Production Of Cellulase-free Xylanase Research Articles

Melioration of Paddy Straw to produce cellulase-free xylanase and bioactives under Solid State Fermentation and deciphering its impact by Life Cycle Assessment

Aiming towards zero waste management of Paddy straw (PS), the study offers a novel route for production of cellulase-free xylanase, using consortia of Trichoderma spp. under Solid State Fermentation (SSF) of PS valorized using nitrogen rich de-oiled neem cake (NC). Life Cycle Assessment (LCA) for enzyme production, performed using SimaPro software, depicted adverse impacts due to electricity consumption (92.84%) and use of ammonium sulphate salt (6.17%). Nonetheless, employing renewable energy and reducing salt consumption could help minimize these impacts. OHR-LCMS study of the partially purified enzyme revealed the presence of β-xylanase and α-L-Arabinofuranosidase. Enzymatic saccharification of various substrates enhanced the release of reducing sugars (mg/g) from corn cob (137.54 ± 0.96), pine needle (41.43 ± 1), sugarcane bagasse (105.17 ± 0.7), and PS (76.66 ± 1.29), demonstrating its applicability in the biofuel domain. LC-MS, ICMPS, and EDX profiling of the residual spent unravelled the manifestation of bioactives, minerals, and silica, playing an essential role as biopesticide and biofertilizer.

Optimization of medium composition for cellulase-free xylanase production by solid-state fermentation on corn cob waste by Aspergillus niger DX-23

Economical and higher production of cellulase-free xylanase(s) is needed for their wider application in paper and pulp industries. For enzyme production at industrial scale, the growth medium contributes up to 40% of the total production cost. The large quantity of xylan-rich corn cob biomass generated worldwide from corn-processing industries can be used as a cheaper substrate for the production of xylanase. In the present study, the optimization of the production of xylanase by Aspergillus niger DX-23 was studied under solid-state fermentation (SSF) using the Plackett-Burman design (PBD) and response surface methodology (RSM). Among eleven factors investigated (each at two levels) in PBD, corn cob powder, KH2PO4, yeast extract, Tween 20, FeSO4.7H2O, MgSO4.7H2O, CoCl2, MnSO4.H2O and ZnSO4∙H2O significantly (p < 0.05) influenced the production of xylanase by A. niger DX-23. Subsequently, RSM involving central composite design (CCD) was adopted to determine the optimum levels of corn cob powder, NaNO3 and KH2PO4 and FeSO4. According to ANOVA for xylanase production, for CCD-I although the quadratic model developed was significant (p = 0.026465), the model also showed significant lack-of-fit (p = 0.004056) and low predicted R2. Hence, CCD-II which included two variables, i.e. corn cob powder and NaNO3, was carried out. For CCD-II, the quadratic model developed was significant (p = 0.007753), lack-of-fit value was non-significant (p = 0.677031) and the predicted R2 was 0.51. Based on CCD-II, the optimum levels of corn cob powder and NaNO3 in the medium were found to be 150.0 g/l and 5.1 g/l, respectively. To confirm the accuracy of the model, when xylanase production by A. niger DX-23 was studied in an optimized medium under SSF conditions, the xylanase yield reached 306.12 ± 7.4 U/g after 10 days of growth which agreed fairly well with the predicted value (290.15 U/g). Hence, the quadratic model created could be considered to be accurate and reliable for predicting the production of xylanase by A. niger DX-23 under SSF. Moreover, when the effect of pH and inoculum concentration on xylanase production was investigated, in an optimized medium, the maximum production of xylanase was obtained at pH 5.0 and at an inoculum level of 5.0 × 106 spores/ml. When the time course of the fermentation was followed, A. niger DX-23 produced maximum xylanase (496.9 ± 3.0 U/g) after 6 days of fermentation.

Partial purification and characterization of Cellulase-free xylanase production from bacillus licheniformis

A new isolate of Bacillus licheniformis SC produced extracellular xylanase when grown on 3% wheat bran, without xylanase multiplicity. The enzyme was partially purified by ammonium sulphate and gel filtration using Sephadex G-100 column. The purified xylanase showed optimum pH at 8.0 and half maximum activity at pH 4.5. The pH stability profile showed that it was more stable in the pH range of 6 to 8. Its activity increased with rise in temperature and maximum upto 60 0 C. The molecular mass for xylanase was 24.6 kDa respectively. The SDS-PAGE of the purified xylanase migrated as a single band. The Km was found to be 4.8 g/ml and the Vmax was 0.58 µmol/minute/ml for xylanase. In addition, the present xylanase was capable of producing high-quality xylo-oligosaccharides, indicating its application potential for biobleaching processes of pulp.

Statistical optimization of thermo-alkali stable xylanase production from Bacillus tequilensis strain ARMATI

Background: Xylanase from bacteria finds use in prebleaching process and bioconversion of lignocelluloses into feedstocks. The xylanolytic enzyme brings about the hydrolysis of complex biomolecules into simple monomer units. This study aims to optimize the cellulase-free xylanase production and cell biomass of Bacillus tequilensis strain ARMATI using response surface methodology (RSM). Results: Statistical screening of medium constituents and the physical factors affecting xylanase and biomass yield of the isolate were optimized by RSM using central composite design at N = 30, namely 30 experimental runs with 4 independent variables. The central composite design showed 3.7 fold and 1.5 fold increased xylanase production and biomass yield of the isolate respectively compared to ‘one factor at a time approach', in the presence of the basal medium containing birchwood xylan (1.5% w/v) and yeast extract (1% w/v), incubated at 40oC for 24 h. Analysis of variance (ANOVA) revealed high coefficient of determination (R2) of 0.9978 and 0.9906 for the respective responses at significant level (p Conclusions: The cellulase-free xylanase showed an alkali-tolerant and thermo-stable property with potentially applicable nature at industrial scale. This statistical approach established a major contribution in enzyme production from the isolate by optimizing independent factors and represents a first reference on the enhanced production of thermo-alkali stable cellulase-free xylanase from B. tequilensis.

Utilization of Corn Cob Waste for Cellulase-Free Xylanase Production by Aspergillus niger DX-23: Medium Optimization and Strain Improvement

Aspergillus niger DX-23 produces a cellulase-free xylanase which showed efficient deinking of old newspaper pulp for recycled paper production. Towards economical production of the above xylanase when various agro-waste biomass were evaluated as substrates, under shake flask conditions, A. niger DX-23 produced highest amount of xylanase (59.5 ± 5.0 U/mL) using corn cob powder as substrate. A central composite design was used to optimize concentration of corn cob powder, NaNO3 and KH2PO4 in the medium for maximum xylanase production. The optimum concentration of the above nutrients were determined to be 37.0 g/L corn cob powder, 2.5 g/L of NaNO3 and 1.0 g/L of KH2PO4 at which level xylanase yield of 110.4 U/mL was obtained, which was 82.9 % more than the yield obtained in unoptimized medium. Moreover, under shake flask conditions, 5.0 % (v/v) of inoculum, pH of 5.0 and incubation time of 84 h was found to be suitable for maximum xylanase production. At laboratory fermentor level, A. niger DX-23 produced 79.4 U/mL (after 96 h) and 117.9 U/mL (after 72 h) xylanase using untreated corncob powder and alkali treated corn cob powder, respectively. In order to obtain higher xylanase procuring strains, A. niger DX-23 was mutagenized using UV rays. In optimized medium mutant strain of A. niger produced 150.9 ± 3.4 U/mL of xylanase which was 118.0 % higher than the xylanase yield obtained for parent type in unoptimized medium (60.2 ± 2.6 U/mL). The above results suggested effectiveness of the combined strategy of medium optimization and mutation towards enhanced production of xylanase by A. niger.

Optimization of alkali-thermostable and cellulase-free eylanase production from Bacillus Sp.

Context: To analyze the nutritional and physicochemical parameters for the production of alkali-thermostable and cellulase free xylanase from bacteria. Objectives: The aim of this study was to isolation and identification and of alkali-thermostable and cellulase free xylanase producing bacteria from soil as well as optimization of process parameters for xylanase production. Materials and Methods: The bacterium Bacillus sp. was isolated from soil by serial dilution technique on xylan agar medium and identified by morphological and biochemical studies. The production of xylanase was carried out on xylan broth medium and xylanase activity was assayed by dinitrosalicylic acid (DNS) method. The effect of cultural parameters on the production of xylanase was determined by measuring the activity of xylanase. The effect of temperature and pH on the activity of partially purified xylanase as well as substrate specificity of xylanase were examined. Results: The maximum xylanase production (4000 U/L) by a Bacillus sp. was attained when the medium containing 0.5% wheat bran xylan and peptone at pH 8.0 and 50-55°C within 48-60 h. The partially purified xylanase was optimally active at pH 9.0 and 55°C. The xylanase showed high substrate activity towards wheat bran xylan but no activity towards cellulose, carboxymethyl cellulose and starch. Thus the enzyme was alkali-thermostable and cellulase free xylanase. Conclusion: The results obtained in this study suggest that the Bacillus sp. used is highly potential and useful for the production of cellulase free xylanase. DOI: http://dx.doi.org/10.3329/jbs.v19i0.12994 J. bio-sci. 19: 7-14, 2011

Optimization of cellulase-free xylanase production by alkalophilic Cellulosimicrobium sp. CKMX1 in solid-state fermentation of apple pomace using central composite design and response surface methodology

Microbial xylanases and associated enzymes degrade the xylans present in lignocellulose in nature. Xylanase production by Cellulosimicrobium sp. CKMX1, isolated from mushroom compost, produced a cellulase-free extracellular endo-1, 4-β-xylanase (EC 3.2.1.8) at 35 °C and pH 8.0. Apple pomace—an inexpensive and abundant source of carbon—supported maximal xylanase activity of 500.10 U/g dry bacterial pomace (DBP) under solid state fermentation. Culture conditions, e.g., type of medium, particle size of carbon source, incubation period, temperature, initial pH, and inoculum size, were optimized and xylanase activity was increased to 535.6 U/g DBP. CMCase, avicelase, FPase and β-glucosidase activities were not detected, highlighting the novelty of the xylanase enzyme produced by CKMX1. Further optimization of enzyme production was carried out using central composite design following response surface methodology with four independent variables (yeast extract, urea, Tween 20 and carboxymethyl cellulose), which resulted in very high levels of xylanase (861.90 U/g DBP). Preliminary identification of the bacterial isolate was made on the basis of morphological and biochemical characters and confirmed by partial 16Sr RNA gene sequencing, which identified CKMX1 as Cellulosimicrobium sp. CKMX1. A phylogenetic analysis based on the 16Sr RNA gene sequence placed the isolate within the genus Cellulosimicrobium, being related most closely to Cellulosimicrobium cellulans strain AMP-11 (97% similarity). The ability of this strain to produce cost-effective xylanase from apple pomace on a large scale will help in the waste management of apple pomace.

Optimization of Cellulase-Free Xylanase Production by Thermophilic Streptomyces thermovulgaris TISTR1948 through Plackett-Burman and Response Surface Methodological Approaches

Cellulase-free xylanase production by thermophilic Streptomyces thermovulgaris TISTR1948 was cultivated in a basal medium with rice straw as sole source of carbon and as an inducible substrate. Variable medium components were selected in accordance with the Plackett-Burman experimental design. The optimization conditions of physical factors (pH and temperature levels) were then combined in further studies through the response surface methodology approach. Only two significant components, rice straw and yeast extract, were chosen for the optimization studies. A second-order quadratic model was constructed by central composite design (CCD). The model revealed that both pH and temperature levels were significant, and were dependent on xylanase production. Under these experimental designs, the xylanase yield increased from 51.11 to 274.49 U/mL (3,400 to 10,000 U/g of rice straw) or about 537% higher than an unoptimized basal medium. The optimum conditions to achieve maximum yield of xylanase were 27.45 g/L of rice straw and 5.42 g/L of yeast extract under relatively neutral conditions of pH 7.11, 50.03 °C, and a incubation period.

Thermoactive cellulase-free xylanase production from alkaliphilic Bacillus strains using various agro-residues and their potential in biobleaching of kraft pulp

The four bacterial strains were isolated on media containing xylan and screened for xylanase activity. The bacterial strains (Ag12, Ag13, Ag20 and Ag32) were characterized based on morphological, biochemical and physiological characters and identified as belonging to the genus Bacillus. The effects of different factors such as pH (7.0 – 10.0), temperature (25.0 – 50.0°C) and inexpensive agro-residues (wheat straw, wheat bran and corncob) on xylanase production of strains were studied under shake flask conditions. Maximal enzyme activities were obtained by cultivation in birch-wood xylan, but high enzyme production was also obtained on wheat straw and corncob when cultivated at pH 8.5. Under optimized fermentation conditions, no cellulolytic activity were detected on the crude extracts. The effects of temperature (40.0 – 80.0°C), pH (6.0 – 10.0) and salt concentration (1.0, 5.0 and 10.0%) on the xylanases activity were determined. The maximum activity was obtained temperature 60.0°C and pH at 9.0. The enzyme was stable at 60.0°C for more than 60 min, suggesting that the xylanases of Bacillus strains are thermoactive and being of interests for biobleaching processes. The effectiveness of crude xylanases from the strains Ag12, Ag20 and Ag32 on kraft pulp were carried out at pH 9.0 at 60.0°C. Biobleaching studies of kraft pulp with xylanases and its subsequent treatment with 1.0% EDTA (30 min at 50.0°C) and peroxide (80 min at 70.0°C), showed that the enzymes reduced the kappa number by 27.4, 61.7 and 75.3% and enhanced the brightness by 1.0, 1.5 and 3.0% from xylanases produced by strains Ag12, Ag20 and Ag32, respectively. These results suggest that the application of this xylanases to the paper and pulp industry may be very promising.

Hyper production of cellulase-free xylanase by Thermomyces lanuginosus SSBP on bagasse pulp and its application in biobleaching

A cellulase-free xylanase production by Thermomyces lanuginosus SSBP using bagasse pulp was examined under submerged (SmC) and solid-state cultivation (SSC). Higher level of xylanase activity (19,320 +/- 37 U g(-1) dried carbon source) was obtained in SSC cultures than in SmC (1,772 +/- 15 U g(-1) dried carbon source) after 120 h with 10% inoculum. The biobleaching efficacy of crude xylanase was tested on bagasse pulp, and the maximum brightness of 46.1 +/- 0.06% was observed with 50 U of crude xylanase per gram of pulp, which was 3.8 points higher than the brightness of untreated samples. Reducing sugars (26 +/- 0.1 mg g(-1)) and UV-absorbing lignin-derived compounds in the pulp filtrates were observed as maximum in 50 U of crude xylanase-treated samples. T. lanuginosus SSBP has potential applications due to its high productivity of xylanase and its efficiency in pulp bleaching.

Production of Cellulase-Free Xylanase from a Novel Yeast Strain Used for Biobleaching in Paper Industry

Production of Cellulase-Free Xylanase from a Novel Yeast Strain Used for Biobleaching in Paper Industry

Production of cellulase-free xylanase by Trichoderma reesei SAF3

A xylanase producing fungi has been isolated from soil and identified as Trichoderma reesei SAF3. Maximum growth of the organism was found at 48 h under submerged condition in xylan containing enriched medium, whereas highest enzyme production (4.75U/mL) was recorded at 72 h. No detectable cellulase activity was noted during whole cultivation period. The partially purified enzyme hydrolyzed xylan into xylopentose and xylose. All these properties of xylanase highlighten its promising uses in industrial scale.

Alkali-thermostable and cellulase-free xylanase production by an extreme thermophile Geobacillus thermoleovorans

The optimization of cultural variables resulted in a marked enhancement in the secretion of cellulase-free and alkali-thermostable xylanase (EC 3.2.1.8) by an extreme thermophile Geobacillus thermoleovorans. The enzyme secretion was enhanced when the medium was supplemented with xylan (0.15%) and Tween-80 (0.1% v/v). In wheat bran-tryptone medium, the peak in enzyme production was attained within 42 h in a fermenter as compared to 72 h in shake flasks. Optimization of the culture conditions resulted in a 7.72-fold enhancement in enzyme production. The cellulase-free xylanase was optimally active at pH 8.5 and 80°C, and it was found to be useful in the pre-bleaching process of paper pulps.

Production of Cellulase-Free Xylanase from Bacillus megaterium by Solid State Fermentation for Biobleaching of Pulp

Xylanase production from B. megaterium was enhanced using solid state fermentation with respect to the use of solid substrate, moistening solution, moisture content, inoculum, sugars, soyabean meal, amino acids, and extraction with surfactant. An increase of approximately 423-fold in xylanase production and complete suppression of CMCase production was achieved over submerged liquid fermentation. Biobleaching using this cellulase-free xylanase, 8 U/g of oven dried pulp of 10% consistency, showed 8.12% and 1.16% increase in brightness and viscosity, 13.67% decrease in kappa number, and 31% decrease in chlorine consumption at the CD stage.

High-level of xylanase production by the thermophilic Paecilomyces themophila J18 on wheat straw in solid-state fermentation

The production of extracellular xylanase by a newly isolated thermophilic fungus, Paecilomyces themophila J18, on the lignocellulosic materials was studied in solid-state fermentation (SSF). The strain grew well at 50 °C and produced a high-level of xylanase activity using the selected lignocellulosic materials, especially wheat straw. Production of xylanase by P. themophila J18 on wheat straw was enhanced by optimizing the particle size of wheat straw, nitrogen source, initial moisture level, growth temperature and initial pH of the culture medium. Under the optimized conditions, yield as high as 18 580 U g −1 of carbon source of xylanase was achieved. No CMCase activity was observed. The xylanase exhibited remarkable stability and retained more than 50% of its original activity at 70 °C for 4 h at pH 7.0–8.0. Therefore, P. themophila J18 could to be a promising microorganism for thermostable, cellulase-free xylanase production in SSF.

Optimization of Aeration and Agitation Rates to Improve Cellulase-Free Xylanase Production by Thermotolerant Streptomyces sp. Ab106 and Repeated Fed-Batch Cultivation Using Agricultural Waste

Thermostable cellulase-free xylanase was produced by Streptomyces sp. Ab106 using agricultural waste, sugar cane bagasse, as the substrate at 50°C and pH 7.0. The central composite faccentered experimental design was applied to evaluate the optimal agitation and aeration rates in a 5-l fermentor. The highest activity (16.0±0.5 IU/ml) was obtained at an aeration rate of 1 vvm and an agitation rate of 150 rpm (kL a = 351 h−1). Using the repeated fed-batch cultivation technique, the maximum xylanase activity of 32±1 IU/ml was obtained during the second cycle of repeated fed-batch culture.

Optimization of aeration and agitation rates to improve cellulase-free xylanase production by thermotolerant Streptomyces sp. Ab106 and repeated fed-batch cultivation using agricultural waste

Thermostable cellulase-free xylanase was produced by Streptomyces sp. Ab106 using agricultural waste, sugar cane bagasse, as the substrate at 50°C and pH 7.0. The central composite faccentered experimental design was applied to evaluate the optimal agitation and aeration rates in a 5- l fermentor. The highest activity (16.0±0.5 IU/ml) was obtained at an aeration rate of 1 vvm and an agitation rate of 150 rpm ( k L a = 351 h −1). Using the repeated fed-batch cultivation technique, the maximum xylanase activity of 32±1 IU/ml was obtained during the second cycle of repeated fed-batch culture.

Optimization of thermostable and alkaline-tolerant cellulase-free xylanase production from agricultural waste by thermotolerant Streptomyces sp. Ab106, using the central composite experimental design

Cellulase-free xylanase was produced by Streptomyces sp. Ab106 from the agricultural waste cane bagasse. The effect of external factors pH and temperature on the xylanase production was studied and the optimization by using the central composite experimental design was investigated. The effects of pH and temperature were significant for the xylanase production. A second-order quadratic model and a response surface method showed that the optimum condition for xylanase production was 50 °C and pH 7. The maximum yield of xylanase was about 15 IU without cellulase and manannase activities.

Production of cellulase-free xylanase by a thermotolerant Streptomyces sp. grown on agricultural waste and media optimization using mixture design and Plackett–Burman experimental design methods

Cellulase-free xylanase was produced by Streptomyces sp. Ab106 on finely ground cane bagasse at 55 °C. The optimal medium composition was developed by applying the mixture design and linear mathematical program, and evaluated using the Plackett–Burman experimental design. The best composition of basal medium was found by using the mixture design method. The highest xylanase activity, 10.6 IU, was obtained after 6 days of fermentation in shaked flask at 100 rpm, 55 °C, pH 7. Both experimental designs showed that trace elements induced xylanase production. With fermentation in a 5-l fermenter, xylanase activity of 12.5 IU was achieved.

Thermostable, alkalophilic and cellulase free xylanase production by Thermoactinomyces thalophilus subgroup C

Thermoactinomyces thalophilus produced cellulase free extracellular endo-1,4-β-xylanase (EC 3.2.1.8) at 50°C and pH 8.5. Maximum xylanase production was achieved in fermentation medium using birchwood xylan as substrate after 96 h of growth at 50°C. Other agricultural substrates such as wheat bran, wheat straw, sugarcane bagasse and cornstover produced less xylanase. The crude enzyme preparation from mutant T. thalophilus P2 grown under optimised fermentation conditions showed no cellulase contamination and maximum xylanase activity of 42 U/ml at 65%deg;C and pH 8.5–9.0. This enzyme with initial xylanase activity of 42 U/ml was found thermostable up to 65°C and retaining 50% of its activity after its incubation for 125 min at 65°C.