Characterization of a Neocallimastix patriciarum xylanase gene and its product

A xylanase gene (xynC) isolated from the anaerobic ruminal fungus Neocallimastix patriciarum was characterized. The gene consists of an N-terminal catalytic domain that exhibited homology to family 11 of glycosyl hydrolases, a C-terminal cellulose binding domain (CBD) and a putative dockerin domain in between. Each domain was linked by a short linker domain rich in proline and alanine. Deletion analysis demonstrated that the CBD was essential for optimal xylanase activity of the enzyme, while the putative dockerin domain may not be required for enzyme function.     

Molecular cloning and characterization of a bifunctional xylanolytic enzyme from Neocallimastix patriciarum

A cDNA encoding a bifunctional acetylxylan esterase/xylanase, XynS20E, was cloned from the ruminal fungus Neocallimastix patriciarum. A putative conserved domain of carbohydrate esterase family 1 was observed at the N-terminus and a putative conserved domain of glycosyl hydrolase family 11 was detected at the C-terminus of XynS20E. To examine the enzyme activities, XynS20E was expressed in Escherichia coli as a recombinant His₆ fusion protein and purified by immobilized metal ion-affinity chromatography. Response surface modeling combined with central composite design and regression analysis was then applied to determine the optimal temperature and pH conditions of the recombinant XynS20E. The optimal conditions for the highest xylanase activity of the recombinant XynS20E were observed at a temperature of 49°C and a pH of 5.8, while those for the highest carbohydrate esterase activity were observed at a temperature of 58°C and a pH of 8.2. Under the optimal conditions for the enzyme activity, the xylanase and acetylxylan esterase specific activities of the recombinant XynS20E toward birchwood xylan were 128.7 and 873.1 U mg⁻¹, respectively. To our knowledge, this is the first report of a bifunctional xylanolytic enzyme with acetylxylan esterase and xylanase activities from rumen fungus.     

An acetylxylan esterase and a xylanase expressed from genes cloned from the ruminal fungus Neocallimastix patriciarum act synergistically to degrade acetylated xylans

A Neocallimastix patriciarum acetylxylan esterase (BnaA) was expressed from the cloned gene in Escherichia coli. Purified recombinant BnaA efficiently released acetate from soluble acetylated birchwood xylan (ABX), with a specific activity of 76 U mg⁻¹. In contrast, release of acetate was very inefficient from the insoluble substrates, spear grass and delignified spear grass. Addition of a recombinant xylanase, XynA, also expressed from a cloned N. patriciarum gene, had no effect on the release of acetate from ABX. However, the combination of recombinant BnaA and XynA released more acetate from spear grass and delignified spear grass than did BnaA alone. Significantly more reducing sugar was also released from all three substrates by the combination of recombinant XynA and BnaA than by XynA alone. Thus the extent of digestion of acetylated xylans by XynA appears to be limited by the acetylation. In this system BnaA does not appear to increase the rate of cleavage of insoluble substrates by XynA, but probably allows the release of shorter xylose oligomers from already solubilised acetylated xylan polymers. Received: 11 January 1999 / Accepted: 28 February 1999     

Expression of a modified Neocallimastix patriciarum xylanase in Butyrivibrio fibrisolvens digests more fibre but cannot effectively compete with highly fibrolytic bacteria in the rumen

AIMS: This study investigated the competitive abilities of two Neocallimastix patriciarum-derived xylanases constructs in Butyrivibrio fibrisolvens H17c (xynA and pUMSX) and their ability to compete in vivo. METHODS AND RESULTS: The digestibility of neutral detergent fibre (NDF) increased during co-culture of xynA or pUMSX and weakly cellulolytic, but not with highly cellulolytic, ruminococci. Competition studies among xynA, pUMSX and cellulolytic consortia demonstrated that xynA was the fittest. XynA did not persist at high levels in the rumen and was undetectable after 22 days. CONCLUSION: The construction of recombinant xylanolytic B. fibrisolvens does improve the digestibility of fibre above that of the native, but digestibility is still less than that of the most potent fibre digesters such as ruminococci. SIGNIFICANCE AND IMPACT OF THE STUDY: Fibre digestion may be improved by genetic manipulation of ruminal bacteria but ecological parameters, such as persistence in vivo and the niche of the organism, must be taken into account.     

The effects of co-cultivation with the acetogen Acetitomaculum ruminis on the fermentative metabolism of the rumen fungi Neocallimastix patriciarum and Neocallimastix sp. strain L2

Abstract The effects of co-cultivation with the hydrogen-utilizing acetogenic bacterium Acetitomaculum ruminis on the fermentative activities of the rumen fungi Neocallimastix patriciarum or Neocallimastix sp. L2 were investigated. In both co-cultures acetate production increased, making it the predominant fermentation product, as the accumulation of lactate, formate, ethanol, H₂ and (in the case of Neocallimastix sp. L2) succinate all decreased. The effects of co-cultivation with Methanobrevibacter smithii were more pronounced. Decreased activities of lactate dehydrogenase, alcohol dehydrogenase and (in the case of Neocallimastix sp. L2) fumarate reductase accompanied the shift in fermentation product formation. The rate of glucose utilization and the fungal biomass yield were also increased in the co-culture.     

Purification of keratinase from poultry farm isolate-Scopulariopsis brevicaulis and statistical optimization of enzyme activity

The fungus Scopulariopsis brevicaulis was isolated from poultry farm soil at Namakkal, India. The extracellular keratinase from this fungus was purified to homogeneity by ammonium sulphate precipitation and procedure involving DEAE-Cellulose and Sephadex G-100 chromatographic techniques. The purified enzyme was formed from a monomeric protein with molecular masses of 39 and 36 kDa by SDS–PAGE and gel filtration, respectively. The optimum pH at 40 °C was 8.0 and the optimum temperature at pH 8.0 was 40 °C. The activity of purified keratinase with respect to pH, temperature and salt concentration was optimized by Box–Behnken design experiment. It was shown that a second-order polynominal regression model could properly interpret the experimental data with an R²-value of 0.9957 and an F-value of 178.32, based on the maximum enzyme activity examined. Calculated optimum conditions were predicted to confer a 100% yield of keratinase activity with 5 mM CaCl₂, pH 8.0 and at a temperature of 40 °C. The enzyme was strongly inhibited by PMSF, which suggests a serine residue at or near an active site. The purified keratinase was examined with its potential for dehairing the skin.     

Kinetic characterization of enhanced lipase activity on oil bodies

Enzyme access, kinetic behavior, and protein–protein interactions are critical for explaining reaction of the metabolites contained within the myriad compartments of biological systems. To explore these relationships, the reaction kinetics of oil bodies versus oil emulsions as substrates for lipolytic reactions were measured. The initial rate of hydrolysis for the oil body system was comparatively very low due to a brief latency period. However, the complete activation of the lipase at the interface resulted in an enzyme–membrane complex that was catalytically enhanced 3–15-fold over the emulsion system for substrate concentrations in the measured range of approximately 1–5.5 mM. This disparity is explained by the availability of substrate to the enzyme active site (defined as the availability parameter “A”) which varies between the two substrates by 40-fold. A simple hyperbolic kinetic mechanism is proposed with K _m replaced by the parameter, A, to account for this phenomenon, leading to a maximum rate of approximately 1450 IU/mg protein. The interaction is verified through separation of the enzyme–membrane complex which shows nearly double the activity towards an emulsified soybean oil substrate (activity ratio of 5:3) when compared to the native enzyme.     

Statistical optimization of thermo-tolerant xylanase activity from Amazon isolated Bacillus circulans on solid-state cultivation

A 2(2) factorial design was performed to find the best conditions of pH and temperature for xylanolytic activity of Bacillus circulans BL53 isolated from the Amazon environment. Solid-state cultivation was carried out on an inexpensive, abundant agro-industrial soybean residue. The central composite design (CCD) used for the analysis of treatment combinations showed that a second-order polynomial regression model was in good agreement with experimental results, with R(2) = 0.9369 (P < 0.05). The maximum activity was obtained at a high temperature (80 degrees C) and over a large pH range (4.0-7.0). Enzymatic activity was maintained in heated extracts up to 50 degrees C, suggesting that the xylanases of B. circulans BL53 are thermo-tolerant biocatalysts, being of interest for industrial processes. The crude enzyme extract hydrolyzed rice straw, sugar cane bagasse and soybean fiber and its activity was stimulated by Co(2+), Fe(3+), and beta-mercaptoethanol but inhibited by Mn(2+), Cu(2+), Ca(2+), Zn(2+), Ba(2+), Mg(2+) and by EDTA.     

High activity xylanase from thermotolerantStreptomyces T7: Cultural conditions and enzyme properties

Summary A thermotolerantStreptomyces T₇ produced 70–72 U/ml of extracellular xylanase activity when grown at 50°C in submerged culture, in à medium containing 5% wheat bran as a carbon source. Among the various sugars tested, maltose showed the highest activity of 8 U/ml. Pure xylan was less effective as an inducer as compared to wheat bran. Ammonium sulphate at a concentration of 0.7% was found to be optimum for maximum yield of the enzyme. The optimum period and pH for maximum production were 72th and 7.0, respectively. The culture filtrate was devoid of amylase, cellulase and B-xylosidase activity. The xylanase was exceptionally stable and did not show any loss in activity after storage at 50°C at pH 5.0 for 6 days.     

Temperature influence on fluorescence intensity and enzyme activity of the fusion protein of GFP and hyperthermophilic xylanase

By constructing the expression system for fusion protein of GFPmut1 (a green fluorescent protein mutant) with the hyperthermophilic xylanase obtained from Dictyoglomus thermophilum Rt46B.1, the effects of temperature on the fluorescence of GFP and its relationship with the activities of GFP-fused xylanase have been studied. The fluorescence intensities of both GFP and GFP-xylanase have proved to be thermally sensitive, with the thermal sensitivity of the fluorescence intensity of GFP-xylanase being 15% higher than that of GFP. The lost fluorescence intensity of GFP inactivated at high temperature of below 60°C in either single or fusion form can be completely recovered by treatment at 0°C. By the fluorescence recovery of GFP domain at low temperature, the ratios of fluorescence intensity to xylanase activity (R _gfp/A _xyl) at 15°C and 37°C have been compared. Even though the numbers of molecules of GFP and xylanase are equivalent, the R _gfp/A _xyl ratio at 15°C is ten times of that at 37°C. This is mainly due to the fact that lower temperature is more conducive to the correct folding of GFP than the hyperthermophilic xylanase during the expression. This study has indicated that the ratio of GFP fluorescence to the thermophilic enzyme activity for the fusion proteins expressed at different temperatures could be helpful in understanding the folding properties of the two fusion partners and in design of the fusion proteins.     

一株产木聚糖酶菌株的筛选鉴定及产酶条件初步优化

本研究从造纸厂的污泥及污水筛选获得了一株产木聚糖酶能力较强的菌株,经鉴定该菌株为异硫链霉菌(Streptomyces althioticus),专利保藏号为CCTCC M 2014110。通过单因素试验初步优化了碳源、氮源、温度、初始p H、发酵周期、摇床转速、接种量及装液量。试验结果显示该菌株最佳产酶条件为:以为玉米芯为碳源,用量30 g/L;以大豆粉与硝酸钾复合物为氮源,用量分别为20g/L和10 g/L;发酵周期120 h,发酵温度40℃,接种量6%,初始p H 5.0,装液量50 m L/250 m L三角瓶,摇床转速为160 r/min。     

Immobilization of flavoproteins on silicon: effect of cross-linker chain length on enzyme activity.

The effect of cross-linker chain length on the activities of choline oxidase (ChO) and glucose oxidase (GOx) immobilized on oxidized silicon wafers has been investigated for the cross-linkers N-succinimidyl 4-maleimido-butyrate (GMBS) and N-succinimidyl 6-maleimidocaproate (EMCS). Enzyme activities were determined with an indirect fluorometric assay based on the production of hydrogen peroxide. Immobilization of ChO or GOx onto oxidized silicon with either cross-linker resulted in an 86-99% loss in enzymatic activity relative to the soluble form of the flavoprotein. However, the different cross-linkers had distinctly different effects on enzyme activity: EMCS-immobilized GOx was four times more active than GMBS-immobilized GOx; EMCS-immobilized ChO had a sevenfold higher activity than GMBS-immobilized ChO.     

Accurate prediction of enzyme mutant activity based on a multibody statistical potential

MOTIVATION: An important area of research in biochemistry and molecular biology focuses on characterization of enzyme mutants. However, synthesis and analysis of experimental mutants is time consuming and expensive. We describe a machine-learning approach for inferring the activity levels of all unexplored single point mutants of an enzyme, based on a training set of such mutants with experimentally measured activity. RESULTS: Based on a Delaunay tessellation-derived four-body statistical potential function, a perturbation vector measuring environmental changes relative to wild type (wt) at every residue position uniquely characterizes each enzyme mutant for model development and prediction. First, a measure of model performance utilizing area (AUC) under the receiver operating characteristic (ROC) curve surpasses 0.83 and 0.77 for data sets of experimental HIV-1 protease and T4 lysozyme mutants, respectively. Additionally, a novel method is introduced for evaluating statistical significance associated with the number of correct test set predictions obtained from a trained model. Third, 100 stratified random splits of the protease and T4 lysozyme mutant data sets into training and test sets achieve 77.0% and 80.8% mean accuracy, respectively. Next, protease and T4 lysozyme models trained with experimental mutants are used to predict activity levels for all remaining mutants; a subsequent search for publications reporting on dozens of these test mutants reveals that experimental results are matched by 79% and 86% of predictions, respectively. Finally, learning curves for each mutant enzyme system indicate the influence of training set size on model performance. AVAILABILITY: Prediction databases at http://proteins.gmu.edu/automute/     

Regulation and optimization of xylanase production inClostridium thermolacticum

Summary In substrate-limited continuous or fed-batch cultures,Clostridium thermolacticum excreted high yield of xylanases even when readily metabolizable compounds such as glucose were used as substrate. These results demonstrated that theC. thermolacticum xylanases were constitutive and were catabolite repressed. Optimization of culture conditions showed that the highest yields were obtained in fed-batch culture.     

Elevating bioavailability of cyclosporine a via encapsulation in artificial oil bodies stabilized by caleosin

To elevate its bioavailability via oral administration, cyclosporine A (CsA), a hydrophobic drug, was either incorporated into olive oil directly or encapsulated in artificial oil bodies (AOBs) constituted with olive oil and phospholipid in the presence or absence of recombinant caleosin purified from Escherichia coli. The bioavailabilities of CsA in these formulations were assessed in Wistar rats in comparison with the commercial formulation, Sandimmun Neoral. Among these tests, CsA-loaded AOBs stabilized by the recombinant caleosin exhibited better bioavailability than the commercial formulation and possessed the highest maximum whole blood concentration (C(max)), 1247.4 +/- 106.8 ng/mL, in the experimental animals 4.3 +/- 0.7 h (t(max)) after oral administration. C(max) and the area under the plasma concentration-time curve (AUC(0-24)) were individually increased by 50.8% and 71.3% in the rats fed with caleosin-stabilized AOBs when compared with those fed with the reference Sandimmun Neoral. The results suggest that constitution of AOBs stabilized by caleosin may be a suitable technique to encapsulate hydrophobic drugs for oral administration.     

Development and enzyme activity of protein bodies in proteinoplasts of tobacco root cells

Summary The development of protein bodies in proteinoplasts of tobacco (Nicotiana tabacum L. var. Wis. 38) roots was investigated with TEM, HVEM, and enzyme cytochemistry. These plastids contain a three-dimensional network of fenestrated tubules which originate from invaginations of the inner membrane of the plastid envelope. Elaboration of the network occurs in parallel with cell differentiation: slender tubules common to plastids in meristematic cells undergo dilation as protein accumulates during cell differentiation; proteinoplasts of vacuolate and root cap cells usually contain a large protein body. The contents of the peripheral tubules, originating from the inner membrane, are less electron dense than the tubules making up the central network. Localized dilations within the tubular network result in the formation of dense spheroidal structures, protein bodies, apparently as a result of continued protein accumulation via tubules connecting to the central network. Protein might be imported from segments of rough ER attached to or apposed to the outer membrane of the proteinoplast envelope.The presence of catalase (E.C. 1.11. 1.6), peroxidase (E.C. 1.11.1.7), and cytochrome oxidase (E.C. 1.9.3.1) was demonstrated by cytochemistry with diaminobenzidine (DAB) as substrate. Oxidized DAB was found in protein bodies after incubation in each of the specific reaction media. While aminotriazole and sodium azide inhibited oxidation of DAB by catalase and peroxidase, respectively, only potassium cyanide completely inhibited oxidation of DAB in protein bodies. We conclude that protein bodies of proteinoplasts in tobacco roots are not sites for storage of protein, rather protein bodies contain heme protein(s) with strong oxidase activity that may convey a specific function to proteinoplasts.Abbreviations used CAT catalase - CYT-OX cytochrome oxidase - DAB diaminobenzidine - ER endoplasmic reticulum - f filaments - HVEM high voltage electron microscopy - M mitochondrion - MT microtubule - P peroxisome - PB protein body - PER peroxidase - Pl plastid - Pg plastoglobuli - RER rough endoplasmic reticulum - RuBPcase ribulose-1,5-bisphosphate carboxylase - S starch - T tubule - V vacuole Scientific Article No. A3997, Contribution No. 6981, of the Maryland Agricultural Experiment StationThe scale bar on each micrograph is 0.1 , unless indicated otherwise