Purification and properties of extracellular xylan hydrolases ofStreptomyces exfoliatus

Fractionation of extracellular xylan hydrolases of a strain ofStreptomyces exfoliatus MC₁ (by salting out, molecular sieving and ion exchange chromatography) revealed the presence of five species of the enzyme. Three major fractions could be purified to homogeneity; two were apparently endohydrolases and the third an exo-xylan hydrolase. The three fractions showed different degrees of affinity to the substrate and differed considerably in their substrate specificities. One of the endo-enzymes was specific to xylan while the other could also attack cellulose, inulin and pectin. The exo-enzyme showed xylanolytic and cellulolytic functions only. The three fractions further differed in their response to the presence of metal ions, mercapto reagents and compounds. Although the pH and temperature optima were different, the three fractions functioned synergistically in the hydrolysis of xylan.     

Kinetic modeling of brewery's spent grain autohydrolysis

Isothermal autohydrolysis treatments of brewery's spent grain were used as a method for hemicellulose solubilization and xylo-oligosaccharides production. The time course of the concentrations of residual hemicelluloses (made up of xylan and arabinan) and reaction products were determined in experiments carried out at temperatures in the range from 150 to 190 degrees C using liquid-to-solid ratios of 8 and 10 g/g. To model the experimental findings concerning to brewery's spent grain autohydrolysis several kinetic models based on sequential pseudo-homogeneous first-order reactions were tested. Xylan and arabinan were assumed to yield oligosaccharides, monosaccharides (xylose or arabinose), furfural, and other decomposition products in consecutive reaction steps. The models proposed provide a satisfactory interpretation of the hydrolytic conversion of xylan and arabinan. An additional model merging the two proposed models for xylan and arabinan degradation assuming that furfural was formed from both pentoses was developed and the results obtained are discussed. The dependence of the calculated kinetic coefficients on temperature was established using Arrhenius-type equations.     

Bacillus pumilus WL-11木聚糖酶A的纯化、鉴定及其底物降解方式

对一株BacilluspumilusWL_11木聚糖酶的纯化、酶学性质及其底物降解模式进行了研究。经过硫酸铵盐析、CM_Sephadex及SephadexG_75层析分离纯化,获得一种纯化的WL_11木聚糖酶A ,其分子量为2 6 0kD ,pI值9 5 ,以燕麦木聚糖为底物时的表观Km 值为16 6mg mL ,Vmax值为12 6 3μmol (min·mg)。木聚糖酶A的pH稳定范围为6 0至10 4 ,最适作用pH范围则在7 2至8 0之间,是耐碱性木聚糖酶;最适作用温度为4 5℃～5 5℃,在37℃、4 5℃以下时该酶热稳定性均较好;5 0℃保温时,该酶活力的半衰期大约为2h ,在超过5 0℃的环境下,该酶的热稳定较差,5 5℃和6 0℃时的酶活半衰期分别为35min和15min。WL_11木聚糖酶A对来源于燕麦、桦木和榉木的可溶性木聚糖的酶解结果发现,木聚糖酶A对几种不同来源的木聚糖的降解过程并不一致。采用HPLC法分析上述底物的降解产物生成过程发现木聚糖酶A为内切型木聚糖酶,不同底物的降解产物中都无单糖的积累,且三糖的积累量都较高;与禾本科的燕麦木聚糖底物降解不同的是,木聚糖酶A对硬木木聚糖降解形成的五糖的继续降解能力较强。采用TLC法分析了WL_11粗木聚糖酶降解燕麦木聚糖的过程,结果表明燕麦木聚糖能够被WL_11粗木聚糖酶降解生成系列木寡糖,未检出木糖,这说明WL_11主要合成内切型木聚     

The use of N,N'-diallylaldardiamides as cross-linkers in xylan derivatives-based hydrogels

N,N′-Diallylaldardiamides (DA) were synthesized from galactaric, xylaric, and arabinaric acids, and used as cross-linkers together with xylan (X) derivatives to create new bio-based hydrogels. Birch pulp extracted xylan was derivatized to different degrees of substitution of 1-allyloxy-2-hydroxy-propyl (A) groups combined with 1-butyloxy-2-hydroxy-propyl (B) and/or hydroxypropyl (HP) groups. The hydrogels were prepared in water solution by UV induced free-radical cross-linking polymerization of derivatized xylan polymers without DA cross-linker (xylan derivative hydrogel) or in the presence of 1 or 5 wt % of DA cross-linker (DA hydrogel). Commercially available cross-linker (+)-N,N′-diallyltartardiamide (DAT) was also used. The degree of substitution (DS) of A, B, and HP groups in xylan derivatives was analyzed according to ¹H NMR spectra. The DS values for the cross-linkable A groups of the derivatized xylans were 0.4 (HPX-A), 0.2 (HPX-BA), and 0.4 (X-BA). The hydrogels were examined with FT-IR and elemental analysis which proved the cross-linking successful. Water absorption of the hydrogels was examined in deionized water. Swelling degrees up to 350% were observed. The swollen morphology of the hydrogels was assessed by scanning electron microscopy (SEM). The presence of cross-linkers in DA hydrogels had only a small impact on the water absorbency when compared to xylan derivative hydrogels but a more uniform pore structure was achieved.     

Purification and characteristic of endo-(1--4)-beta-xylanase from Geotrichum candidum 3C

A method of purification of endo-(1-->4)-beta-xylanase (endoxylanase; EC 3.2.1.8) from the culture liquid of Geotrichum candidum 3C, grown for three days, is described. The enzyme purified 23-fold had a specific activity of 32.6 U per mg protein (yield, 14.4%). Endoxylanase was shown to be homogeneous by SDS-PAGE (molecular weight, 60 to 67 kDa). With carboxymethyl xylan as substrate, the optimum activity (determined viscosimetrically) was recorded at pH 4.0 (pI 3.4). The enzyme retained stability at pH 3.0-4.5 and 30-45 degrees C for 1 h. With xylan from beach wood, the hydrolytic activity of the enzyme (ability to saccharify the substrate) was maximum at 50 degrees C. In 72 h of exposure to 0.2 mg/ml endoxylanase, the extent of saccharification of xylans from birch wood, rye grain, and wheat straw amounted to 10, 12, and 7.7%, respectively. At 0.4 mg/ml, the extent of saccharification of birch wood xylan was as high as 20%. In the case of birch wood xylan, the initial hydrolysis products were xylooligosaccharides with degrees of polymerization in excess of four; the end products were represented by xylobiose, xylotriose, xylose, and acid xylooligosaccharides.     

Chronic hypoxia increases the NO contribution of acetylcholine vasodilation of the fetal guinea pig heart

To investigate the effect of chronic hypoxia (HPX) on vasodilation of the fetal heart, we exposed pregnant guinea pigs to room air or 12% O(2) for 4, 7, or 10 days. We excised hearts from anesthetized fetuses (60 +/- 3 days; 65-day gestation = term) and measured changes in both the coronary artery pressure of the isolated constant-flow preparation and endothelial nitric oxide synthase (eNOS) mRNA of fetal ventricles. Dilator responses to cumulative addition (10(-9)-10(-5) M) of acetylcholine and sodium nitroprusside in prostaglandin F(2alpha) (5 x 10(-6) M)-constricted hearts were similar among normoxia (NMX), 4-, 7-, and 10-day HPX (control). Nitro-L-arginine (L-NA, 10(-4)M), a NOS inhibitor, inhibited maximal acetylcholine dilation of hearts exposed to 10-day HPX greater than NMX, 4-, and 7-day HPX. Hypoxia (after 7 and 10 days) increased eNOS mRNA of fetal ventricles compared with NMX and 4-day HPX. 4-Aminopyridine (3 mM), a voltage-dependent K(+)-channel inhibitor, inhibited acetylcholine- but not sodium nitroprusside-induced dilation of NMX and 10-day HPX hearts to a similar magnitude. Glibenclamide (10(-5) M), an ATP-sensitive K(+)-channel inhibitor, had no effect on vasodilation. We conclude that chronic HPX increases the contribution of NO but does not alter K(+)-channel activation in response to acetylcholine-stimulated coronary dilation. Thus increases in NO production via upregulation of eNOS gene expression may be an adaptive response to chronic HPX in the fetal coronary circulation.     

Separation and purification of xylose oligomers using centrifugal partition chromatography

Xylose oligomers, which have a prebiotic effect, have been used as additives to human and animal food. These oligomers are also the primary intermediate in hemicellulose degradation during the pretreatment of biomass. Centrifugal partition chromatography (CPC) was used in this study to separate and purify xylan-derived oligomers from birchwood xylan. The xylan was partially hydrolyzed to achieve varying degrees of polymerization at 130°C using 0.98% aqueous sulfuric acid for 20 min with a 2.5% solid loading. The CPC solvent system consisting of dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), and water in a 1:6:3 volumetric ratio was used because of its ability to dissolve xylose oligomers of different degrees of polymerization. The CPC was operated in the ascending mode with the water- and DMSO-rich bottom phase acting as the stationary phase, while the THF-rich top phase was the eluent. This paper delineates a method for the production and purification of xylose monomer and xylose oligomers (up to xylopentaose) using CPC. The amount and purity of compounds collected from the CPC fractionation based on 1 g of birchwood xylan were 25.26 mg of xylose at 91.86% purity, 10.71 mg of xylobiose at 85.07% purity, 4.15 mg of xylotriose at 54.71% purity, 5.03 mg of xylotetraose at 38.33% purity and 3.31 mg of xylopentaose at 30.43% purity.     

Degradation and utilization by Butyrivibrio fibrisolvens H17c of xylans with different chemical and physical properties.

Hemicelluloses, mainly xylans, can be a major component of diets consumed by ruminants and undergo various degrees of microbial digestion in the rumen. The ability of Butyrivibrio fibrisolvens, a major xylanolytic ruminal species, to degrade and utilize nine chemically and physically different xylans for growth was examined. The arabinoxylans used included two isolated from corncobs (CCX-A and CCX-B), a native xylan excreted by corn cell tissue cultures (CX), an oxalic acid-treated, arabinose-depleted CX, and oat spelt xylan. Except for CCX-A, these xylans were extensively converted within 3 h of growth to acid-alcohol-soluble forms that remained at high levels for the duration of culture growth. These xylans contain mainly xylose and arabinose with small amounts of uronic acids. For a given xylan, all three components were used at about the same rate and extent. During the early stages of growth B. fibrisolvens also rapidly solubilized glucuronoxylans from birchwood, larchwood, 4-O-methylglucuronoxylan, and the xylose homopolymer xylan isolated from beechwood (BEWX). In contrast to the findings for the arabinoxylans, little acid-alcohol-soluble carbohydrate remained in these cultures after 9 h of growth, except for BEWX. Initially, with birchwood, larchwood, and 4-O-methylglucuronoxylan the uronic acid components were preferentially used over the xylose. Final xylan utilization measured at 72 h for all xylans varied from 57% for CCX-A to 92% for BEWX and was correlated with the initial 12-h utilization rate for a given xylan. Since CCX-A and BEWX are both highly water insoluble, this aspect did not appear to influence overall utilization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic studies of low-abundance human plasma proteins. VI. Polymorphism of hemopexin.

An analytical isoelectric focusing method in 3 M urea followed by immunoblotting has been devised to detect genetic and biochemical variation in the glycoprotein hemopexin (HPX) in human plasma or serum. HPX reveals extensive microheterogeneity with multiple major and minor components that are susceptible to neuraminidase treatment, suggesting that the observed biochemical variation is due to differences in sialic acid content between HPX isoproteins. However, charge differences that persist in HPX isoproteins following neuraminidase treatment suggest the presence of genetically determined HPX variation, and this is confirmed by population and family studies. HPX was found to be monomorphic, with an invariant pattern, in U.S. whites; but it is polymorphic in U.S. blacks, with three alleles controlled by a single locus, a situation that demonstrates an autosomal codominant pattern of inheritance. The HPX 1, HPX 2, and HPX 3 allele frequencies in U.S. blacks are .941, .018, and .041, respectively.     

A comparison of chemical pretreatment methods for improving saccharification of cotton stalks

The effectiveness of sulfuric acid (H(2)SO(4)), sodium hydroxide (NaOH), hydrogen peroxide (H(2)O(2)), and ozone pretreatments for conversion of cotton stalks to ethanol was investigated. Ground cotton stalks at a solid loading of 10% (w/v) were pretreated with H(2)SO(4), NaOH, and H(2)O(2) at concentrations of 0.5%, 1%, and 2% (w/v). Treatment temperatures of 90 degrees C and 121 degrees C at 15 psi were investigated for residence times of 30, 60, and 90 min. Ozone pretreatment was performed at 4 degrees C with constant sparging of stalks in water. Solids from H(2)SO(4), NaOH, and H(2)O(2) pretreatments (at 2%, 60 min, 121 degrees C/15 psi) showed significant lignin degradation and/or high sugar availability and hence were hydrolyzed by Celluclast 1.5L and Novozym 188 at 50 degrees C. Sulfuric acid pretreatment resulted in the highest xylan reduction (95.23% for 2% acid, 90 min, 121 degrees C/15 psi) but the lowest cellulose to glucose conversion during hydrolysis (23.85%). Sodium hydroxide pretreatment resulted in the highest level of delignification (65.63% for 2% NaOH, 90 min, 121 degrees C/15 psi) and cellulose conversion (60.8%). Hydrogen peroxide pretreatment resulted in significantly lower (p相似文献   

Heme-hemopexin: a 'chronosteric' heme-protein

Hemopexin (HPX) serves as scavenger and transporter of toxic plasma heme to the liver. HPX is formed by two four-bladed beta-propeller domains, resembling two thick disks that lock together at a 90 degrees angle. The heme is bound between the two beta-propeller domains in a pocket formed by the interdomain linker peptide. Residues His213 and His266 coordinate the heme iron atom giving a stable bis-histidyl complex. The HPX-heme geometry is reminiscent of heme-proteins endowed with ligand binding and (pseudo-)enzymatic properties. HPX-heme binds reversibly CO, (*)NO, and cyanide by detaching His213; however, O(2) induces HPX-heme(II) oxidation. Furthermore, HPX-heme(II) facilitates (*)NO/O(2) and (*)NO/peroxynitrite scavenging. Heme sequestering by HPX prevents heme-mediated activation of oxidants which induce the low-density lipoprotein oxidation. Here, ligand binding and (pseudo-)enzymatic properties of HPX-heme are reviewed. HPX, acting not only as a heme carrier but also displaying transient heme-based ligand binding and (pseudo-)enzymatic properties, could be considered a 'chronosteric' heme-protein.     

Acetylation and characterization of xylan from hardwood kraft pulp

Alkaline treatment of eucalyptus hardwood kraft pulp with 10% NaOH yielded 6-8% xylan. The acetylation of the extracted xylan was carried in DMAC/LiCl/pyridine system to obtain a series of xylan acetates with different degrees of substitution (DS). Structure elucidation of xylan and xylan acetate was obtained by ¹H and ¹³C NMR spectroscopy and other homonuclear and heteronuclear 2D-NMR techniques. Inverse-gated ¹³C NMR was employed to determine the DS of xylan acetate. Furthermore, results also revealed equal reactivities at the C-2 and C-3 positions of xylan towards acetylation. Thermal stability, solubility behavior and nanofiber formation of xylan acetate were influenced by its DS values. The mechanical properties of xylan acetate propionate were also investigated.     

Preliminary study on the effects of heavy metals on the growth and some antioxidant enzymes in Arthrospira platensis‐M2 strain

In this study, we have analyzed superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR) activities, biomass accumulation and chlorophyll‐a content in the Arthrospira platensis ‐M2 strain grown at different concentrations of zinc (Zn), tin (Sn) and mercury (Hg). We found that there is a close relationship between chlorophyll‐a content and biomass accumulation in A. platensis ‐M2 strain as a result of Zn, Sn and Hg exposures. Sn was found to be the most toxic heavy metal among others because of the continious inhibition of both biomass and chlorophyll‐a accumulation at 500 and 1000 μg mL^?1 concentrations after the third day of the study, while they represented continuous increases at each Zn and Hg concentration over 7 days. Lower concentrations of Zn and Sn stimulate SOD and GR activities remarkably, probably due to oxidative stress caused by heavy metal toxicity. APX activity was significantly lowered by higher concentrations of the three metals used in this study. Our results suggest that higher heavy metal concentrations inhibited SOD, APX and GR activities but biomass and chlorophyll‐a accumulation endured in a time‐dependent manner, possibly due to some different defence mechanisms, which remain to be investigated.     

Expression of the <Emphasis Type="Italic">Apx</Emphasis> gene family during leaf senescence of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Oxygen-free radicals are thought to play an essential role in senescence. Therefore, the expression patterns of the small gene family encoding the H₂O₂ scavenging enzymes ascorbate peroxidase (APX; EC 1.11.1.11) were analyzed during senescence of Arabidopsis thaliana (L.) Heinh. Applying real-time RT-PCR, the mRNA levels were quantified for three cytosolic (APX1, APX2, APX6), two chloroplastic types (stromal sAPX, thylakoid tAPX), and three microsomal (APX3, APX4, APX5) isoforms identified in the genome of Arabidopsis. The genes of chloroplastic thylakoid-bound tAPX and the microsomal APX4 exhibit a strong age-related decrease of mRNA level in leaves derived from one rosette as well as in leaves derived from plants of different ages. In contrast to the tAPX, the mRNA of sAPX was only reduced in old leaves of old plants. The microsomal APX3 and APX5, and the cytosolic APX1, APX2, and APX6 did not show remarkable age-related changes in mRNA levels. The data show that expression of the individual APX genes is differentially regulated during senescence indicating possible functional specialization of respective isoenzymes. The hydrogen peroxide levels seem to be controlled very precisely in different cell compartments during plant development.     

Effect of carbon source, growth and temperature on the expression of the sec genes of Streptomyces lividans 1326

The mRNA level in sec genes of Streptomyces lividans was studied as a function of growth temperature, glucose effect, and growth using two different carbon sources. Glucose and xylan, a complex hemicellulose, were used as carbon sources for the growth of S. lividans. For both substrates, the mRNA levels of secA, secD, secE, secF, and secY genes were almost constant during the early and log phases, but showed a marked decrease at the beginning of the stationary phase followed by a full recovery of mRNA level in the late stationary phase. This indicates that the sec genes are actively transcribed during the differentiation process. The mRNA level in xylan was generally from 1.5- to 2-fold that in glucose. At growth temperatures of 28 degrees C, 34 degrees C, or 40 degrees C, there was no significant difference in the sec gene mRNA levels.     

Characterization of the degree of polymerization of xylooligomers produced by flowthrough hydrolysis of pure xylan and corn stover with water

Mechanisms that control xylan removal during pretreatment of lignocellulosic biomass are not well understood. For example, although hemicellulose hydrolysis is virtually always assumed to follow first-order homogeneous kinetics, the increase in xylan removal with flow rate for flowthrough pretreatment systems is inconsistent with the predictions for such models, and better information is needed to understand the causes of such discrepancies. Thus, new methods were developed to follow the fate of xylooligomers with degrees of polymerization of up to 30, a range not possible before, for water-only flowthrough pretreatment of oat spelt xylan and corn stover for temperatures of 200–240 °C. Material balances based on the oligomer release profiles produced by batch and flowthrough operations could be closed, suggesting the methods were quite accurate. However, the results also showed that increasing the flow rate from 0 to 2 and then 25 mL/min affected the size distribution of the xylan oligomers (DP < 30) released from corn stover but not from oat spelt xylan and also increased overall hemicellulose sugar solubilization. One explanation for these difference is that lignin and lignin–xylan compounds in particular play an important role in the hydrolysis of lignocellulosic biomass.     

Purification and properties of three endo-beta-1,4-xylanases produced by Streptomyces sp. strain S38 which differ in their ability to enhance the bleaching of kraft pulps*(2)

In the presence of xylan, Streptomyces sp. strain S38 secretes three xylanases (Xyl1, Xyl2, and Xyl3) that were purified to protein homogeneity and characterized. When used in bleach boosting tests on kraft hardwood and softwood, Xyl1, a family-11 enzyme, was more effective than Xyl2 and Xyl3 that belonged to family-10. Xyl1 was fully responsible for the biodelignification potential of the culture supernatants with a minimal effective amount of 10 IU per gram of dry pulp for both softwood and hardwood pulp. Complete conventional CEDED bleaching sequences showed that enzymatic pretreatment (20 IU/g dry pulp) could result in active chlorine savings of 8.6 and 4.9 kg/ton of dry pulp with hardwood and softwood, respectively. The purified enzymes were totally devoid of cellulase activity on CM-cellulose and their activities were optimal at about 60 degrees C and pH 6. Moreover, the V(max) value of Xyl1 at 50 degrees C measured on birchwood xylan (5,700 μmoles/min/mg prot.) was significantly higher than those of Xyl2 and Xyl3 whereas their K(m) values were similar. Their half-lives at 50 degrees C were larger than 16 h but sharply decreased at 60 degrees C where the family-11 Xyl1 was less stable (t(1/2)(60 degrees C) = 10 min) than both family-10 enzymes Xyl2 (t(1/2)(60 degrees C) = 30 min) and Xyl3 (t(1/2)(60 degrees C) = 70 min).     

Hydrothermal and pulp processing of Eucalyptus

Hydrothermal processing of Eucalyptus wood was performed at operation temperature of 181 degrees C, processing time or 37.5 min and solid water ratio of 1/6 to ensure a maximum loss of xylan recuperation with minimum cellulose fibre degradation. Under those conditions, the loss of xylan was 22% less than that achieved with the conditions 196 degrees C, 50.6 min and 1/8 (solid/water). IN In addition, an experimental design was used to study the influence of process variables: temperature (145-175 degrees C), pulping time (40-120 min) and ethanol concentration (40-70% weight concentration), on the properties of pulps (yield, kappa number, viscosity, cellulose, xylan, lignin acetyl groups contents and brightness) and paper sheets (stretch index, burst index and tear index) obtained from the solid fraction after hydrothermal treatment of Eucalyptus globulus. Pulps with acceptably high physical and chemical properties can be obtained operating at 175 degrees C for 90 min with 55% ethanol concentration.     

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.