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Interlaboratory Testing of Methods for Assay of Xylanase Activity
Abstract
Twenty laboratories participated in a collaborative investigation of assays for endo-1,4-β-xylanase activity based on production of reducing sugars from polymeric 4-O-methyl glucuronoxylan. The substrates and methods already in use in the different laboratories were first recorded and the apparent activities obtained using these methods in the analysis of a distributed enzyme sample were compared. The standard deviation of the results reported in this analysis was 108% of the mean. Significant reduction in interlaboratory variation was obtained when all the participants used the same substrate for activity determination, each with their own assay procedure. The level of agreement was further improved when both the substrate and the method procedure were standardized.In a round robin testing of a single substrate and method, including precise instructions for enzyme dilution, the standard deviation between the results after the rejection of two outliers was 17% of the mean. This figure probably reflects the inherently poor reproducibility of results when using only partially soluble, poorly defined and rather impure polymeric substrates. The final level of variation was however low enough to allow meaningful comparison of results obtained in different laboratories when using the standardized assay substrate and method procedure.Fifteen laboratories also participated in preliminary testing of an assay based on the release of dyed fragments from 4-O-methyl glucuronoxylan dyed with Remazol Brilliant Blue dye. High values of the coefficients of correlation indicated good linearity between the amount of dyed fragments released and enzyme concentration. The relative standard deviations of the results obtained by fifteen laboratories were about 30% for an optimum range of xylanase activity in the reaction mixture.
... Measurements were conducted in triplicate using a UV-VIS spectrophotometer (UV-1280, Shimadzu, Kyoto, Japan) and the results were expressed in U/gdb. The activity of xylanase (endo-1,4-β-xylanase) and cellulose (endoglucanases and exoglucanases) was determined by the DNS method [20,21], β-glucosidase activity was determined according to the study of [22], and invertase activity was determined by the method of [23]. The activities of MnP and laccase were determined by monitoring the oxidation of the substrate 2,6dimethoxyphenol (DMP) at 469 nm for 120 s according to the method of [24]. ...
Grape pomace (GP) is considered a natural source of bioactive compounds. To improve the ex-tractability of bioactive compounds, in this work GP was biologically treated for 15 days with the white-rot fungus Trametes versicolor in laboratory jars and a tray bioreactor under solid-state fermentation (SSF) conditions. During SSF, the activity of lignolytic (laccase, manganese peroxi-dase) and hydrolytic (xylanase, cellulase, β-glucosidase and invertase) enzymes was measured, with the activity of laccase (2.66 U/gdb in jars and 0.96 U/gdb in the bioreactor) and xylanase (346.04 U/gdb in jars and 200.65 U/gdb in the bioreactor) being the highest. The effect of the com-plex enzyme system was reflected in the changes in the chemical composition of GP with in-creasing ash, crude protein and free fat content: 28%, 10% and 17% in the laboratory jars and 29%, 11% and 7% in the bioreactor, respectively. In addition, the biological treatment had a positive effect on the extractability of 13 individual phenolic compounds. Therefore, the applied SSF technique represents an effective strategy to improve the profile of phenolic compounds and the nutritional composition of GP, promoting their valorization and opening the door for potential application in the food industry and other sectors.
... Xylanase assay Xylanase assay was performed by Dinitrosalicylic acid (DNSA) method (Bailey et al. 1992). The assay mixture for xylanase assay contained 50 µl of enzyme extract and 1 ml of 1 mM Birchwood xylan in buffer (pH 7). ...
To overcome the human and animal survivability risk, sustainable development is the only option on earth that can be achieved through the maximum use of renewable environmental resources. Recycling of waste paper is an emerging waste management approach to conserve natural resources. Herein, we studied enzyme-mediated process to recycle the xerographic paper by using the crude fungal extract from indigenously isolated fungi identified as Aspergillus assiutensis. The fungal enzyme cocktail has been characterized for the production of multiple enzymes namely cellulase, amylase, xylanase, pectinase, and protease. All these enzymes have pH optima in the acidic range and except cellulase and all the enzymes are stable from 10 to 80 C. In the zymogram analysis, pectinase, xylanase, amylase, and cellulase were detected at 68 kDa, ~ 54 kDa, 38 kDa, and 30 kDa, respectively. Also, the presence of protease was confirmed by the clear zone at 68, 31, and 16 kDa. A 26% decrease in the kappa number and reduction in Hex A of the pulp was observed on the treatment of the pulp with enzyme as compared to the control pulp without any treatment. The physical and chemical properties of the pulp were also improved by enzyme-mediated pulping as compared to the control The physiochemical parameter of the effluent like TDS was reduced (397 ppm) significantly in comparison to chemical deinking process and it was within the permissible limit. BOD and alkalinity were reduced when the enzymes and chemical dosage were used in combination. These results indicate that chemi-enzymatic deinking is most promising to reduce or remove the pollution parameters including ink and this approach can be used in the paper and pulp industry for sustainable development.
... The standard curve was generated using xylose solutions with concentrations ranging from 0 to 10 µmol/mL. The concentration of released reducing sugars was quantified by referring to a xylose standard curve (Bailey et al. 1992). ...
Nowadays, natural resources like lignocellulosic biomass are gaining more and more attention. This study was conducted to analyse chemical composition of dried and ground samples (500 μm) of various Algerian bioresources including alfa stems (AS), dry palms (DP), olive pomace (OP), pinecones (PC), and tomato waste (TW). AS exhibited the lowest lignin content (3.60 ± 0.60%), but the highest cellulose (58.30 ± 2.06%), and hemicellulose (20.00 ± 3.07%) levels. DP, OP, and PC had around 30% cellulose, and 10% hemicellulose. OP had the highest lignin content (29.00 ± 6.40%), while TW contained (15.70 ± 2.67% cellulose, 13.70 ± 0.002% hemicellulose, and 17.90 ± 4.00% lignin). Among 91 isolated microorganisms, nine were selected for cellulase, xylanase, and/or laccase production. The ability of Bacillus mojavensis to produce laccase and cellulase, as well as B. safensis to produce cellulase and xylanase, is being reported for the first time. In submerged conditions, TW was the most suitable substrate for enzyme production. In this conditions, T. versicolor K1 was the only strain able to produce laccase (4,170 ± 556 U/L). Additionally, Coniocheata hoffmannii P4 exhibited the highest cellulase activity (907.62 ± 26.22 U/L), and B. mojavensis Y3 the highest xylanase activity (612.73 ± 12.73 U/L). T. versicolor K1 culture showed reducing sugars accumulation of 18.87% compared to initial concentrations. Sucrose was the predominant sugar detected by HPLC analysis (13.44 ± 0.02 g/L). Our findings suggest that T. versicolor K1 holds promise for laccase production, while TW represents a suitable substrate for sucrose production.
... Xylanase activity was determined according to Bailey et al. (1992). A test tube containing one ml of the enzyme extract and one ml of 1% w/v birchwood xylan (in 0.5 M sodium acetate buffer, pH 5.0) was incubated at 50°C for 10 minutes. ...
Xylan is a second widely available polysaccharide in nature and can be enzymatically degraded for the production of sugars. The complete biodegradation of xylan needs synergistic action of different xylanolytic enzymes. In this research, potential xylanolytic activities of the wood rot basidiomycete fungi from Dagaga and Gambo forests were screened. Xylanases of the potential fungi were also quantitatively determined. Clear zones round the cultures on xylan supplemented agar media of isolate 011-1D (Trametes gibbosa) and 030-1D (Phellinus tremulae) were significantly wider than other isolates. The fungal isolates differently responded to the incubation days both in submerged and solid-state fermentations and most isolates gave higher xylanase yield in solid-state fermentation than in submerged fermentation. The enzymes were active and stable in the temperature range of 40 to 55oC and pH range of 4.0 to 6.0. Incubation temperature of 30 to 35oC and pH of 5.0 to 7.0 were found to be suitable for production of the xylanases from the fungal isolates. Among the supplemented carbon sources, carboxymethylcellulose, xylan and sucrose were found suitable for xylanase productions but most isolates differently responded to the nitrogen source supplementations. MgSO4, ZnSO4 and CaCl2 were also found to be suitable divalent metallic ions for the productions of the enzymes. The isolates could be used for hydrolysis lignocellulosic xylan to 5-carbon sugars by optimizing their growth conditions.
... Endoglucanase activity was determined at 50 °C and pH 5 (i) by hydrolysis of a 2.0% solution of sodium carboxymethyl cellulose (Na-CMC) after 30 min of incubation of 0.5 mL of culture filtrate with 0.5 mL of substrate; (ii) exoglucanase -by hydrolysis of filter paper (1 x 6 cm) for 60 min; and (iii) xylanase -by hydrolysis of 1% xylan solution for 5 min (Bailey at al., 1992;Meng et al., 2021). The amount of reducing sugars formed in the reaction mixture after the enzymatic hydrolysis of Na-CMC, filter paper or xylan was determined by the method using 3,5-dinitrosalicylic acid (DNS) (Miller, 1959). ...
The second generation (2G) or cellulosic ethanol can help with diversification of the use of fossil energy sources. However, as bioconversion of plant waste into 2G bioethanol requires expensive additional steps of pre-treatment/hydrolysis of lignocellulosic materials, and this technology has not yet reached the technological readiness level which would allow it to be scaled-up, this process needs more interdisciplinary and comprehensive studies. This work was aimed at experimental study of a full cycle of successive processes of pre-treatment/saccharification using cellulolytic enzymes of filamentous fungi and fermentation of obtained syrups by xylose-fermenting yeast, using selected natural microorganisms for the fungal-based bioconversion of lignocellulosic agricultural waste to 2G ethanol. Using the Plackett-Burman and Box-Behnken methods of mathematical statistics, the optimal conditions for pre-treatment and enzymatic hydrolysis of wheat straw by a hemi- and cellulolytic multi-enzyme complex of the selected fungal strain Talaromyces funiculosus UCM F-16795 were established: microwave-assisted alkali pre-treatment with sodium hydroxide (NaOH) solutions (concentration range 4.6–4.8%), and saccharification conditions of medium pH 4, temperature 40 °С, hydrolysis duration 18 hours, and dilution of culture liquid with a buffer solution 1:1. The total energy of microwave irradiation 1.2 kJ and the ratio of substrate/enzyme solution 100 mg/1 mL were used. Under optimized conditions, wheat straw hydrolysates contained 5.0–7.5 g/L of reducing sugars, which, according to HPLC assessment, contained 0.7–1.0 g/L of glucose, 2.2–2.9 g/L of xylose and 0.7–0.8 g/L cellobiose. We used the selected strain of xylose-fermenting yeast in fermentation of mixtures of the most important monosaccharides in hydrolysates, xylose and glucose, in the concentration range relevant for syrups obtained by us during the optimized saccharification of lignocellulosic substrates with T. funiculosus enzymes. Based on sequencing and phylogenetic analysis, strain UCM Y-2810 was confirmed as Scheffersomyces stipitis; its nucleotide sequences of ITS region and 28S gene rDNA were deposited in GenBank under the accession numbers OP931914 and OP931915, respectively. The ethanologenic process for S. stipitis UCM Y-2810 was studied according to Box-Behnken design, assessing ethanol concentration by gas chromatography-mass spectrometry. Yeast fermentation under static microaerophilic conditions showed a 1.5 times higher rate of bioethanol production and 1.7 times greater efficiency of ethanologenesis per yeast biomass than for submerged cultivation. Optimization of the process of ethanologenesis resulted in the maximum rate of fermentation mixture of sugars, being 11.30 ± 0.36 g/L of ethanol, with optimal values of factors: 30 g/L of xylose, 5.5 g/L of glucose and cultivation for 5.5 days. It was revealed that the tested glucose concentrations did not significantly affect the process of xylose-fermentation by yeast, and non-competitive inhibition of xylose transport by glucose into yeast cells did not occur. This study demonstrated the potential of a full cycle bioconversion of lignocellulosic waste to 2G ethanol based on use of natural fungal strains and optimization of conditions for all steps.
... The total cellulase (expressed in filter paper unit (FPU)), the endoglucanase, and the β-glucosidase activity were assayed using 50 mg Whatman No. 1 filter paper, 2% carboxymethyl cellulose and 15 mM cellobiose as substrates, respectively, following the protocol of (Ghose, 1987). The xylanase activity was determined by the method adopted by (Bailey et al., 1992) using 1% beech wood xylan as the substrate. Reducing sugars were quantified with the DNS reagent (Miller, 1959), and protein estimation was conducted using the Bradford assay with Bovine Serum Albumin (BSA) as the standard (Kruger, 2009). ...
Hemicellulose plays a key role in both the production of cellulose nanofibrils (CNF) and their properties as suspensions and films. While the use of enzymatic and chemical pre-treatments for tailoring hemicellulose levels are well-established, post-treatment methods using enzymes remain relatively underexplored and hold significant promise for modifying CNF film properties. This study aimed to investigate the effects of enzymatic xylan removal on the properties of CNF film for packaging applications. The enzymatic post-treatment was carried out using an enzymatic cocktail enriched with endoxylanase (EX). EX post-treated-CNFs films were characterized in terms of physical, morphological, optical, thermal, mechanical, and barrier properties. Employing varying levels of EX facilitated the hydrolysis of 8 to 35 % of xylan, yielding CNFs with different xylan contents. Xylan was found to be vital for the stability of CNF suspensions, as its removal led to the agglomeration of nanofibrils. Nanostructures with preserved crystalline structures and different morphologies, including nanofibers, nanorods, and their hybrids were observed. The EX post-treatment contributed to a smoother film surface, improved thermostability, and better moisture barrier properties. However, as the xylan content decreased, the films became lighter (lower grammage), less strong, and more brittle. Thus, the enzymatic removal of xylan enabled the customization of CNF films' performance without affecting the inherent crystalline structure, resulting in materials with diverse functionalities that could be explored for use in packaging films.
Two endo-1,4-β-d-xylanases (1,4-β-d-xylan xylanohydrolase, EC 3.2.1.8) from Trichoderma harzianum E58 have been purified by ultrafiltration and chromatography on carboxymethyl-Sepharose, phenyl-Sepharose and Sephadex G-75. The d-xylanases were shown to be homogeneous by the criteria of dodecyl sulphate polyacrylamide gel electrophoresis and isoelectric focusing. The molecular weights were estimated to be 20 000 and 29 000, with pl values of 9.4 and 9.5, respectively. Typically, 456 mg of the 20 000 dalton and 1.9 mg of the 29 000 dalton d-xylanases were purified from 4.2 litre of culture filtrate with specific activities of 370 and 75 U mg−1, respectively. Optimum d-xylanase activities were obtained when the enzymes were incubated at pH 5, 50°C, for the 20 000 dalton protein and pH 5, 60°C for the 29 000 dalton protein.
Methods are described for measurement of carboxymethylcellulase activity involving action of the enzyme on carboxymethylcellulose followed by determination of reducing power of resulting sugars with the aid of dinitrosalicylic acid or iodine-potassium iodide. Results obtained with the different reagents and with carboxymethylcellulases from different sources are compared. The applicability of the methods is discussed.
The effects of carbon source, substrate concentration, culture pH, and spore inoculum concentration on production of extracellular xylanase and cellulase were examined. Very low enzyme activities were obtained with growth on glucose, xylose, and cellobiose, while significantly higher levels were produced from lactose and arabinose. Higher levels of both enzymes were generated from alpha cellulose, wood pulp, and fibrous paper waste than from purified xylan. However, the ratio of xylanase to cellulase activity was higher with pure xylan. High levels of both xylanase and cellulase activity were obtained when the culture was grown on lactose plus xylan. A factorial experiment showed that spore and substrate concentration had significant effects on xylanase yield, and that an interaction existed between these two variables. Highest levels of xylanase were generated with cultures grown on 1% wood pulp at pH 7.0 using an inoculum of 105 spores ml-1. Maximal xylanase activity was observed at pH 4.8-5.5 at 55°C using a 30-min assay. The type of xylan used as substrate and the method of reducing sugar detection significantly affected measured xylanase activity.