Comparative study of xylose(glucose) isomerase synthesis in Arthrobacter strains

The substrate specificity of isomerases produced by six strains of Arthrobacter sp. was studied. The role of utilizable carbon sources in controlling enzyme biosynthesis was established. All of the strains studied were found to produce xylose isomerases efficiently, converting D-xylose into D-xylulose and D-glucose into D-fructose. All but A. ureafaciens B-6 strains showed low activity toward D-ribose, Arthrobacter sp. B-5 was slightly active toward L-arabinose, and A. ureafaciens B-6 and Arthrobacter sp. B-2239, toward L-rhamnose. In Arthrobacter sp. B-5, the synthesis of xylose/glucose isomerase was constitutive (i.e., it was not suppressed by readily metabolizable carbon sources). The synthesis of xylose/glucose isomerase induced by D-xylose in Arthrobacter sp. strains B-2239, B-2240, B-2241, and B-2242 and by D-xylose and xylitol in A. ureafaciens B-6 was suppressed by readily metabolizable carbon sources in a concentration-dependent manner. The data obtained suggest that D-xylose and/or its metabolites are involved in the regulation of xylose/glucose isomerase synthesis in the Arthrobacter sp. strains B-5, B-2239, B-2240, and B-2241.     

Action of Arthrobacter ureafaciens sialidase on sialoglycolipid substrates. Mode of action and highly specific recognition of the oligosaccharide moiety of ganglioside GM1.

A new bacterial sialidase (N-acetylneuraminate glycohydrolase, EC 3.2.1.18) isolated from the culture filtrate of Arthrobacter ureafaciens was characterized in detail with respect to its action on sialoglycolipids. Strong electrolytes had a reversible inhibitory effect on the action of the enzyme on brain gangliosides in accordance with Debye-Hückel effect of ionic environment on ionic activity, and resulted in an acidic shift and a broadening of the pH optimum. Both ionic and non-ionic detergents markedly enhanced the enzymic activity on the gangliosides, and caused an acidic shift on the pH optimum of this enzyme. Sulfhydryl groups seemed to be involved in its active site. This enzyme had a highly specific action on sialidase-resistant ganglioside GM1, showing about 100-fold higher activity on GM1 than Clostridium perfringens sialidase, the only sialidase so far reported to cleave the lipid substrate in the presence of bile salts. In the absence of detergents, the activity of A. ureafaciens sialidase on GM1 was very low. Ganglioside GM1 in either the monomeric or micelar form was hydrolyzed to asialo-GM1 by A. ureafaciens sialidase most efficiently in the presence of sodium cholate of about three times the GM1 molar concentration. The presence of detergents increased both the Km and Vmax values for ganglioside GM1. The oligosaccharide prepared from GM1 by ozonolysis was cleaved well by this sialidase in the absence of detergents, and no detergent was found to affect the hydrolysis. The Km value for the sugar substrate was about two orders of magnitude greater than that for the corresponding lipid substrate. It is suggested that the hydrophobic ceramide moiety increases affinity of the lipid substrate to the enzyme, but inhibits hydrolysis of the substrate, possibly due to its hydrophobic interaction with hydrophobic portions of the enzyme molecule (resulting in lower Km and Vmax for lipid substrates). This inhibition may be released by detergent due to formation of mixed micelles of sialoglycolipid and detergent molecules. It is also indicated that recognition of the specific saccharide structure of GM1 by individual sialidases is essential for release of the resistant sialyl residue, and that A. ureafaciens sialidase seemed to have an isoenzymic or oligomeric structure.     

Fermentation of D-xylose by free and immobilized Saccharomyces cerevisiae

With D-xylose (50 g l ) as sole carbon substrate, aerobic cultures of S. cerevisiae consumed significant amounts of sugar (26.4 g l ), producing 4.0 g xylitol l but no ethanol. In the presence of a mixture of glucose (35 g l ) and xylose (15 g l ), yeasts consumed 1.6 g xylose l that was converted nearly stoichiometrically to xylitol. Anaerobic conditions lessened xylose consumption and its conversion into xylitol. Traces of ethanol (0.4 g l ) were produced when xylose was the only carbon source, however. Agar-entrapped yeasts behaved as anaerobically-grown cultures but with higher specific rates of xylose consumption and xylitol production.     

Sugar fermentation by <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> to produce ethanol

As a first step in the research on ethanol production from lignocellulose residues, sugar fermentation by Fusarium oxysporum in oxygen-limited conditions is studied in this work. As a substrate, solutions of arabinose, glucose, xylose and glucose/xylose mixtures are employed. The main kinetic and yield parameters of the process are determined according to a time-dependent model. The microorganism growth is characterized by the maximum specific growth rate and biomass productivity, the substrate consumption is studied through the specific consumption rate and biomass yield, and the product formation via the specific production rate and product yields. In conclusion, F. oxysporum can convert glucose and xylose into ethanol with product yields of 0.38 and 0.25, respectively; when using a glucose/xylose mixture as carbon source, the sugars are utilized sequentially and a maximum value of 0.28 g/g ethanol yield is determined from a 50% glucose/50% xylose mixture. Although fermentation performance by F.␣oxysporum is somewhat lower than that of other fermenting microorganisms, its ability for simultaneous lignocellulose-residue saccharification and fermentation is considered as a potential advantage.     

Biodegradation of 4-chlorophenol via a hydroquinone pathway by Arthrobacter ureafaciens CPR706

Abstract A newly isolated Arthrobacter ureafaciens , strain CPR706, could degrade 4-chlorophenol via a new pathway, in which the chloro-substituent was eliminated in the first step and hydroquinone was produced as a transient intermediate. Strain CPR 706 exhibited much higher substrate tolerance and degradation rate than other strains that degraded 4-chlorophenol by the hydroxylation at the second carbon position to form chlorocatechol. Strain CPR706 could also degrade other para -substituted phenols (4-nitro-, 4-bromo-, 4-iodo-, and 4-fluoro-phenol) via the hyroquinone pathway.     

A model of xylitol production by the yeast Candida mogii

Production of xylitol from xylose in batch fermentations of Candida mogii ATCC 18364 is discussed in the presence of glucose as the cosubstrate. Various initial ratios of glucose and xylose concentrations are assessed for their impact on yield and rate of production of xylitol. Supplementation with glucose at the beginning of the fermentation increased the specific growth rate, biomass yield and volumetric productivity of xylitol compared with fermentation that used xylose as the sole carbon source. A mathematical model is developed for eventual use in predicting the product formation rate and yield. The model parameters were estimated from experimental observations, using a genetic algorithm. Batch fermentations, which were carried out with xylose alone and a mixture of xylose and glucose, were used to validate the model. The model fitted well with the experimental data of cell growth, substrate consumption and xylitol production.     

利用木糖-葡萄糖为原料产乙醇酵母的筛选、鉴定及发酵试验

建立筛选利用木糖为碳源产乙醇酵母模型,获得一株适合利用木质纤维素为原料产乙醇的酵母菌株。样品经麦芽汁培养基培养后,以木糖为唯一碳源的筛选培养基初筛,再以重铬酸钾显色法复筛。通过生理生化和26D1/D2区对筛选得到的菌株进行分析和鉴定,该菌初步鉴定为Pichia caribbica。经过筛选得到的菌株Y2-3以木糖（40g/L）为唯一碳源发酵时：生物量为23.5g/L,木糖利用率为94.7 %,乙醇终产量为4.57 g/L;以混合糖（葡萄糖40 g/L,木糖20 g/L）发酵时：生物量为28.6 g/L,木糖利用率为94.2 %,葡萄糖利用率为95.6%,乙醇终产量为20.6 g/L。Pichia caribbica是可以转化木糖及木糖-葡萄糖混合糖为乙醇的酵母菌株,为利用木质纤维素发酵乙醇的进一步研究奠定了基础。     

insilico Characterization and Homology Modeling of Arabitol Dehydrogenase (ArDH) from Candida albican

Background: Arabitol dehydrogenase (ArDH) is involved in the production of different sugar alcohols like arabitol, sorbitol, mannitol, erythritol and xylitol by using five carbon sugars as substrate. Arabinose, d-ribose, d-ribulose, xylose and d-xylulose are known substrate of this enzyme. ArDH is mainly produced by osmophilic fungi for the conversion of ribulose to arabitol under stress conditions. Recently this enzyme has been used by various industries for the production of pharmaceutically important sugar alcohols form cheap source than glucose. But the information at structure level as well as its binding energy analysis with different substrates was missing. Results: The present study was focused on sequence analysis, insilico characterization and substrate binding analysis of ArDH from a fungus specie candida albican. Sequence analysis and physicochemical properties showed that this protein is highly stable, negatively charged and having more hydrophilic regions, these properties made this enzyme to bind with number of five carbon sugars as substrate. The predicted 3D model will helpful for further structure based studies. Docking analysis provided free energies of binding of each substrate from a best pose as arabinose -9.8224calK/mol, dribose -11.3701Kcal/mol, d-ribulose -8.9230Kcal/mol, xylose -9.7007Kcal/mol and d-xylulose 9.7802Kcal/mol. Conclusion: Our study provided insight information of structure and interactions of ArDH with its substrate. These results obtained from this study clearly indicate that d-ribose is best substrate for ArDH for the production of sugar alcohols. This information will be helpful for better usage of this enzyme for hyper-production of sugar alcohols by different industries.     

Biosynthetic features and properties of xylose isomerases from Arthrobacter nicotianae, Escherichia coli, and Erwinia carotovota subsp. atroseptica

The characteristics of xylose isomerase biosynthesis in the bacteria Arthrobacter nicotianae BIM B-5, Erwinia carotovota subsp atroseptica jn42xylA, and Escherichia coli HB101xylA have been studied. The bacteria formed the enzyme constitutively. Out of the carbon sources studied, D-glucose and D-xylose were most favorable for the biosynthesis of xylose isomerase in E. carotovota subsp atroseptica, but the least appropriate in terms of the enzyme production efficiency in E. coli. Minimum and maximum levels of xylose isomerase formation in A. nicotianae were noted, respectively, during D-xylose and sucrose utilization. An addition to the nutrient medium of 0.1-1.5% D-glucose (together with D-xylose) did not affect the enzyme synthesis in A. nicotianae, but suppressed it in Erwinia carotovota subsp atroseptica (by 7% at the highest concentration) and Escherichia coli (by 63 and 75% at concentrations of 0.1 and 1.0%, respectively). The enzyme proteins produced by the bacteria exhibited the same substrate specificity and electrophoretic mobility (PAGE) as xylose isomerase A. nicotianae, although insignificant differences in the major physicochemical properties were noted.     

Carbon material and bioenergetic balances of xylitol production from corncobs by Debaryomyces hansenii

The effect of oxygenation on xylitol production by the yeast Debaryomyces hansenii has been investigated in this work using the liquors from corncob hydrolysis as the fermentation medium. The concentrations of consumed substrates (glucose, xylose, arabinose, acetate and oxygen) and formed products (xylitol, arabitol, ethanol, biomass and carbon dioxide) have been used, together with those previously obtained varying the hydrolysis technique, the level of adaptation of the microorganism, the sterilization procedure and the initial substrate and biomass concentrations, in carbon material balances to evaluate the percentages of xylose consumed by the yeast for the reduction to xylitol, alcohol fermentation, respiration and cell growth. The highest xylitol concentration (71 g/L) and volumetric productivity (1.5 g/L.h) were obtained semiaerobically using detoxified hydrolyzate produced by autohydrolysis-posthydrolysis, at starting levels of xylose (S(0)) and biomass (X(0)) of about 100 g/L and 12 g(DM)/L, respectively. No less than 80% xylose was addressed to xylitol production under these conditions. The experimental data collected in this work at variable oxygen levels allowed estimating a P/O ratio of 1.16 mol(ATP)/mol(O). The overall ATP requirements for biomass production and maintenance demonstrated to remarkably increase with X(0) and for S(0) >or= 130 g/L and to reach minimum values (1.9-2.1 mol(ATP)/C-mol(DM)) just under semiaerobic conditions favoring xylitol accumulation.     

Thermoanaerobacter ethanolicus Growth and Product Yield from Elevated Levels of Xylose or Glucose in Continuous Cultures

The performance of Thermoanaerobacter ethanolicus was evaluated in continuous culture with media containing concentrations of xylose (8 to 20 g/liter) greater than those previously reported. The ethanol yield declined from to 0.42 to 0.29 g of ethanol per g of xylose consumed when input xylose was increased from 4 to 20 g/liter. Yields of both total C₂ and C₃ products from consumed xylose and of cell biomass from ATP produced declined as the input xylose concentration was increased, which was not the case when glucose was the substrate. This suggested that yeast extract functioned as a significant energy and carbon source for cells in fermentations of xylose but not of glucose. The feasibility of this interpretation was confirmed by (i) the calculation of the products theoretically obtainable from yeast extract and (ii) the observation of significant quantities of fermentation products in inoculated sugar-free media. Markedly different patterns of metabolism for the two sugar substrates were also evidenced by the cell yield for glucose being twice that of xylose at elevated sugar concentrations. It was noted that caution must be exerted when results obtained at low xylose concentrations are extrapolated to predict those which can be obtained at higher concentrations.     

Sugar cane bagasse as a possible source of fermentable carbohydrates. II. Optimization of the xylose isomerase reaction for isomerization of xylose as well as sugar cane bagasse hydrolyzate to xylulose in laboratory-scale units

Both the forward and backward reactions of xylose isomerase (Sweetzyme Q) with xylose and glucose as substrates have been studied in terms of kinetics and thermodynamics. The relationship between the two reactions can thus be determined. Much attention has been given to the reaction with xylose as substrate. The optimal conditions of the xylose reaction in terms of pH, buffer, metal ions, substrate concentration, temperature, and ionic strength have been determined. These findings did not differ much from those reported for the glucose reaction. Equilibrium constants for the aldose to ketose conversion were more favorable in the case of glucose. The results obtained with continuous isomerization of xylose in columns packed with either Sweetzyme Q or Taka-Sweet were very similar to those obtained from batch isomerization processes. Particle size had a definite effect on reaction rate, which indicates that diffusion limitations do occur with the immobilized enzyme particles. Heat stability of Sweetzyme Q was good with t(1/2) of 118, 248, and 1200 h at 70, 55, and 40 degrees C, respectively. A novel method for the separation of xylose-xylulose mixtures with water as eluant on a specially prepared Dowex 1 x 8 column was developed. This technique has the capability of producing pure xylulose for industrial or research applications. A writ for a patent regarding this technique is at present prepared.     

Absence of diauxie during simultaneous utilization of glucose and Xylose by Sulfolobus acidocaldarius

Sulfolobus acidocaldarius utilizes glucose and xylose as sole carbon sources, but its ability to metabolize these sugars simultaneously is not known. We report the absence of diauxie during growth of S. acidocaldarius on glucose and xylose as co-carbon sources. The presence of glucose did not repress xylose utilization. The organism utilized a mixture of 1 g/liter of each sugar simultaneously with a specific growth rate of 0.079 h(-1) and showed no preference for the order in which it utilized each sugar. The organism grew faster on 2 g/liter xylose (0.074 h(-1)) as the sole carbon source than on an equal amount of glucose (0.022 h(-1)). When grown on a mixture of the two carbon sources, the growth rate of the organism increased from 0.052 h(-1) to 0.085 h(-1) as the ratio of xylose to glucose increased from 0.25 to 4. S. acidocaldarius appeared to utilize a mixture of glucose and xylose at a rate roughly proportional to their concentrations in the medium, resulting in complete utilization of both sugars at about the same time. Gene expression in cells grown on xylose alone was very similar to that in cells grown on a mixture of xylose and glucose and substantially different from that in cells grown on glucose alone. The mechanism by which the organism utilized a mixture of sugars has yet to be elucidated.     

NADP(+)-dependent D-xylose dehydrogenase from pig liver. Purification and properties.

An NADP(+)-dependent D-xylose dehydrogenase from pig liver cytosol was purified about 2000-fold to apparent homogeneity with a yield of 15% and specific activity of 6 units/mg of protein. An Mr value of 62,000 was obtained by gel filtration. PAGE in the presence of SDS gave an Mr value of 32,000, suggesting that the native enzyme is a dimer of similar or identical subunits. D-Xylose, D-ribose, L-arabinose, 2-deoxy-D-glucose, D-glucose and D-mannose were substrates in the presence of NADP+ but the specificity constant (ratio kcat./Km(app.)) is, by far, much higher for D-xylose than for the other sugars. The enzyme is specific for NADP+; NAD+ is not reduced in the presence of D-xylose or other sugars. Initial-velocity studies for the forward direction with xylose or NADP+ concentrations varied at fixed concentrations of the nucleotide or the sugar respectively revealed a pattern of parallel lines in double-reciprocal plots. Km values for D-xylose and NADP+ were 8.8 mM and 0.99 mM respectively. Dead-end inhibition studies to confirm a ping-pong mechanism showed that NAD+ acted as an uncompetitive inhibitor versus NADP+ (Ki 5.8 mM) and as a competitive inhibitor versus xylose. D-Lyxose was a competitive inhibitor versus xylose and uncompetitive versus NADP+. These results fit better to a sequential compulsory ordered mechanism with NADP+ as the first substrate, but a ping-pong mechanism with xylose as the first substrate has not been ruled out. The presence of D-xylose dehydrogenase suggests that in mammalian liver D-xylose is utilized by a pathway other than the pentose phosphate pathway.     

Glucose repression of xylose utilisation by Lactococcus lactisIO-1

In dual substrate (5 g glucose l , 5 g xylose l ) batch fermentation of L. lactis IO-1 a classic diauxie was observed. In batch fermentations (5 g xylose l ) xylose isomerase activity was only detected in xylose grown cells. In mixed-substrate, carbon limited chemostat cultures (5 g glucose l , 5 g xylose l ) xylose utilisation was partially repressed by glucose at dilution rates above 0.01 h and completely repressed at 0.50 h .     

The effect of glycerol as a sole and secondary substrate on the growth and fatty acid composition of Rhodotorula glutinis

Rhodotorula glutinis is a yeast that produces copious quantities of lipids in the form of triacylglycerols (TAG) and can be used to make biodiesel via a transesterification process. The ester bonds in the TAG are broken leaving behind two products: fatty acid methyl esters and glycerol that could provide an inexpensive carbon source to grow oleaginous yeast R. glutinis. Described here are the effects of different growth substrates on TAG accumulation and fatty acids produced by R. glutinis. Yeast cultured 24h on medium containing dextrose, xylose, glycerol, dextrose and xylose, xylose and glycerol, or dextrose and glycerol accumulated 16, 12, 25, 10, 21, and 34% TAG on a dry weight basis, respectively. Lipids were extracted from R. glutinis culture and transesterified to form fatty acid methyl esters. The results show a difference in the degree of saturation for the carbon sources tested. Cells cultivated on glycerol alone had the highest degree of unsaturated fatty acids at 53% while xylose had the lowest at 25%. R. glutinis can be cultivated on all sugars tested as single carbon substrates or in mixtures. Glycerol may be used as secondary or primary carbon substrate.     

Conversion of xylose to ethanol by recombinant Saccharomyces cerevisiae: importance of xylulokinase (XKS1) and oxygen availability.

The yeast Saccharomyces cerevisiae efficiently ferments hexose sugars to ethanol, but it is unable to utilize xylose, a pentose sugar abundant in lignocellulosic materials. Recombinant strains containing genes coding for xylose reductase (XR) and xylitol dehydrogenase (XDH) from the xylose-utilizing yeast Pichia stipitis have been reported; however, such strains ferment xylose to ethanol poorly. One reason for this may be the low capacity of xylulokinase, the third enzyme in the xylose pathway. To investigate the potential limitation of the xylulokinase step, we have overexpressed the endogenous gene for this enzyme (XKS1) in S. cerevisiae that also expresses the P. stipitis genes for XR and XDH. The metabolism of this recombinant yeast was further investigated in pure xylose bioreactor cultivation at various oxygen levels. The results clearly indicated that overexpression of XKS1 significantly enhances the specific rate of xylose utilization. In addition, the XK-overexpressing strain can more efficiently convert xylose to ethanol under all aeration conditions studied. One of the important illustrations is the significant anaerobic and aerobic xylose conversion to ethanol by the recombinant Saccharomyces; moreover, this was achieved on pure xylose as a carbon. Under microaerobic conditions, 5.4 g L(-1) ethanol was produced from 47 g L(-1) xylose during 100 h. In fed-batch cultivations using a mixture of xylose and glucose as carbon sources, the specific ethanol production rate was highest at the highest aeration rate tested and declined by almost one order of magnitude at lower aeration levels. Intracellular metabolite analyses and in vitro enzyme activities suggest the following: the control of flux in a strain that overexpresses XKS1 has shifted to the nonoxidative steps of the pentose phosphate pathway (i.e., downstream of xylose 5-phosphate), and enzymatic steps in the lower part of glycolysis and ethanol formation pathways (pyruvate kinase, pyruvate decarboxylase, and alcohol dehydrogenase) do not have a high flux control in this recombinant strain. Furthermore, the intracellular ATP levels were found to be significantly lower for the XK strain compared with either the control strain under similar conditions or glucose-grown Saccharomyces. The ATP : ADP ratios were also lower for the XK strain, especially under microaerobic conditions (0.9 vs 6.4).     

Characteristics of levan fructotransferase from Arthrobacter ureafaciens K2032 and difructose anhydride IV formation from levan

A microorganism producing levan fructotransferase was isolated from sugar-disclosed soil and it was identified as Arthrobacter ureafaciens. The major product from levan by enzyme reaction was identified as di-D-fructofuranose 2,6':6,2' dianhydride by mass spectrometry, nuclear magnetic resonance, and chemical analyses. Small amounts of several oligosaccharides and free fructose were also formed by enzyme reaction. An extracellular enzyme that produces di-D-fructofuranose 2,6':6,2' dianhydride from levan was purified from the culture broth of A. ureafaciens K2032. The enzyme had optimum activity around pH 5.8 and 45 degrees C and had a dimeric form in solution. The N-terminal amino acid residues of the purified enzyme were SAPGSLRAVYHMTPPSGXLXDPQ. The enzyme has narrow substrate range and converts the levan to di-D-fructofuranose 2,6':6,2' dianhydride with around 62.5% conversion yield.     

Distribution of neuraminidase in Arthrobacter and its purification by affinity chromatography

Neuraminidase [sialidase, EC 3.2.1.18] was found to be widely distributed in bacteria belonging to Arthrobacter. Among these bacteria, Arthrobacter ureafaciens, A. oxydans, and A. aurescens produced relatively potent neuraminidase activities. For the production of this enzyme, not only colominic acid, a homopolymer of N-acetylneuraminic acid, but also N-acetylneuraminic acid, the reaction product of this enzyme, are effective as sources of carbon. An affinity adsorbent specific for neuraminidase was prepared by cross-linking colominic acid with soluble starch by means of epichlorohydrin. Neuraminidase from A. ureafaciens could be purified on this affinity column. The purified neuraminidase was shown to be free from protease, N-acetylneuraminic acid aldolase, phospholipase C, and glycosidases. Aminoff's assay procedure for sialic acid was modified to avoid the centrifugation step. The modified procedure gave a higher molecular extinction coefficient.