy balance, as dictated by the production from pyruvate of "high-energy" molecules such as adenosine triphosphate or acetyl-coenzyme A. The apparent involvement of pyruvate metabolism in both the energetics and the expression of catabolite repression in E. coli is consistent with this view.     

Cytochrome P-450 and alkane hydroxylase activity in Candida guilliermondii.

In the investigated Candida guilliermondii strain after growth on n-alkanes as the only carbon and energy source 5--10 nMol cytochrome P-450 per g cells (wet weight) could be detected. Cytochrome P-450 and alkane hydroxylase activity was found in the 100 000 xg pellet. Cofactor studies and inhibition experiments revealed the existence of a NADPH-dependent cytochrome P-450 alkane hydroxylase system.     

Induction and Catabolite Repression of Glucosidases in Pseudomonas maltophilia

Intracellular α-and β-glucosidases were induced in cell suspensions of Pseu-domonas maltophilia by maltose or cellobiose, and the synthesis of these enzymes was sensitive to apparent catabolite repression by α-ketoglutarate.     

Cloning and expression of Candida guilliermondii xylose reductase gene (xyl1) in Pichia pastoris

A xylose reductase gene (xyl1) of Candida guilliermondii ATCC 20118 was cloned and characterized. The open reading frame of xyl1 contained 954 nucleotides encoding a protein of 317 amino acids with a predicted molecular mass of 36 kDa. The derived amino acid sequence of C. guilliermondii xylose reductase was 70.4% homologous to that of Pichia stipitis. The gene was placed under the control of an alcohol oxidase promoter (AOX1) and integrated into the genome of a methylotrophic yeast, Pichia pastoris. Methanol induced the expression of the 36-kDa xylose reductase in both intracellular and secreted expression systems. The expressed enzyme preferentially utilized NADPH as a cofactor and was functional both in vitro and in vivo. The different cofactor specificity between P. pastoris and C. guilliermondii xylose reductases might be due to the difference in the numbers of histidine residues and their locations between the two proteins. The recombinant was able to ferment xylose, and the maximum xylitol accumulation (7.8 g/l) was observed when the organism was grown under aerobic conditions. Received: 26 August 1997 / Received revision: 6 November 1997 / Accepted: 21 November 1997     

Fermentation performance of Candida guilliermondii for xylitol production on single and mixed substrate media

Semidefined media fermentation simulating the sugar composition of hemicellulosic hydrolysates (around 85 g l^-1 xylose, 17 g l^-1 glucose, and 9 g l^-1 arabinose) was investigated to evaluate the glucose and arabinose influence on xylose-to-xylitol bioconversion by Candida guilliermondii. The results revealed that glucose reduced the xylose consumption rate by 30%. Arabinose did not affect the xylose consumption but its utilization by the yeast was fully repressed by both glucose and xylose sugars. Arabinose was only consumed when it was used as a single carbon source. Xylitol production was best when glucose was not present in the fermentation medium. On the other hand, the arabinose favored the xylitol yield (which attained 0.74 g g^-1 xylose consumed) and it did not interfere with xylitol volumetric productivity (Q _P=0.85 g g^-1), the value of which was similar to that obtained with xylose alone.     

PHB Biosynthesis in Catabolite Repression Mutant of Burkholderia sacchari

Due to the effect of catabolite repression, sugar mixtures cannot be metabolized in a rapid and efficient way implicating in lower productivity in bioprocesses using lignocellulosic hydrolysates. In gram-negative bacteria, this mechanism is mediated by the phosphotransferase system (PTS), which concomitantly internalizes and phosphorylates sugars. In this study, we isolated a UV mutant of Burkholderia sacchari, called LFM828, which transports hexoses and pentoses by a non-PTS uptake system. This mutant presented released glucose catabolite repression over the pentoses. In mixtures of glucose, xylose, and arabinose, specific growth rates and the specific sugar consumption rates were, respectively, 10 and 23% higher in LFM828, resulting in a reduced time to exhaust all sugars in the medium. However, in polyhydroxybutyrate (PHB) biosynthesis experiments it was necessary the supplementation of yeast extract to maintain higher values of growth rate and sugar consumption rate. The deficient growth in mineral medium was partially recovered by replacing the ammonium nitrogen source by glutamate. It was demonstrated that the ammonium metabolism is not defective in LFM828, differently from ammonium, glutamate can also be used as carbon and energy allowing an improvement on the carbohydrates utilization for PHB production in LFM828. In contrast, higher rates of ammonia consumption and CO(2) production in LFM828 indicate altered fluxes through the central metabolism in LFM828 and the parental. In conclusion, PTS plays an important role in cell physiology and the elimination of its components has a significant impact on catabolite repression, carbon flux distribution, and PHB biosynthesis in B. sacchari.     

In vitro modification of bovine lens aldose reductase activity

Bovine lens aldose reductase can be activated in crude extracts upon incubation at 37 degrees C at relatively high ionic strength. This phenomenon shows a seasonal occurrence, the enzyme being susceptible to activation only in lenses of animals sacrified in summer. Systems generating oxygen activated species induce the enzyme activation, whereas scavengers of "oxygen radicals" preserve the activated state of the enzyme. Glutathione and other thiol compounds appear to prevent the enzyme activation.     

Multiplicity of dog kidney high-Km aldose reductase and conversion mechanism into aldose reductase

• 1.1. High-K_m, aldose reductase purified from dog kidney inner medulla was easily converted into aldose reductase by incubation in the neutral buffer solution.
• 2.2. High-K_m, aldose reductase was found to be in multiple forms, and was separated into three kinds of species designated as a-, b- and c-forms by HPLC.
• 3.3. The a-form observed as a single peak by HPLC was assumed to be present in three forms (al-, a2- and a3-forms), one was aldose reductase (a 1-form) and the others were the precursors of aldose reductase (a2- and a3-form).
• 4.4. The b-form was rapidly converted into the a3-form, followed slowly by the a2-form and finally into the a 1-form.
• 5.5. The c-form was either directly converted into the al-form, or indirectly into the a2-form followed by the al-form.
• 6.6. Four kinds of species (a2-, a3-, b- and c-forms) of high-Ap, aldose reductase were finally converted into aldose reductase (al-form).

     

Structural and functional properties of aldose xylose reductase from the D-xylose-metabolizing yeast Candida tenuis

The primary structure of the aldose xylose reductase from Candida tenuis (CtAR) is shown to be 39% identical to that of human aldose reductase (hAR). The catalytic tetrad of hAR is completely conserved in CtAR (Tyr51, Lys80, Asp46, His113). The amino acid residues involved in binding of NADPH by hAR (D.K. Wilson, et al., Science 257 (1992) 81-84) are 64% identical in CtAR. Like hAR the yeast enzyme is specific for transferring the 4-pro-R hydrogen of the coenzyme. These properties suggest that CtAR is a member of the aldo/keto reductase superfamily. Unlike hAR the enzyme from C. tenuis has a dual coenzyme specificity and shows similar specificity constants for NADPH and NADH. It binds NADP(+) approximately 250 times less tightly than hAR. Typical turnover numbers for aldehyde reduction by CtAR (15-20 s(-1)) are up to 100-fold higher than corresponding values for hAR, probably reflecting an overall faster dissociation of NAD(P)(+) in the reaction catalyzed by the yeast enzyme.     

Regulation of Aconitase Synthesis in Bacillus subtilis: Induction, Feedback Repression, and Catabolite Repression

The synthesis of aconitase in Bacillus subtilis wild-type and different citric acid cycle mutants has been studied and the influence of various growth conditions examined. Aconitase is induced by citrate and precursors of citrate and repressed by glutamate. Induction and repression counteract each other, and at equimolar concentrations of citrate and glutamate, aconitase synthesis is unaffected. Induction by citrate can partly overcome catabolite repression of aconitase. Isocitrate dehydrogenase show endogenous induction of aconitase due to citrate accumulation. Leaky mutants defective in citrate synthase and aconitase cannot be induced by citrate, which indicates that they carry a regulatory mutation. The complex regulation of aconitase is discussed with reference to the participation of this enzyme in glutamate biosynthesis and energy metabolism.     

Synthesis and aldose reductase inhibitory activity of 5-arylidene-2,4-thiazolidinediones

Several (Z)-5-arylidene-2,4-thiazolidinediones were synthesized and tested as aldose reductase inhibitors (ARIs). The most active of the N-unsubstituted derivatives (2) exerted the same inhibitory activity of Sorbinil. The introduction of an acetic side chain on N-3 of the thiazolidinedione moiety led to a marked increase in lending inhibitory activity, conducting to the discovery of a very potent ARI (4c), whose activity level (IC50=0.13 microM) was in the same range of Tolrestat. Moreover, the corresponding methyl esters (3), devoid of any acidic functionality, showed appreciable inhibitory activity similar to that of the N-unsubstituted compounds. It was also found that the substitution pattern on the 5-benzylidene moiety markedly influenced the activity of N-unsubstituted 2,4-thiazolidinediones 2, compounds with substituents at the meta position being generally more effective than the para-substituted ones; however, this SAR was not evidenced in acetates 3 and acids 4.     

Effect of bovine small intestine thioredoxin on aldose reductase activity

Under oxidative stress mediated by H(2)O(2), significant activation of purified aldose reductase from bovine small intestine was observed in the presence of purified thioredoxin from bovine small intestine.