Maximum Xylanase Activity Research Articles

Alkalistable endo-β-1,4-xylanase production from a newly isolated alkalitolerant Penicillium sp. SS1 using agro-residues

Thermostable and alkalitolerant xylanases have got intense research focus due to their vast applications in various industries including pulp and paper, food, feed, textile, biofuel, etc. In the present investigation, a Penicillum sp. SS1 isolated from degrading woody material was found to produce moderately thermoactive and alkalistable endo-β-1,4-xylanase (xylanase). Maximum xylanase production was observed after fourth day of fermentation (43.84 IU/ml). The organism produced substantial quantities of xylanase using agricultural residues like wheat bran (20.6 IU/ml), rice bran (21.8 IU/ml) and sawdust (10.7 IU/ml) as carbon sources. The enzyme preparation was totally free of filter paper activity (FPase) and possessed negligible carboxymethyl cellulase (CMCase) activity; this could be an important feature of enzyme if the intended application of enzyme is in pulp and paper industries. Among nitrogen sources examined, yeast extract supported maximum xylanase production (45.74 IU/ml), and was followed by soybean meal (22.2 IU/ml) and ammonium sulphate (20 IU/ml). Maximum xylanase production was observed at initial medium pH 9 (25.6 IU/ml); however, at pH 8 and 10 also significantly high enzyme titre was observed (24 and 21.2 IU/ml, respectively). Thus, Penicillium sp. SS1 displayed capability of growing and producing xylanase at high alkaline pH (8–10). Maximum xylanase activity was reported at 50 °C, however, significantly high activity was observed at 60 °C (65.4%), however, at 70–80 °C activity was lost considerably. At 50–60 °C the enzyme retained very high activity up to 30–60 min (91–100%), however, prolonged incubation (90 min) caused considerable activity reduction (residual activity 63–68%).

Production of xylanase by Promicromonospora sp MARS with rice straw under non sterile conditions

Quantitative differentiation of the inoculated Promicromonospora sp MARS and indigenous microbial flora of rice straw is described using ERIC-PCR during the production of xylanase in non sterile solid state fermentation (NS-SSF) to reduce the cost of enzyme production. Under non-sterile solid state fermentation with rice straw, Promicromonospora sp MARS produced 85.0 IU/g of xylanase. DNA fingerprints of unknown bacterial isolates obtained on BHA + xylan exactly matched with that of Promicromonospora sp MARS. Using PCR based enumeration; population of Promicromonospora sp MARS was 67.5 × 10 5 cfu/g after 48 h which increased to 23 × 10 6 cfu/g after 96 h which coincided with the maximum xylanase activity as compared to 14 × 10 4 cfu/g in control after 96 h. Under submerged fermentation (SmF), after 48 h, Promicromonospora sp MARS produced maximum xylanase (42.2 IU/ml) using rice straw as substrate followed by 19.9 and 20.4 IU/ml when Promicromonospora sp MARS was grown on rice husk and wheat straw, respectively. Optimum pH and temperature of the crude xylanase were 8.0 and 65 °C. Crude enzyme was 95% stable for 180 min of incubation at pH 8.0 and was stable at 65 °C for 240 min.

Studies on the effect of pH and carbon sources on enzyme activities of some pectinolytic bacteria isolated from jute retting water

The effect of pH of the assay media and different carbon sources in the culture media on the activities of 4 different enzymes, namely polygalacturonase (PG), pectin lyase (PNL), xylanase, and cellulase, of some pectinolytic bacteria were studied. The pectinolytic bacterial strains were isolated from the jute retting water bodies of 2 important jute growing areas of North 24 Parganas, West Bengal, India. The highest PG (12.45 to 41.0 IU/g cell wet weight) and PNL (22.55 to 75.7 U/mL) activities occurred at the alkaline pH of 8.0 and 8.5, respectively. Maximum xylanase activity (0.01 to 0.735 \mumol/mL/min) occurred at pH 7.0. An acidic pH of 5.0 favored the cellulase activity (0 to 0.168 \mumol/mL/min). All the enzymes were highly substrate specific and inducible in nature. The highest PG and PNL activities were observed in the presence of pectin, whereas the highest xylanase and cellulase activities were detected in the presence of xylan and cellulose, respectively, as the sole carbon source in the media.

Xylanase production by Aspergillus awamori under solid state fermentation conditions on tomato pomace

In this work, tomato pomace, a waste abundantly available in the Mediterranean and other temperate climates agro-food industries, has been used as raw material for the production of some hydrolytic enzymes, including xylanase, exo-polygalacturonase (exo-PG), cellulase (CMCase) and α-amylase. The principal step of the process is the solid state fermentation (SSF) of this residue by Aspergillus awamori. In several laboratory experiments, maximum xylanase and exo-PG activities were measured during the first days of culture, reaching values around 100 and 80 IU/gds (international units of enzyme activity per gram of dried solid), respectively. For CMCase and α-amylase production remained almost constant along fermentation, with average values of 19 and 21.5 IU/gds, respectively. Experiments carried out in a plate-type bioreactor at lab scale showed a clear positive effect of aeration on xylanase and CMCase, while the opposite was observed for exo-PG and α-amylase. In general, xylanase was the enzyme produced in higher levels, thus the optimum conditions for the determination of the enzyme activity was characterized. The xylanase activity shows an optimum pH of 5 and an optimum temperature of 50 ºC. The enzyme is activated by Mg2+, but strongly inhibited by Hg2+ and Cu2+. The enzymatic activity remains quite high if the extract is preserved in a range of pH from 3 to 10 and a temperature between 30 ºC to 40 ºC.

Biochemical and molecular characterization of thermo-alkali tolerant xylanase producing bacteria from thermal springs of Manikaran.

One hundred ten alkalo-tolerant thermophilic bacteria were isolated from 17 samples (water and sediment) collected from Manikaran. Of 110 isolates, 70 showed the production of xylanases and were further screened for growth and production of xylanases at different temperature ranging from 40 to 75°C. Eleven isolates that showed growth and xylanase production at temperatures ≥50°C were selected for quantitative estimation in modified Reese mineral liquid medium containing wheat bran. Maximum xylanase activity was produced by isolate H-7 followed by H-9 and R-9 and was statistically superior to other isolates. The microscopic observation showed that the isolates possessed the typical rod with endospore, characteristic of genus Bacillus. The isolates were found to be oxidase and catalase positive. Using BIOLOG Microlog 3 software, the isolates H7, H9 and R9 were identified as Paenibacillus ehemensis, Bacillus cereus/B. thuringiensis and B. subtilis respectively, based on utilization of 95 carbon sources. PCR-RFLP analysis of 16S rDNA indicated that the isolates were genetically different from each other. DNA sequencing of the three isolates and phylogenetic analysis revealed that all the isolates obtained from Manikaran thermal springs showed 97 to 100% similarity with the sequences within the GenBank. The closest phylogenetic neighbours according to the 16S rRNA gene sequence data for the three isolates H-7, H-9, and R-9 were Paenibacillus ehemensis, Bacillus cereus and Bacillus subtilis, respectively.

Production of Xylanase From an Alkalitolerant Streptomyces sp. 7b Under Solid-State Fermentation, Its Purification, and Characterization

Streptomyces sp. 7b showed highest xylanase activity among 41 bacterial isolates screened under submerged fermentation. The organism grew over broad pH (5-11) and temperatures range (25-55 degrees C) and displayed maximum xylanase production on wheat bran (1230 U/g) under solid-state fermentation. Xylanase production was enhanced substantially (76%-77%) by inclusion of trypton (2180 U/g) or beef extract (2170 U/g) and moderately (36%-46%) by yeast extract (1800 U/g) or soybean meal (1670 U/g). Inclusion of readily utilizable sugars such as glucose, maltose, fructose, lactose or xylose in the substrate repressed the xylanase production. The optimum initial pH of the medium for maximum enzyme production was 7 to 8; however, appreciable level of activity was obtained at pH 6 (1,680 U/g) and 9 (1,900 U/g). Most appropriate solid to liquid ratio for maximum xylanase production in solid-state fermentation was found to be 1:2.5. The organism produced a single xylanase of molecular weight of approximately 30 kDa as analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis after purification with ammonium sulfate precipitation, and carboxy methyl sephadex chromatography. The enzyme was purified to the extent of 5.68-fold by salt precipitation and ion-exchange chromatography. Optimum temperature and pH for maximum xylanase activity were 50 degrees C and 6, respectively.

Enhanced production of xylanase by recombinant Escherichia coli DH5α through optimization of medium composition using response surface methodology

The effect of medium composition on the production of intracellular xylanase by recombinant Escherichia coli DH5α was studied in shake flask culture. The medium was optimized using response surface methodology (RSM) based on two-level fractional factorial design (2FFD) and central composite design (CCD). Initially, significant factors of the medium composition affecting xylanase production were screened using 2FFD, where the variables consisted of glucose, diammonium hydrogen phosphate [(NH4)2HPO4], dipotassium hydrogen phosphate (K2HPO4), potassium dihydrogen phosphate (KH2PO4) and magnesium sulphate (MgSO4). The significant variables from the 2FFD [(NH4)2HPO4, K2HPO4 and MgSO4] were selected further for CCD to improve the final xylanase yield. The optimum medium composition to achieve maximum xylanase activity was 15 g L−1 glucose, 10 g L−1 (NH4)2HPO4, 18 g L−1 K2HPO4, 3.5 g L−1 KH2PO4 and 1.5 g L−1 MgSO4, with a final xylanase activity of 2,649 U mL−1. Verification experiments confirmed the validity of the proposed model, which led to an increase in xylanase production by about 74% as compared to non-optimized medium (1,526 U mL−1).

Heterologous expression of a gene for thermostable xylanase from Chaetomium thermophilum in Pichia pastoris GS115

We studied heterologous expression of xylanase 11A gene of Chaetomium thermophilum in Pichia pastoris and characterized the thermostable nature of the purified gene product. For this purpose, the xylanase 11A gene of C. thermophilum was cloned in P. pastoris GS115 under the control of AOX1 promoter. The maximum extracellular activity of recombinant xylanase (xyn698: gene with intron) was 15.6 U ml(-1) while that of recombinant without intron (xyn669) was 1.26 U ml(-1) after 96 h growth. The gene product was purified apparently to homogeneity level. The optimum temperature of pure recombinant xylanase activity was 70°C and the enzyme retained its 40.57% activity after incubation at 80°C for 10 min. It exhibited quite lower demand of activation energy, enthalpy, Gibbs free energy, entropy, and xylan binding energy during substrate hydrolysis than that required by that of the donor, thus indicating its thermostable nature. pH-dependent catalysis showed that it was quite stable in a pH range of 5.5-8.5. This revealed that gene was successfully processed in P. pastoris and remained heat stable and may qualify for its potential use in paper and pulp and animal feed applications.

Production and Application of Xylanase from Penicillium sp. Utilizing Coffee By-products

The lignocellulosic coffee by-products such as coffee pulp, coffee cherry husk, silver skin, and spent coffee were evaluated for their efficacy as a sole carbon sources for the production of xylanase in solid-state fermentation using Penicillium sp. CFR 303. Among the residues, coffee cherry husk was observed to produce maximum xylanase activity of 9,475 U/g. The process parameters such as moisture (50%), pH (5.0), temperature (30 °C), particle size (1.5 mm), inoculum size (20%), fermentation time (5 days), carbon source (xylose), and nitrogen source (peptone) were optimized and the enzyme activity was in the range of 19,560–20,388 U/g. The enzyme production was further improved to 23,494 U/g with steam as a pre-treatment. The extracellular xylanase from the fungal source was purified to homogeneity from culture supernatant by ammonium sulfate fractionation, DE32-cellulose with a recovery yield of 25.5%. It appeared as a single band on SDS-PAGE gel with a molecular mass of approximately 27 kDa. It had optimum parameters of 50 °C temperature, pH 5.0, K m 5.6 mg/mL, and V max 925 μmol mg−1 min−1 with brichwood xylan as a substrate. The crude enzyme hydrolysed lignocellulosic substrate as well as industrial pulp. Production of xylanase utilizing coffee by-products constitutes a renewable resource and is reported for the first time.

Statistical Analysis of Main and Interaction Effects to Optimize Xylanase Production under Submerged Cultivation Conditions

In recent years, xylanase has become an essential option for environmental friendly industrial biotechnologicalapplications and there is a rising demand for large scale production. In this study, a Bacillus species 2129 was tested forthe xylanase production under submerged cultivation conditions. Maximum xylanase activities were achieved using oatas the substrate and by optimizing process conditions such as substrate concentration, pH and nitrogen source usingstatistically significant design of experiments, employing the response surface methodology (RSM) concept. Underoptimized conditions there was an 8% increase in the enzyme activity and results from statistical approximation in theform of analysis of variance (ANOVA) shows that the squared effects of the variables were significant than both themain and interaction effects.

English

  The objective of this research was to investigate xylanase production by filamentous fungi (Trichoderma viride) to determine the best cultivation conditions in the process, aiming toward optimization of enzyme production. The best temperature, as well as the best carbon source, for biomass production was determined through an automated turbidimetric method (Bioscreen-C). The enzyme activity of this fungus was separately evaluated in two solid substrates (wheat and soybean bran) and in Vogel medium, pure and by adding other carbon sources. Temperature effects, cultivation time, and spore concentrations were also tested. The best temperature and carbon source for enzyme and biomass production was 25°C and sorbitol, respectively. Maximum xylanase activity was achieved when the fungus was cultivated in wheat bran along with sorbitol (1%, w/v), using a spore concentration of 2 x 106 spores.mL-1, pH 5.0, for 144 h cultivation. The study demonstrated not only the importance of the nature of the substrate in obtaining a system resistant to catabolic repression, but also the importance of the culture conditions for biosynthesis of this enzyme. T. viride showed a high potential for xylanase production under the conditions presented in these assays.   Key words: Trichoderma viride, xylanase activity, enzyme optimization.

BIOMASSES AND XYLANASE PRODUCTION BY STRAINS OF PENICILLIUM ISOLATED FROM BRAZILIAN ATLANTIC FOREST

ABSTRACT Six Penicillium strains were isolated from soil at a depth of 0 15 cm in the Juréia-Itatins Ecology Station (JIES), in the São Paulo State, Brazil. They were evaluated for xylanase production under different temperatures and carbon sources. The best carbon source and temperature were first determined in an automated Bioscreen C system, verifying the growth of microorganisms. Liquid media containing tap water with 2% carbohydrate and/or 1% nitrogen sources were used. Afterwards, Penicillium citrinum, P. fellutanum, P. rugulosum and P. decumbens were cultivated in 250 mL Erlenmeyer flasks with 50 mL of culture medium containing tap water sole 2% carbon source (fructose, glucose, mannitol, sucrose or xylose) and 1% yeast extract as a nitrogen source at pH 5.0 and 28o C, with agitation of 150 rpm for 72 hours. These same strains, except P. decumbens, and P. purpurogenum were cultivated in solid substrate with wheat bran under the same environmental conditions to study the potential of xylanase activity. Maximum xylanase activity was observed in cultures with wheat bran, without the addition of any other carbon source, using inocula containing 1 x 107 spores.mL-1 (28o C, pH 5.0, 72 h). It can be concluded that P. fellutanum and P. citrinumare a good xylanase producers under the conditions of 28º C. The results of xylanase activity were 54% less at 28º C in liquid cultures media cultures than in solid substrate.

Optimization of xylanase production from free and immobilized cells of Fusarium solani F7

The aim of the present investigation was to characterize a xylanase-producing Fusarium solani isolate and to optimize cultural conditions for xylanase enzyme production from free and immobilized cells. Screening of Fusarium solani isolate was based on the diameter of the clear zone formation in oat spelt xylan agar plates. Fusarium solani isolate F7 was selected and optimized for xylanase enzyme production using cheaper substrates such as wheat straw, rice straw, rice bran, and wood husk. Maximum enzyme activity was observed in wheat straw (78.32 U ml-1 for free cells and 94.68 U ml-1 for immobilized cells). Optimum pH and temperature for xylanase activity were found to be 5.5 and 30°C at 3% substrate concentration for free cells and 5.0 and 30°C at 3% substrate concentration for immobilized cells. In the purification step, 75% ammonium sulphate saturation was found to be suitable, giving maximum xylanase activity. Production of xylanase was greater from immobilized cells than from free cells. Purified xylanase from free cells yielded a single band with a molecular weight of 89kDa, while it was 92.8kDa for immobilized cells. The use of wheat straw as a major carbon source is particularly valuable, because oat spelt xylan is very expensive. The Fusarium solani F7 isolate proved to be a promising microorganism for xylanase production.

Parthenium sp. as a plant biomass for the production of alkalitolerant xylanase from mutant Penicillium oxalicum SAUE-3.510 in submerged fermentation

Abstract The use of congress grass ( Parthenium sp.) and water hyacinth ( Eichhornia crassipes ) as low cost raw materials for xylanase production from mutant Penicillium oxalicum SAU E -3.510 in submerged fermentation was investigated. For development of mutant from wild type P. oxalicum SA-8 ITCC 6024, a strategy of mixed mutagenesis was followed using UV-irradiation and ethidium bromide, which had resulted into 1.87 fold increases in the activity of the enzyme. For enzyme production, the fungus was cultivated in mineral medium containing congress grass as carbon source. Considerably higher levels of production (475.2 ± 6.0 IU ml −1 ) were achieved in media containing congress grass, although it was slightly less than that was obtained (488.5 ± 6.5 IU ml −1 ) in presence of commercial oat spelt xylan. This fact confirms the feasibility of using this low cost non-food resource as an alternative carbon source to save costs of the enzyme production process. Maximum xylanase activity was reported at 55 °C with its stability at 80 °C for 2 h. The highest activity of xylanase at pH 9.0 and its stability at similar pH for 24 h denote the alkalitolerant nature of enzyme.

Production, purification and characterization of celullase-free xylanase from Aspergillus terreus UL 4209

Aspergillus terreus, UL 4209 strain, isolated from the soil in South Africa was used to produce an extracellular cellulase-free xylanase in shake flask cultures containing oat spelt and/or birchwood xylans. Maximum xylanase activity (35 U/ml) was observed after 96 h at 35oC and pH 6 in 1% oat spelt xylan. The xylanase was purified to homogeneity by gel filtration on Sephacryl S-200. This enzyme was found to be a single subunit protein of 22 kDa showing optimal activity at 35oC and pH 6. The enzyme retained 95% activity at 35 - 40oC after 4 h incubation at pH 6 and at 50oC the half-life was 5.8 h. The apparent Km and Vmax values were 3.57 mg/ml and 55.5
mol/min per mg protein, respectively. MALDITOF and LC mass spectroscopy gave 8 peptide ions whose sequence alignments showed that the xylanase produced by this strain has homology with those of other Aspergillus strains such as A. terreus and A. versicolor. These observations showed that our strain produced a low molecular weight, acidophilic, and thermostable xylanase that may be considered for processes operated at moderate temperatures and pH such as preparation of baked cereal food, clarification of fruit juices and saccharification of agro-residues.

Statistical optimization and kinetic modeling of xylanase production by Arthrobacter sp.

AbstractXylanases are hydrolytic enzymes which randomly cleave the β‐1, 4 backbone of the complex plant cell wall polysaccharide xylan. Diverse forms of these enzymes exist, displaying varying folds, mechanisms of action, substrate specificities, hydrolytic activities, and physicochemical characteristics. This article focuses on the fermentative production of xylanase from Arthrobacter sp. and the factors affecting the production. The batch‐submerged fermentation was carried out in a 250‐ml Erlenmeyer flask. The inoculum size of 5% v/v, initial pH of 7.0, and shaking speed of 150 rpm at 32 °C were maintained for the optimization of medium components. The cell growth, xylanase activity, cellulase activity, protein, and pH were determined at regular time intervals. Plackett–Burman statistical methodology was adopted to study the effect of media components on xylanase production. The effect of temperature and pH was studied using optimized medium composition determined by Plackett–Burman experimental design. The maximum xylanase activity of 4.0 U ml−1 at 84 h at the optimum temperature of 35 °C and pH of 7.0 was obtained. Copyright © 2008 Curtin University of Technology and John Wiley & Sons, Ltd.

Response surface optimization of fermentation conditions for producing xylanase by Aspergillus niger SL-05

Fermentation conditions were statistically optimized for producing extracellular xylanase by Aspergillus niger SL-05 using apple pomace and cotton seed meal. The primary study shows that culture medium with a 1:1 ratio of apple pomace and cotton seed meal (carbon and nitrogen sources) yielded maximal xylanase activity. Three significant factors influencing xylanase production were identified as urea, KH(2)PO(4), and initial moisture content using Plackett-Burman design study. The effects of these three factors were further investigated using a design of rotation-regression-orthogonal combination. The optimized conditions by response surface analysis were 2.5% Urea, 0.09% KH(2)PO(4), and 62% initial moisture content. The analysis of variance indicated that the established model was significant (P < 0.05), "while" or "and" the lack of fit was not significant. Under the optimized conditions, the model predicted 4,998 IU/g dry content, whereas validation experiments produced an enzymatic activity of xylanase at 5,662 IU/g dry content after 60 h fermentation. This study innovatively developed a fermentation medium and process to utilize inexpensive agro-industrial wastes to produce a high yield of xylanase.

Optimal xylanase production using Penicilium janthinellum NCIM 1169: A model based approach

Xylanases are an industrially important class of hydrolytic enzymes that degrade xylans. Production of xylanase from a fungal culture by submerged fermentation and optimization of the operating conditions for maximum activity are the two aims of the present study. Penicillium janthinellum NCIM 1169 with Mandels–Weber medium, sugarcane bagassse (40#) as a carbon source and beef extract as a nitrogen source were used in the experiments. We did 41 experiments to see the effect of variations in carbon, nitrogen source, pH, and inoculum on xylanase activity. This data was then used to build an input/output model using multiple linear regression, back propagation neural network and lazy learning algorithm. It was found that lazy learning model correlated well in mapping input/output data. This model was then utilized as an objective function in genetic algorithm to find the optimal combination of the operating conditions to get the maximum xylanase activity. It was observed that with carbon source, 1.63%, nitrogen source, 0.16%, pH, 4.1, and inoculum, 5.5%, maximum xylanase activity of 28.98 ± 1.73 U/ml was achieved.

Solid-state Fermentation of Xylanase from Penicillium canescens 10–10c in a Multi-layer-packed Bed Reactor

Xylanase is produced by Penicillium canescens 10-10c from soya oil cake in static conditions using solid-state fermentation. The impact of several parameters such as the nature and the size of inoculum, bed-loading, and aeration is evaluated during the fermentation process. Mycelial inoculum gives more production than conidial inoculum. Increasing the quantity of inoculum enhances slightly xylanase production. Forced aeration induces more sporulation of strain and reduces xylanase production. However, forced moistened air improves the production compared to production obtained with forced dry air. In addition, increasing bed-loading reduces the specific xylanase production likely due to the incapacity of the Penicillium strain to grow deeply in the fermented soya oil cake mass. Thus, the best cultivation conditions involve mycelial inoculum form, a bed loading of 1-cm height and passive aeration. The maximum xylanase activity is obtained after 7 days of fermentation and attains 10,200 U/g of soya oil cake. These levels are higher than those presented in the literature and, therefore, show all the potentialities of this stock and this technique for the production of xylanase.

High-level expression of the xylanase from Thermomyces lanuginosus in Escherichia coli

According to the amino acid sequence, a codon-optimized xylanase gene (xynA1) from Thermomyces lanuginosus DSM 5826 was synthesized to construct the expression vector pHsh-xynA1. After optimization of the mRNA secondary structure in the translational initiation region of pHsh-xynA1, free energy of the 70 nt was changed from −6.56 to −4.96 cal/mol, and the spacing between AUG and the Shine-Dalgarno sequence was decreased from 15 to 8 nt. The expression level was increased from 1.3 to 13% of total cell protein. A maximum xylanase activity of 47.1 U/mL was obtained from cellular extract. The recombinant enzyme was purified 21.5-fold from the cellular extract of Escherichia coli by heat treatment, DEAE-Sepharose FF column and t-Butyl-HIC column. The optimal temperature and pH were 65 °C and pH 6.0, respectively. The purified enzyme was stable for 30 min over the pH range of 5.0–8.0 at 60 °C, and had a half-life of 3 h at 65 °C.