Xylanase production by Trichoderma reesei rut C-30 on rice straw

Xylanase production of Trichoderma reesei Rut C-30 was examined at different initial pH values (4.8, 5.9, and 7.0) on rice straw in shake flasks, and in a fermentor, for the best pH condition. Enzyme performance was tested on ammonia-treated dwarf elephant grass. The maximum xylanase activities, 92 and 122 IU/mL, were obtained at pH 4.8 in the shake flasks and fermentor, respectively, in which good growth of the fungus was observed during the first 24 h and consumption of proteins dissolved from the rice straw caused the pH to rise later to values between 6.4 and 6.7 (optimal for xylanase production). The xylanases from T. reesei were as effective as Multifect XL, a commercial enzyme preparation, in hydrolyzing ammonia-treated elephant grass.     

Xylanase Production by Bacillus circulans D1 Using Maltose as Carbon Source

Bacillus circulans D1 is a good producer of extracellular thermostable xylanase. Xylanase production in different carbon sources was evaluated and the enzyme synthesis was induced by various carbon sources. It was found that d-maltose is the best inducer of the enzyme synthesis (7.05 U/mg dry biomass at 48 h), while d-glucose and d-arabinose lead to the production of basal levels of xylanase. The crude enzyme solution is free of cellulases, even when the microorganism was cultivated in a medium with d-cellobiose. When oat spelt xylan was supplemented with d-glucose, the repressive effect of this sugar on xylanase production was observed at 24 h, only when used at 5.0 g/L, leading to a reduction of 60% on the enzyme production. On the other hand, when the xylan medium was supplemented with d-xylose (3.0 or 5.0 g/L), this effect was more evident (80 and 90% of reduction on the enzyme production, respectively). Unlike that observed in the xylan medium, glucose repressed xylanase production in the maltose medium, leading to a reduction of 55% on the enzyme production at 24 h of cultivation. Xylose, at 1.0 g/L, induced xylanase production on the maltose medium. On this medium, the repressive effect of xylose, at 3.0 or 5.0 g/L, was less expressive when compared to its effect on the xylan medium.     

Determination of the modes of action and synergies of xylanases by analysis of xylooligosaccharide profiles over time using fluorescence‐assisted carbohydrate electrophoresis

The structure of xylan, which has a 1,4‐linked β‐xylose backbone with various substituents, is much more heterogeneous and complex than that of cellulose. Because of this, complete degradation of xylan needs a large number of enzymes that includes GH10, GH11, and GH3 family xylanases together with auxiliary enzymes. Fluorescence‐assisted carbohydrate electrophoresis (FACE) is able to accurately differentiate unsubstituted and substituted xylooligosaccharides (XOS) in the heterogeneous products generated by different xylanases and allows changes in concentrations of specific XOS to be analyzed quantitatively. Based on a quantitative analysis of XOS profiles over time using FACE, we have demonstrated that GH10 and GH11 family xylanases immediately degrade xylan into sizeable XOS, which are converted into smaller XOS in a much lower speed. The shortest substituted XOS produced by hydrolysis of the substituted xylan backbone by GH10 and GH11 family xylanases were MeGlcA²Xyl₃ and MeGlcA²Xyl₄, respectively. The unsubstituted xylan backbone was degraded into xylose, xylobiose, and xylotriose by both GH10 and GH11 family xylanases; the product profiles are not family‐specific but, instead, depend on different subsite binding affinities in the active sites of individual enzymes. Synergystic action between xylanases and β‐xylosidase degraded MeGlcA²Xyl₄ into xylose and MeGlcA²Xyl₃ but further degradation of MeGlcA²Xyl₃ required additional enzymes. Synergy between xylanases and β‐xylosidase was also found to significantly accelerate the conversion of XOS into xylose.     

One‐step zymogram method for the simultaneous detection of cellulase/xylanase activity and molecular weight estimation of the enzyme

Here, we describe a zymographic method for the simultaneous detection of enzymatic activity and molecular weight (MW) estimation, following a single electrophoresis step. This involved separating cellulase and xylanase activities from bacteria and fungi, obtained from different sources, such as commercial extracts, crude extract and purified proteins, under denaturing conditions, by 10% polyacrylamide gel electrophoresis, using polyacrylamide gels copolymerized with 1% (w/v) carboxymethylcellulose or beechwood xylan as substrates. Then, enzymes were refolded by treatment with 2.5% Triton X‐100 in an appropriate buffer for each enzymatic activity, and visualized by Coomassie blue staining for MW estimation. Finally, Congo red staining revealed bio‐active cellulase and xylanase bands after electrophoretic separation of the proteins in the preparations. This method may provide a useful additional tool for screening of particular cellulase and xylanase producers, identification and MW estimation of polypeptides that manifest these activities, and for monitoring and control of fungal and bacterial cellulase and xylanase production.     

Isolation and Identification of a Newly Isolated <Emphasis Type="Italic">Alternaria</Emphasis> sp. ND-16 and Characterization of Xylanase

Alternaria sp. ND-16, a bacterium isolated from soil sample, was identified as a strain of Alternaria mali based on the morphology and comparison of internal transcribed spacer rDNA gene sequence studies. Furthermore, it is demonstrated that this strain has xylanase activity, and the activity can be optimized under suitable growing conditions where wheat bran and urea are the primary sources of carbon and nitrogen. Partially purified xylanase from Alternaria sp. ND-16 is shown to have an optimal pH of 6.0 and optimal temperature of 50 °C, making this enzyme potentially suitable for industrial applications. It is also demonstrated that Na⁺ and Mn²⁺ show strong inhibition of the xylanase while K⁺, Li⁺, Fe²⁺, Cu²⁺, and Zn²⁺ have no significant effect on the activity.     

Hydrolytic Properties of a Hybrid Xylanase and Its Parents

The hydrolytic properties of a hybrid xylanase (ATx) and its parents (reAnxA and reTfxA) were studied using xylans and xylooligosaccharides as substrates. Analysis of reaction mixtures by high-performance liquid chromatograph revealed that xylotriose (X3) was the main product released from birchwood xylan and wheat bran insoluble xylan by ATx and reAnxA, respectively. Xylobiose (X2) was the main product separately released from birchwood xylan and wheat bran insoluble xylan by reTfxA. Xylotetraose (X4), xylopentaose (X5), and xylohexaose (X6) could be hydrolyzed by ATx, which showed no activity on X2 and X3. Therefore, X4 might be the minimum oligomer hydrolyzed by ATx. X2–X6 could be hydrolyzed by reAnxA and reTfxA, respectively. All of ATx, reAnxA, and reTfxA showed transglycosylation activity.     

Xylanase recovery by ethanol and Na2SO4 precipitation

Xylans are the major components of the hemicellulosic fraction of lignocellulosic biomass and their hydrolysis can be obtained using xylanases fromPenicillium janthinellum. In this work, sugarcane bagasse hemicellulosic hydrolysate was used as the substrate for producing xylanase. The precipitation of these enzymes was studied using ethanol and Na₂SO₄ as precipitating agents. Ethanol precipitation experiments were performed batchwise in concentrations ranging from 10 to 80%, pH 4.0 to 7.0, at 4áC. The concentrations used in the precipitations with Na₂SO₄ were from 5 to 60% at pH 5.5 and 25áC. Solubility curves as a function of xylanase activity and total protein for both precipitating agents were made. According to the results, Na₂SO₄ is not appropriate for precipitating xylanases in this medium since at salt concentrations higher than 25%, the enzyme was denaturated and at this concentration less than 80% of the enzyme and total protein were precipitated. Because of differences in xylanase and total protein solubility, a fractionated precipitation using ethanol can be performed, since with 40% ethanol, 49% of the total protein was precipitated and more than 95% of the enzyme was kept in solution. On the other hand approx 100% of the xylanases were recovered by precipitation after adding 80% ethanol.     

Bleachability and characterization by fourier transform infrared principal component analysis of acetosolv pulps obtained from sugarcane bagasse

Sugarcane bagasse Acetosolv pulps were bleached by xylanase and the pulps classified by using Fourier transform infrared (FTIR) spectroscopy and principal component analysis (PCA). Pulp was treated with xylanase for 4–8 h with stirring at 30°C. Some samples were further extracted with NaOH for 1 h at 65°C. FTIR spectra were recorded directly from the dried pulp samples by using the diffuse reflectance technique. Reduction in kappa number of 69% was obtained after sequence xylanase (4 h)-alkaline extraction. During bleaching the viscosity decreased only 12%. FTIR-PCA showed that the first three principal components (PCs) explained more than 90% of the total variance of the pulp spectra. PC2×PC1 plot showed that the points related to pulps from sequence xylanase (4 h)-alkaline extraction are different from the other. This group isenlarged by plotting PC3×PC1 or PC3×PC2 containing all pulps submitted to alkaline extraction. PC2 and PC3 are the principal factor for differentiation of the pulps. These PCs suffer influence of the ester bands (1740 and 1244 cm⁻¹). On the other hand, the pulps bleached only with xylanase could not be differentiated from the nonbleached pulps.     

Computational approach for identification,characterization, three-dimensional structure modelling and machine learning-based thermostability prediction of xylanases from the genome of Aspergillus fumigatus

Identification of thermostable and alkaline xylanases from different fungal and bacterial species have gained an interest for the researchers because of its biotechnological relevance in many industries, such as pulp, paper, and bioethanol. In this study, we have identified and characterized xylanases from the genome of the thermophilic fungus of Aspergillus fumigatus by in silico analysis. Genome data mining revealed that the A fumigatus genome has six xylanase genes that belong to GH10, GH11, GH43 glycoside hydrolase families. In general, most of the bacterial and fungal GH11 xylanases are alkaline, and GH10 xylanases are acidic; however, we found that one identified xylanase from A fumigatus that belongs to the GH10 family is alkaline while the rest are acidic. Moreover, physicochemical properties also stated that most of the xylanases identified have lower molecular weight except one that belongs to the GH43 family. Structure prediction by homology modelling gave optimized structures of the xylanases. It suggests that GH10 family structure models adapt (β∕α) 8 barrel type, GH11 homology models adapt β-jelly type, and the GH43 family has a fivefold β-propeller type structure. Molecular docking of identified xylanases with xylan revealed that GH11 xylanases have strong interaction (-9.6 kcal/mol) with xylan than the GH10 (-8.5 and -9.3 kcal/mol) and GH43 (-8.8 kcal/mol). We used the machine learning approach based TAXyl server to predict the thermostability of the xylanases. It revealed that two GH10 xylanases and one GH11 xylanase are thermo-active up to 75ᵒC. We have explored the physiochemical properties responsible for maintaining thermostability for bacterial and fungal GH10 and GH11 xylanases by comparing crystal structures. All the analyzed parameters specified that GH10 xylanases from both the fungi and bacteria are more thermostable due to higher hydrogen bonds, salt bridges, and helical content.     

In situ demonstration and quantitative analysis of the intrinsic properties of glycoside hydrolases

Based on digital image analysis techniques and a series of optimizations in native electrophoresis, a new direct method to simultaneously detect the intrinsic properties of each active component in the enzymatic system of glycoside hydrolase was established. The key technique is that the concentration changes of substrate (or product) on the gel can be determined quantitatively by the gray value changes of the corresponding band after electrophoretic separation. In this manner, the catalytic characteristics of each glycoside hydrolase component were demonstrated in situ and were easily determined after immersing the gel in a series of solutions containing substrates or their derivatives. Because of its high throughput, great sensitivity, and convenient operation, this method can be used to demonstrate the natural diversity of glycoside hydrolases and to study spatial and temporal regulation in multienzyme expression systems. Thus, it is an effective approach to study the functional proteomics of glycoside hydrolases.