Highest Xylanase Production Research Articles

Enhanced production of cellulase-free, thermo-alkali-solvent-stable xylanase from Bacillus altitudinis DHN8, its characterization and application in sorghum straw saccharification

Abstract A newly isolated Bacillus altitudinis DHN8 was assessed for xylanase production by utilizing sorghum straw. The highest xylanase production was recorded at sorghum straw 3% w/v, inoculum size 1% v/v, inoculum age 18 h, incubation time 42 h, pH 7.0, temperature 35 °C and agitation speed 250 rpm. Moreover, xylose 0.5%, gelatine 0.5% and KNO3 0.3% (w/v) further enhanced the production. The detailed optimization study resulted in a 3.74-fold increase in xylanase production as compared to that of the initial conditions. The partially purified xylanase showed ~70% pH stability after 18 h at pH range of 6–10. Thermostability study revealed more than 60% xylanase activity at temperature range 45–65 °C after 60 min. The presence of metal ions (10 mM CaCl2, MnCl2 and FeCl3) and solvents (10% v/v isopropanol, methanol, ethanol and acetone) were increased xylanase activities remarkably. During saccharification study, 3% alkaline hydrogen peroxide treatment was found to be beneficial for the maximum enzymatic hydrolysis of sorghum straw (34.94 mg/g reducing sugar) after 36 h. As such, this xylanase could be considered as a cellulase-free, thermo-alkali-solvent stable biocatalyst being important tool for many biotechnological industries. To the best of our knowledge, this is the first report on the production of xylanase by this Bacillus species.

Xylanase and itaconic acid production by Aspergillus terreus NRRL 1960 within a biorefinery concept

Production of two industrially important products, xylanase and itaconic acid (IA), by Aspergillus terreus NRRL 1960 from agricultural residues was investigated within a biorefinery concept. Biological pretreatment was applied to lignocellulosic materials by using A. terreus, which produced xylanase while growing on agricultural residues. For IA production, already grown cells were transferred into a new medium. The first step provided not only the pretreatment of lignocellulosic material in order to be used as feedstock but also production of xylanase. For this purpose, cotton stalk, sunflower stalk and corn cob were used as carbon sources as lignocellulosic material. Among them, the highest xylanase production was obtained on corn cob. By application of two-step fermentation, about 70 IU/mL xylanase and 18 g/L IA production levels were achieved. This study shows the stepwise usage potential of the microorganism as a tool in a biorefinery concept.

Enhancing xylanases production by a new Amazon Forest strain of Aspergillus oryzae using solid-state fermentation under controlled operation conditions

Bioprocess studies towards improving the production of xylanases are of great value due to the importance of these enzymes on several industrial applications. This work evaluates the effects of operational conditions on xylanase production by a new strain of Aspergillus oryzae (P6B2) isolated from the Amazon Rain Forest. The fungus was cultivated under solid-state fermentation (SSF) using an instrumented lab-scale bioreactor equipped with an on-line automated monitoring and control system. The effects of initial substrate moisture content and temperature on xylanase production were evaluated and compared to static conditions. It was outstanding the positive effect on xylanase production by SSF cultivations under controlled conditions of forced over static aeration conditions. The conditions selected for further characterization resulted in the highest production of xylanase (2830.7IU/g), which was achieved using an initial substrate moisture content of 80%, at 28°C with an air flow rate of 20mL/min and an inlet air humidity of 80%. Results of respirometric analysis showed good agreement with enzyme production, suggesting that these data could be very useful for optimization and scale-up studies.

Produksi Xilanase dari Isolat Sumber Air Panas Sonai, Sulawesi Tenggara, menggunakan Limbah Pertanian

Xylanase is the enzyme with prospec for hydrolysis hemicellulases. Many industry use xylanase thermostable. This processes require enzymes which are operationally stable at high temperature thus allowing e.g. easy mixing, better substrate solubility, high mass transfer rate, and lower risk of contamination. Thermophiles have often been proposed as sources of industrially relevant thermostable enzymes. Thermophilic bacteria, live at hot-springs, are can produced thermostable enzymes. In this work, we studied the bacteria strains isolated from water collected in the Sonai hot-springs of Southeast Sulawesi region and condition production enzyme. Screaning bacteria xylanolitic use semi-quantitative detection at agar plate with xilan, and quantitative detection activity enzyme. Result experinces in the 28 isolates screened, isolat IIA-3 (Pseudomonas sp.) showed the highest xylanase production. Organism efficiently used 2% rice straw as substrates. Pseudomonas sp. was used to hydrolyses rice straw at 50oC, agitation 150 rpm and pH 9.

Highly thermo–halo–alkali-stable β-1,4-endoxylanase from a novel polyextremophilic strain of Bacillus halodurans

A novel bacterial isolate, capable of producing extracellular highly thermostable, halo-alkali-stable and cellulase-free xylanase, was isolated from soil and identified as Bacillus halodurans TSPV1 by polyphasic approach. The Plackett-Burman design identified wheat bran, lactose, tryptone and NaCl as the factors that significantly affect xylanase production, and thus, these were optimized by response surface methodology. The data analysis suggested that optimum levels of wheat bran (15-20gL(-1)), lactose (1.0-1.5gL(-1)), tryptone (2-2.5gL(-1)) and NaCl (7.0-8.0gL(-1)) support 6.75-fold higher xylanase production than that in the un-optimized medium. The xylanase is optimally active at 90°C and pH 10, and stable for 4h at 90°C (T 1/2 60h) over a broad range of NaCl concentrations (0-29%). This is the first report on the isolation of polyextremophilic B. halodurans strain that produces thermo-halo-alkali-stable xylanase in submerged fermentation. This enzyme efficiently saccharifies agro residues like wheat bran and corncobs. Fifty-six percent of hemicellulose of wheat bran could be hydrolyzed by xylanase (100Ug(-1)substrate) along with cellulase (22UFPase and 50UCMCaseg(-1)). The xylanase, being thermo-alkali stable and cellulase free, can find applications in pre-bleaching of paper pulps and hydrolysis of xylan in agricultural residues.

Improved xylanase production using apple pomace waste by Aspergillus niger in koji fermentation

Xylanase production by Aspergillus niger NRRL‐567 in solid‐state fermentation (koji fermentation) was optimized using 24 factorial design and response surface methodology. The evaluated variables were the initial moisture level and concentration of inducers [veratryl alcohol (VA), copper sulphate (CS), and lactose (LAC)], leading to the response of xylanase production. Initial moisture level and LAC were found to be the most significant variable for xylanase production (p<0.05). The highest xylanase production was observed with 3578.8 ± 65.3 IU/gds (gram dry substrate) under optimal conditions using initial moisture of 85% (v/w), pH 5.0 and inducers VA (2 mM/kg), LAC 2% (w/w), and CS (1.5 mM/kg) after 48 h of incubation time. Higher xylanase activity of 3952 ± 78.3 IU/gds was attained during scale‐up of the process in solid‐state tray fermentation under optimum conditions after 72 h of incubation time. The present study demonstrates that A. niger NRRL‐567 can efficiently be used to achieve xylanase production with an economical and environmental benefit in solid‐state tray fermentation. The developed process can be used to develop an effective process for commercially feasible bioproduction of xylanases for speciality applications, such as conversion of lignocellulosic biomass to biofuels and other value‐added products.

Examining the Potential of Plasma-Assisted Pretreated Wheat Straw for Enzyme Production by Trichoderma reesei

Plasma-assisted pretreated wheat straw was investigated for cellulase and xylanase production by Trichoderma reesei fermentation. Fermentations were conducted with media containing washed and unwashed plasma-assisted pretreated wheat straw as carbon source which was sterilized by autoclavation. To account for any effects of autoclavation, a comparison was made with unsterilized media containing antibiotics. It was found that unsterilized washed plasma-assisted pretreated wheat straw (which contained antibiotics) was best suited for the production of xylanases (110 IU ml(-1)) and cellulases (0.5 filter paper units (FPU) ml(-1)). Addition of Avicel boosted enzyme titers with the highest cellulase titers (1.5 FPU ml(-1)) found with addition of 50 % w/w Avicel and with the highest xylanase production (350 IU ml(-1)) reached in the presence of 10 % w/w Avicel. Comparison with enzyme titers from other nonrefined feedstocks suggests that plasma pretreated wheat straw is a promising and suitable substrate for cellulase and hemicellulase production.

Paddy Husk as Support for Solid State Fermentation to Produce Xylanase from Bacillus pumilus

To optimize culture conditions for xylanase production by solid state fermentation (SSF) using Bacillus pumilus, with paddy husk as support, solid medium contained 200 g of paddy husk with 800 mL of liquid fermentation medium [xylan, 20.0 g/L; peptone, 2.0 g/L; yeast extract, 2.5 g/L; K 2HPO 4, 2.5 g/L; KH 2PO 4, 1.0 g/L; NaCl, 0.1 g/L; (NH 4) 2SO 4, 2.0 g/L, CaCl 2·2H 2O, 0.005 g/L; MgCl 2·6H 2O, 0.005 g/L; and FeCl 3, 0.005 g/L] at pH 9.0 was applied. The highest xylanase activity (142.0 ±0.47 U/g DM] was obtained on the 6 th day at 30°C. The optimized paddy husk to liquid fermentation medium ratio was 2:9, and the optimized culture temperature was 40°C. When commercial Birchwood xylan was replaced with different concentrations of corncob, xylanase production was maximized (224.2 U/g DM) in the medium with 150 g/L corncob. Xylanase production was increased by sucrose, fructose and arabinose, whereas reduced by glucose, galactose, lactose and amylose. When organic nitrogen sources were replaced with locally available nitrogen sources such as groundnut powder or sesame seedcake powder or coconut seedcake powder or soy meal powder, the highest xylanase production (290.7 U/g DM) was obtained in the medium with soy meal powder and 16.0 g/L of soy meal powder was the optimum (326.5±0.34 U/g DM). Based on the optimization studies, B. pumilus produced 2.3 times higher xylanase activity. The medium cost was reduced from 2 458.3 to 178.3 SLR/kg and the total activity which could be obtained from 1 kg of the medium was increased from 48 624 to 220 253 Units.

Extracellular Xylanase Production by Fusarium Species in Solid State Fermentation

Fusarium sp. has been shown to be a promising organism for enhanced production of xylanases. In the present study, xylanase production by 21 Fusarium sp. isolates (8 Fusarium culmorum, 4 Fusarium solani, 6 Fusarium verticillioides and 3 Fusarium equiseti) was evaluated under solid state fermentation (SSF). The fungal isolate Fusarium solani SYRN7 was the best xylanase producer among the tested isolates. The effects of some agriculture wastes (like wheat straw, wheat bran, beet pulp and cotton seed cake) and incubation period on xylanase production by F. solani were optimized. High xylanase production (1465.8 U/g) was observed in wheat bran after 96 h of incubation. Optimum pH and temperature for xylanase activity were found to be 5 and 50 degrees C, respectively.

Thermostable xylanase production and partial purification by solid-state fermentation using agricultural waste wheat straw

A locally isolated strain of Trichoderma harzianum was studied for production of xylanase (EC 3.2.1.8) using lignocellulosic substrates for solid-state fermentation. Among the different substrates used, wheat straw produced the highest yields (146 IU/ml). The influence of temperature, pH, moistening agents, moisture level, carbon sources, nitrogen sources, pretreatments and metal ions were evaluated with respect to xylanase production. Highest xylanase production was obtained using wheat straw after 12 days of incubation. Different substrate combination ratios and the effect of particle size were also checked against xylanase production. Maximum xylanase production was observed in a wheat straw/rice straw ratio of 1:1, with a particle size of 0.45–0.5 mm. Media supplementation with xylose as a carbon source in a ratio 1:5 (carbon source/substrate) gave maximum activity (157 IU/ml). Nitrogen supplementations from yeast extract produced xylanase activity of 141 IU/ml. Optimum xylanase production was observed at 30°C and pH 5.0. Xylanase production was enhanced in the presence of Ca2+ and Zn2+ ions. Ammonium sulfate fractionation (20–80% saturation) of partially purified xylanase yielded 76.5% of the enzyme with a 3.53-fold purification. The molecular weight of xylanase was found to be ca. 29,000 Da by SDS–PAGE.

Production of Cellulase from Kraft Paper Mill Sludge by Trichoderma Reesei Rut C-30

Paper mill sludge is a solid waste material generated from pulping and papermaking operations. Because of high glucan content and its well-dispersed structure, paper mill sludges are well suited for bioconversion into value-added products. It also has high ash content originated from inorganic additives used in papermaking, which causes hindrance to bioconversion. In this study, paper mill sludges from Kraft process were de-ashed by a centrifugal cleaner and successive treatment by sulfuric acid and sodium hydroxide, and used as a substrate for cellulase production. The treated sludge was the only carbon source for cellulase production, and predominantly inorganic nutrients were used as the nitrogen source for this bioprocess. The cellulase enzyme produced from the de-ashed sludge exhibited cellulase activity of 8 filter paper unit (FPU)/mL, close to that obtainable from pure cellulosic substrates. The yield of cellulase enzyme was 307 FPU/g glucan of de-ashed sludge. Specific activity was 8.0 FPU/mg protein. In activity tests conducted against the corn stover and alpha-cellulose, the xylanse activity was found to be higher than that of a commercial cellulase. Relatively high xylan content in the sludge appears to have induced high xylanase production. Simultaneous saccharification and fermentation (SSF) was performed using partially de-ashed sludge as the feedstock for ethanol production using Sacharomyces cerevisiae and the cellulase produced in-house from the sludge. With 6% (w/v) glucan feed, ethanol yield of 72% of theoretical maximum and 24.4 g/L ethanol concentration were achieved. These results were identical to those of the SSF using commercial cellulases.

Kinetics of Xylanase Fermentation by Recombinant <i>Escherichia coli</i> DH5α in Shake Flask Culture

Problem statement: Interest in xylanase enzyme application has markedly increased in pulp and paper processing industries. The switch to xylanase-producing recombinant Escherichia coli DH5α pTP510 is seen here as an economic alternative towards higher productivity and easier downstream purification. Modeling of E. coli DH5α growth and enzyme secretion is thus desired for future optimization in fermentation process. Approach: Kinetics of intracellular xylanase fermentation by a recombinant E. coli DH5α was studied in shake flask culture. The effect of different medium formulations (complex, minimal and defined), initial pH (6.5, 7.0, 7.4 and 8.0) and agitation speeds (150, 200 and 250 rpm) on cell growth and xylanase production were evaluated. Mathematical models based on Logistic and Luedeking-Piret equations had been proposed to describe the microbial growth and xylanase production. Results: Highest xylanase production was obtained in defined medium. Based on medium formulation, the highest cell concentration (4.59 g L-1) and xylanase production (2, 122.5 U mL-1) was obtained when (NH4)2HPO4 was used as the main nitrogen source, with an adjustment of the initial pH to 7.4 and agitation speed of 200 rpm. The maximum specific growth rate (µmax), growth associated xylanase production coefficient (α) and non-growth associated xylanase production coefficient (β) was 0.41 h-1, 474.26 U mg cell-1and 0 U mg cell-1 h-1, respectively. Conclusion: Xylanase production was growth associated process and the enzyme secretion was greatly dependent on cell concentration and the specific growth rate of E. coli DH5α.

털두꺼비하늘소 (Moechotypa diphysis)로부터 Xylanase를 생산하는 Paenibacillus sp. HY-8 균주의 분리 및 특성

Xylan이 풍부한 식물체를 먹이로 하는 하늘소의 장내에 존재하는 xylanase 생산 미생물의 탐색 과정에서 털두꺼비하늘소 (Moechotypa diphysis) 성충의 장으로부터 우수한 xylanase 생산균주 Paenibacillus sp. HY-8을 분리하였다. 생화학적, 계통학적 분석결과를 바탕으로 이 분리균은 Paenibacillus 속에 속하는 종으로 분석되었다. HY-8 균주에서 xylanase 생산은 제한배지에 xylan을 첨가함으로써 유도되는 특성을 나타내었고 1% 의 yeast extract와 0.5%의 birchwood xylan이 포함된 M9 배지에서 <TEX>$25^{\circ}C$</TEX>, 24시간의 배양에 의해 xylanase의 생산이 최대치에 도달하였다. HY-8 균주가 생산하는 xylanase는 pH6.0에서 여러 가지 식물성 사료의 원료에 대하여 대조구로 사용된 Tricoderma sp. 유래의 xylanase에 비해 우수한 분해능을 나타내었다. From the course of screening of useful xylanase producing microorganism from a phytophagous longicorn beetle, we isolated an extra-cellular xylanase producing strain, Paenibacillus sp. HY-8 from the intestine of Moechotypa diphysis adult. On the basis of morphological, biochemical and phylogenetic studies of the new isolate was identified as a Paenibacillus species. Production of xylanase in this strain was strongly induced by adding xylan to the growth medium and repressed by glucose or xylose. The highest xylanase production was attained in the M9 media containing 1% yeast extract and 0.5% birchwood xylan when cultured at <TEX>$25^{\circ}C$</TEX> for 24 hrs. HY-8 producing xylanase showed superior hydrolytic activities against various plant source feedstuff than control xylanase produced by Tricoderma sp. at pH 6.0.

Production and biochemical characterization of xylanase from an alkalitolerant novel species Aspergillus niveus RS2

The novel fungus Aspergillus niveus RS2 isolated from rice straw showed relatively high xylanase production after 5 days of fermentation. Of the different xylan-containing agricultural by-products tested, rice husk was the best substrate; however, maximum xylanase production occurred when the organism was cultured on purified xylan. Yeast extract was found to be the best nitrogen source for xylanase production, followed by ammonium sulfate and peptone. The optimum pH for maximum enzyme production was 8 (18.2 U/ml); however, an appreciable level of activity was obtained at pH 7 (10.9 U/ml). Temperature and pH optima for xylanase were 50°C and 7.0, respectively; however the enzyme retained considerably high activity under high temperature (12.1 U/ml at 60°C) and high alkaline conditions (17.2 U/ml at pH 8 and 13.9 U/ml at pH 9). The enzyme was strongly inhibited by Hg2+, while Mn2+ was slight activator. The half-life of the enzyme was 48 min at 50°C. The enzyme was purified by 5.08-fold using carboxymethyl-sephadex chromatography. Zymogram analysis suggested the presence of a single candidate xylanase in the purified preparation. SDS-PAGE revealed a molecular weight of approximately 22.5 kDa. The enzyme had K m and V max values of 2.5 and 26 μmol/mg per minute, respectively.

Mutant of a Xylanase-producing Strain of Aspergillus niger in Solid State Fermentation by Low Energy Ion Implantation

In order to obtain an industrial strain with higher xylanase production, the original strain of Aspergillus niger was mutated by N+ implantation (10 keV, 1.04 · 1015–2.08 · 1015 ions/cm2), and a mutant P602 was obtained. The mutant showed xylanase production increased from 2989 to 6320 IU/g and the fermentation time was decreased from 72 h to 64 h. The fermentation temperature range was 28–32 °C which was a little wider than the original strain.

Xylanolytic complex from Aspergillus giganteus: production and characterization.

An Aspergillus giganteus strain was isolated as an excellent producer of xylanase associated with low levels of cellulase. Optimal xylanase production was obtained in liquid Vogel medium containing xylan as carbon source, pH 6.5 to 7.0, at 25 degrees C and under shaking at 120 rpm during 84 h. Among the several carbon sources tested, higher xylanase production was verified in xylan, xylose, sugar-cane bagasse, wheat bran and corn cob cultures, respectively. Optimal conditions for activity determination were 50 degrees C and pH 6.0. The xylanolytic complex of A. giganteus showed low thermal stability with T(50) of 2 h, 13 min and 1 min when it was incubated at 40, 50 and 60 degrees C, respectively, and high stability from pH 4.5 to 10.5, with the best interval between 7.0 to 7.5. This broad range of stability in alkali pH indicates a potential applicability in some industrial processes, which require such condition. Xylanolytic activity of A. giganteus was totally inhibited by Hg(+2), Cu(+2) and SDS at 10 mM. The analysis of the products from the oat spelts xylan hydrolysis through thin-layer chromatography indicated endoxylanase activity, lack of debranching enzymes and beta-xylosidase activity in assay conditions.