Xylan-degrading enzymes of the yeast Cryptococcus albidus. Identification and cellular localization

During growth on wood beta-1,4-xylans the yeast Cryptococcus albidus produced at least two enzymes which convert the polysaccharide to xylose catabolized by the cells. The enzyme almost completely secreted into culture fluid was identified as an endo-1,4-beta-xylanase. The function of the extracellular beta-xylanase is to hydrolyze xylan to oligosaccharides, mainly to xylobiose and xylotriose, which enter the cell where they are split by the second identified enzyme, a cell-bound beta-xylosidae (xylobiase). Aryl beta-xylosidase activity detected in the culture fluid was snown to be due to low affinity of beta-xylanase for p-nitrophenyl beta-D-xylopyranoside. This property of beta-xylanase was preserved after purification of the enzyme by chromatography on DEAE-cellulose, CM-Sephadex and Biogel A 1.5 m or Biogel P 100. Purified beta-xylanase exhibited certain microheterogeneity after polyacrylamide gel electrophoresis. Both extracellular beta-xylanase and intracellular beta-xylosidase were produced in much lower amounts by the cells grown on glucose than by the cells grown on xylan. This suggested that they are not produced constitutively. The investigated strain was not able to grow on cellulose and the crude and purified beta-xylanase were unable to hydrolyze cellulose or its soluble derivatives.     

Beta-Xylosidases and a nonspecific wall-bound beta-glucosidase of the yeast Cryptococcus albidus

Cryptococcus albidus grown on wood xylans possesses a soluble intracellular beta-xylosidase (EC 3.2.1.37) as an additional constituent of the xylan-degrading enzyme system of this yeast. The enzyme attacks linear 1,4-beta-xylooligosaccharides in an exo-fashion, liberating xylose from the non-reducing ends. The activity of the enzyme increases in the cells during growth on xylan and incubation with xylobiose or methyl beta-D-xylopyranoside which are the best inducers of extracellular beta-xylanase (EC 3.2.1.8). Various alkyl-,alkyl-1-thio- and aryl beta-D-xylopyranosides were excellent inducers of a different beta-xylosidase of Cryptococcus albidus. This enzyme is localized outside the plasma membrane and is principally associated with cell walls. Unlike the soluble intracellular beta-xylosidase, the wall-bound enzyme does not hydrolyze xylooligosaccharides. Evidence has been obtained that beta-xylosidase activity in the cell walls is not due to the presence of a specific aryl beta-xylosidase, but is exhibited by a nonspecific beta-glucosidase (EC 3.2.1.21) inducible by beta-D-xylopyranosides. The ratio of beta-glucosidase and beta-xylosidase activity in the cells and isolated cell walls from yeast induced by various beta-xylopyranosides and beta-glucopyranosides was very similar. Both wall-bound activities were inhibited in a similar pattern by inhibitors of beta-glucosidases, 1,5-gluconolactone and nojirimycin. This bifunctional enzyme does not bear any relationship to the utilization of xylans in Cryptococcus albidus.     

Modification of the carbohydrate composition of sulfite pulp by purified and characterized beta-xylanase and beta-xylosidase of Aureobasidium pullulans

Both beta-xylanase and beta-xylosidase were purified to homogeneity from a xylose-grown culture of Aureobasidium pullulans. Cellular distribution studies of enzyme activities revealed that beta-xylanase was an extracellular enzyme, during both the exponential and stationary phases, whereas beta-xylosidase was mostly periplasmic associated. The beta-xylanase exhibited very high specificity for xylan extracted from Eucalyptus grandis dissolving pulp, whereas the beta-xylosidase was only active on p-nitrophenyl xyloside and xylobiose. Comparison of kcat/Km ratios showed that the beta-xylanase hydrolyzed xylan from dissolving pulp 1.3, 2.1, and 2. 3 times more efficiently than Eucalyptus hemicellulose B, Eucalyptus hemicellulose A, and larchwood xylan, respectively. The beta-xylosidase exhibited a transxylosylation reaction during the hydrolysis of xylobiose. When applied on acid sulfite pulp, both enzymes released xylose and hydrolyzed xylan to a different extent. Although beta-xylosidase (0.4 U/g pulp) liberated more xylose from pulp than beta-xylanase (4.7 U/g pulp), it was responsible for only 3% of xylan solubilization. Treatment of pulp with beta-xylanase liberated 51.7 microgram of xylose/g and hydrolyzed 10% of xylan. The two enzymes acted additively on pulp and removed 12% of pulp xylan. A synergistic effect in terms of release of xylose from pulp was observed when the enzyme mixture of beta-xylanase and beta-xylosidase was supplemented with beta-mannanase. However, this did not result in further enzymatic degradation of pulp xylan. Both beta-xylanase and beta-xylosidase altered the carbohydrate composition of sulfite pulp by increasing the relative cellulose content at the expense of reduced hemicellulose content of pulp.     

纤维素降解产物对Kluyveromyces marxianus 1727共发酵葡萄糖和木糖的影响

研究纤维素酸水解产生的4种副产物乙酸、甲酸、糠醛、5-羟甲基糠醛及发酵产物乙醇对Kluyveromyces marxianus 1727共发酵葡萄糖和木糖的影响。结果表明:5.0 g/L乙酸和1.0 g/L甲酸对葡萄糖和木糖共发酵具有明显的抑制作用;1.0 g/L糠醛和5-羟甲基糠醛基本不影响K.marxianus 1727发酵葡萄糖,且能够被K.marxianus1727转化为毒性相对较低的物质。由于5-羟甲基糠醛的转化速率慢,对K.marxianus 1727发酵木糖的抑制程度大于糠醛。乙醇对K.marxianus 1727发酵木糖具有抑制作用,当乙醇质量浓度大于20 g/L时,生物量及木糖利用率约是对照的44%和70%。     

Characterization of a bacterial xylanase resistant to repression by glucose and xylose

Summary Bacillus subtilis CD4, when grown in nutrient broth or minimal medium in presence of xylan, produced extracellular xylanase that hydrolyzed xylan optimally at pH 5. The enzyme was induced by xylan, xylose and glucose. Addition of xylose or glucose in xylan containing medium did not affect enzyme production. The structural gene encoding xylanase was cloned and expressed in E. coli. The recombinant enzyme exhibited similar properties like that of native enzyme including resistance to repression by xylose and glucose.     

Production of Glucose Isomerase by Streptomyces flavogriseus

A microorganism that produces glucose isomerase was isolated from soil and identified as a strain of Streptomyces flavogriseus. The organism produced a large quantity of glucose isomerase when grown on straw hemicellulose, xylan, xylose, and H₂SO₄ hydrolysate of ryegrass straw. The organism produced glucose isomerase both intra- and extra-cellularly. The highest level of intracellular glucose isomerase (3.5 U/ml) was obtained in about 36 h by a culture grown on straw hemicellulose; the extracellular enzyme (1.5 U/ml) appeared in cultures grown for about 72 h. About equal levels of enzyme were produced in cultures grown on straw hemicellulose, xylan, xylose, and H₂SO₄ hydrolysate of straw, but production of the enzyme was drastically reduced when the organism was grown on other carbon sources. As a nitrogen source, corn steep liquor produced the best results. Soy flour extract, yeast extract, and various peptones also were adequate substrates for glucose isomerase production. Addition of Mg²⁺, Mn²⁺, or Fe²⁺ to the growth medium significantly enhanced enzyme production. The organism, however, did not require Co²⁺, which is commonly required by microorganisms used in the production of glucose isomerase.     

Ethanol production from D-xylose and other sugars by the yeastPachysolen tannophilus

Summary D-Xylose was fermented to ethanol by a strain ofPachysolen tannophilus in yields greater than 0.3g ethanol per g xylose consumed. Ethanol production was influenced by xylose concentration and was at a maximum at 10%, w/v. Ethanol formation occurred at pH 2.75-2.50 but the yeast would not grow at this pH when the initial pH of the medium was less than 3.0. Ethanol was consumed by the yeast when the xylose concentration became limiting. L-Arabinose, D-glucose, D-fructose, cellobiose, D-glucuronic acid, but not sucrose,were also fermented to ethanol byPachysolen tannophilus. Kinetic studies on xylose fermentation established various parameters involved in growth, substrate utilization and ethanol formation when the yeast was fermenter grown.     

Fermentation of glucose and xylose in ruminal strains of Butyrivibrio fibrisolvens

Metabolism of glucose and xylose and parameters of growth were investigated in strains of Butyrivibrio fibrisolvens ATCC 19171 and CE 51. In the strain ATCC 19171, the composition of fermentation end-products was the same in cultures supplied with glucose and xylose. The strain CE 51 produced more volatile fatty acids and less lactate from xylose than from glucose. Cells of this strain grown on xylose possessed phosphoketolase activity (EC 4.1.2.9). In both strains the production of cell dry matter and growth rate were higher in cultures supplied with glucose. In xylose-grown cultures butyrivibrios tended to convert more substrate carbon into metabolites and less into cellular material than in cultures grown on glucose.     

Carbohydrate preferences of Bifidobacterium species isolated from the human gut

The growth of nine species of Bifidobacterium on media containing glucose, xylose, xylooligosaccharides (XOS), xylan or fructooligosaccharides (FOS) as the sole carbon source were compared in pure culture. The bifidobacteria differed in fermentation profiles when tested on different carbohydrates. All species grew to their highest final optical density (OD) on a glucose containing medium, with the exception of B. catenulatum which demonstrated a preference for xylose over glucose, and XOS over FOS. B. bifidum grew to the highest OD on XOS compared to xylose suggesting a specific transport system for the oligosaccharide over the monomer. This is consistent with a lack of beta-xylosidase activity present in the culture medium. Lactate, formate and acetate levels were determined and the ratios of these metabolites altered between and within species growing on different carbohydrates. In general, high lactate production correlated with low formate production and low lactate concentrations were obtained at higher levels of formate. Bifidobacteria may alter their metabolic pathways based upon the carbohydrates that are available for their use.     

Repression of xylose-specific enzymes by ethanol in Scheffersomyces (Pichia) stipitis and utility of repitching xylose-grown populations to eliminate diauxic lag

During the fermentation of lignocellulosic hydrolyzates to ethanol by native pentose-fermenting yeasts such as Scheffersomyces (Pichia) stipitis NRRL Y-7124 (CBS 5773) and Pachysolen tannophilus NRRL Y-2460, the switch from glucose to xylose uptake results in a diauxic lag unless process strategies to prevent this are applied. When yeast were grown on glucose and resuspended in mixed sugars, the length of this lag was observed to be a function of the glucose concentration consumed (and consequently, the ethanol concentration accumulated) prior to the switch from glucose to xylose fermentation. At glucose concentrations of 95 g/L, the switch to xylose utilization was severely stalled such that efficient xylose fermentation could not occur. Further investigation focused on the impact of ethanol on cellular xylose transport and the induction and maintenance of xylose reductase and xylitol dehydrogenase activities when large cell populations of S. stipitis NRRL Y-7124 were pre-grown on glucose or xylose and then presented mixtures of glucose and xylose for fermentation. Ethanol concentrations around 50 g/L fully repressed enzyme induction although xylose transport into the cells was observed to be occurring. Increasing degrees of repression were documented between 15 and 45 g/L ethanol. Repitched cell populations grown on xylose resulted in faster fermentation rates, particularly on xylose but also on glucose, and eliminated diauxic lag and stalling during mixed sugar conversion by P. tannophilus or S. stipitis, despite ethanol accumulations in the 60 or 70 g/L range, respectively. The process strategy of priming cells on xylose was key to the successful utilization of high mixed sugar concentrations because specific enzymes for xylose utilization could be induced before ethanol concentration accumulated to an inhibitory level.     

Regulation of invertase in Aspergillus nidulans: effect of different carbon sources

Aspergillus nidulans produces an extracellular beta-D-fructofuranoside fructohydrolase (invertase) when grown on a medium containing the beta-fructofuranosides sucrose or raffinose, indicating that synthesis is subject to induction by the substrate. On a growth medium containing sucrose, production was maximal at 15 h in cultures incubated at 28 C degrees. After this time the level of detectable invertase in the cultures declined. A proportion of the enzyme was secreted during the linear growth phase of the fungus. Various sugars were investigated for induction of invertase, but only the two beta-fructofuranosides induced high production levels; with the other sugars, the enzyme was produced only at a low constitutive level. Mycelium grown under repressive conditions (1% glucose), rapidly produced invertase when transferred to sucrose-containing medium. After 80 min the invertase level in these cultures was 26-fold higher than the constitutive level. The repressive effect of other sugars, e.g. glucose and xylose, on invertase production was also demonstrated in this experimental system.     

beta-Xylosidases in the yeast Cryptococcus albidus var. aerius.

beta-xylosidase activity has been detected in cell-free extracts and in culture fluids when Cryptococcus albidus var. aerius was grown on glucose as the sole carbon source. The enzyme appears to be constitutive. Mild acid treatment of whole cells suggested that the total activity is located in the periplasmic space and some experiments indicated that it is partially associated with the cell walls. DEAE-Sephadex A50 chromatography has shown that there are two different forms of beta-xylosidase in the cell-free extracts, but only one form is present in the supernatants of culture.     

Purification and properties of thermostable xylanase and beta-xylosidase produced by a newly isolated Bacillus stearothermophilus strain.

We isolated a thermophilic bacterium that produces both xylanase and beta-xylosidase. Based on taxonomical research, this bacterium was identified as Bacillus stearothermophilus. Each extracellular enzyme was separated by hydrophobic chromatography by using a Toyopearl HW-65 column, followed by gel filtration with a Sephacryl S-200 column. Each enzyme in the culture was further purified to homogeneity (62-fold for xylanase and 72-fold for beta-xylosidase) by using a fast protein liquid chromatography system with a Mono Q HR 5/5 column. The optimum temperatures were 60 degrees C for xylanase and 70 degrees C for beta-xylosidase. The isoelectric points and molecular masses were 5.1 and 39.5 kDa for xylanase and 4.2 and 150 kDa for beta-xylosidase, respectively. Heat treatment at 60 degrees C for 1 h did not cause inhibition of the activities of these enzymes. The action of the two enzymes on xylan gave only xylose.     

Effect of different cellulase dosages on cell viability and ethanol production by Kluyveromyces marxianus in SSF processes

This study was aimed to study the effect of commercial cellulases (Celluclast 1.5 LFG) on Kluyveromyces marxianus CECT 10875 growth and ethanol production in SSF processes. Preliminary tests carried out in glucose (50 g/L) fermentation medium showed that high enzyme amounts (2.5-3.5 FPU/mL) could cause a negative effect on K. marxianus growth rate and viable cells number. However, the maximum ethanol production was not affected and about 86% of the theoretical (22 g/L) was reached in all cases independently of the enzyme dosage. In SSF experiments, cell viability was always affected by enzyme loading. Nevertheless, slight differences observed on cell viability during glucose fermentation processes with the detected concentrations of the additives did not justify the negative effect observed in SSF experiments.     

The kinetics of end-product inhibition of l-lactate production from xylose and glucose by Lactococcus lactis IO-1

Summary The relative contributions of lactate inhibition and the generation of sterile (undividing) cells to the low xylose utilisation rate of Lactococcus lactis IO-1 was investigated. The lactate inhibition constant of xylose grown cells was shown to be 9.3 times more than that of glucose grown cells. However, the sterile cell production rate and LDH inactivation rate of the xylose cultures were at least 10 times less than the glucose cultures. Thus, it is suggested that the slower substrate consumption rate in xylose medium is caused mainly by the large inhibition constant for the end product.     

Production of xylanase by the ruminal anaerobic fungus Neocallimastix frontalis

Xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) production was investigated in the ruminal anaerobic fungus Neocallimastix frontalis. The enzyme was released principally into the culture fluid and had pH and temperature optima of 5.5 and 55 degrees C, respectively. In the presence of low concentrations of substrate, the enzyme was stabilized at 50 degrees C. Xylobiose was the principal product of xylanase action, with lesser amounts of longer-chained xylooligosaccharides. No xylose was detected, indicating that xylobiase activity was absent. Activities of xylanase up to 27 U ml-1 (1 U represents 1 micromol of xylose equivalents released min-1) were obtained for cultures grown on xylan (from oat spelt) at 2.5 mg ml-1 in shaken cultures. No growth occurred in unshaken cultures. Xylanase production declined with elevated concentrations of xylan (less than 2.5 mg ml-1), and this was accompanied by an accumulation of xylose and, to a lesser extent, arabinose. Addition of either pentose to cultures grown on low levels of xylan in which neither sugar accumulated suppressed xylanase production, and in growth studies with the paired substrates xylan-xylose, active production of the enzyme occurred during growth on xylan only after xylose had been preferentially utilized. When cellobiose, glucose, and xylose were tested as growth substrates for the production of xylanase (each initially at 2.5 mg ml-1), they were found to be less effective than xylan, and use of xylan from different origins (birch wood or larch wood) as the growth substrate or in the assay system resulted in only marginal differences in enzyme activity. However, elevated production of xylanase occurred during growth on crude hemicellulose (barley straw leaf). The results are discussed in relation to the role of the anaerobic fungi in the ruminal ecosystem, and the possible application of the enzyme in bioconversion processes is also considered.     

Optimization of some culture parameters and properties of β-glucosidase and β-xylosidase from Penicillium funiculosum NRRL – 13033

Penicillium funiculosum NRRL 13033 produced β-glucosidase and β-xylosidase activities when grown on wheat straw. The addition of some inducers (individually or in combination) to the fermentation medium were tested for the production of both enzymes. The relation of mycelial bound enzyme to cell free enzyme was studied during incubation period of fermentation. The optimum activity of β-glucosidase and β-xylosidase were found to be in the pH 4.5 using phosphate-citrate buffer at 50°C for 60 min and at 55°C for 40 min respectively. β-Glucosidase lost about 40% of its original activity by heating to 65°C for 60 min, while, β-xylosidase activity was found to be nearly stable with the same treatment. Both enzyme activities were greatly inhibited when 1.0% (w/v) of xylose and glucose were added to the assay mixture.     

Production of xylanase by the ruminal anaerobic fungus Neocallimastix frontalis.

Xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) production was investigated in the ruminal anaerobic fungus Neocallimastix frontalis. The enzyme was released principally into the culture fluid and had pH and temperature optima of 5.5 and 55 degrees C, respectively. In the presence of low concentrations of substrate, the enzyme was stabilized at 50 degrees C. Xylobiose was the principal product of xylanase action, with lesser amounts of longer-chained xylooligosaccharides. No xylose was detected, indicating that xylobiase activity was absent. Activities of xylanase up to 27 U ml-1 (1 U represents 1 micromol of xylose equivalents released min-1) were obtained for cultures grown on xylan (from oat spelt) at 2.5 mg ml-1 in shaken cultures. No growth occurred in unshaken cultures. Xylanase production declined with elevated concentrations of xylan (less than 2.5 mg ml-1), and this was accompanied by an accumulation of xylose and, to a lesser extent, arabinose. Addition of either pentose to cultures grown on low levels of xylan in which neither sugar accumulated suppressed xylanase production, and in growth studies with the paired substrates xylan-xylose, active production of the enzyme occurred during growth on xylan only after xylose had been preferentially utilized. When cellobiose, glucose, and xylose were tested as growth substrates for the production of xylanase (each initially at 2.5 mg ml-1), they were found to be less effective than xylan, and use of xylan from different origins (birch wood or larch wood) as the growth substrate or in the assay system resulted in only marginal differences in enzyme activity. However, elevated production of xylanase occurred during growth on crude hemicellulose (barley straw leaf). The results are discussed in relation to the role of the anaerobic fungi in the ruminal ecosystem, and the possible application of the enzyme in bioconversion processes is also considered.     

A bifunctional beta-xylosidase-xylose isomerase from Streptomyces sp. EC 10

beta-Xylosidase (1,4-beta-D-xylan xylohydrolase EC 3.2.1.37) and xylose isomerase (D-xylose ketol-isomerase EC 5.3.1.5) produced by Streptomyces sp. strain EC 10, were cell-bound enzymes induced by xylan, straw, and xylose. Enzyme production was subjected to a form of carbon catabolite repression by glycerol. beta-Xylosidase and xylose isomerase copurified strictly, and the preparation was found homogeneous by gel electrophoresis after successive chromatography on DEAE-Sephacel and gel filtration on Biogel A. Streptomyces sp. produced apparently a bifunctional beta-xylosidase-xylose isomerase enzyme. The molecular weight of the enzyme was measured to be 163,000 by gel filtration and 42,000 by SDS-PAGE, indicating that the enzyme behaved as a tetramer of identical subunits. The Streptomyces sp. beta-xylosidase was a typical glycosidase acting as an exoenzyme on xylooligosaccharides, and working optimally at pH 7.5 and 45 degrees C. The xylose isomerase optimal temperature was 70 degrees C and maximal activity was observed in a broad range pH (5-8). Enhanced saccharification of arabinoxylan caused by the addition of the enzyme to endoxylanase suggested a cooperative enzyme action. The first 35 amino acids of the N-terminal sequence of the enzyme showed strong analogies with N-terminal sequences of xylose isomerase produced by other microorganisms but not with other published N-terminal sequences of beta-xylosidases.     

Beta-glucosidase activity from the thermophilic fungus Scytalidium thermophilum is stimulated by glucose and xylose

An inducible mycelial beta-glucosidase from Scytalidum thermophilum was characterized. The enzyme exhibited a pI of 6.5, a carbohydrate content of 15%, and an apparent molecular mass of about 40 kDa. Optima of temperature and pH were 60 degrees C and 6.5, respectively. The enzyme was stable up to 1 h at 50 degrees C and exhibited a half-life of 20 min at 55 degrees C. The enzyme hydrolyzed p-nitrophenyl-beta-d-glucopyranoside, p-nitrophenyl-beta-d-xylopyranoside, o-nitrophenyl-beta-d-galactopyranoside, p-nitrophenyl-alpha-arabinopyranoside, cellobiose, laminaribiose and lactose. Kinetic studies indicated that the same enzyme hydrolyzed these substrates. Beta-Glucosidase was activated by glucose or xylose at concentration varying from 50 to 200 mM. The apparent affinity constants (K0.5) for glucose and xylose were 36.69 and 43.24 mM, respectively. The stimulatory effect of glucose and xylose on the S. thermophilum beta-glucosidase is a novel characteristic which distinguish this enzyme from all other beta-glucosidases so far described.