Xylanase Production Research Articles

Comparison of common carbon sources with unhydrolyzed, dilute acid and steam hydrolyzed distillers’ dried grains with solubles for lignocellulolytic enzyme productions by fungal strains

Distillers’ dried grains with solubles (DDGS) are the byproduct of first-generation bioethanol production. DDGS has a high fiber content, which can be used for hydrolytic enzyme production after pretreatment by physical or chemical methods such as dilute acid or semi-continuous steam explosion. This study compares the treated DDGS samples to common carbon sources such as glucose, Avicel (crystalline cellulose), and untreated DDGS along with other cellulosic materials during the production of cellulases and xylanases by selected four fungal strains. Acid treated DDGS without filtration produced up to 0.72 IU/ml of cellulases and 36.44 IU/ml of xylanases. The results showed that acid hydrolysis was a better treatment than untreated, and steam treated DDGS for both cellulase and xylanase productions (p < 0.05). The comparison of enzyme productions with acid hydrolyzed DDGS to common carbon sources also showed that acid hydrolyzed DDGS was a significantly better carbon source than others. Among four fungal strains, two Aspergillus niger strains (NRRL 567 and NRRL 330) were better than the others in enzyme activity levels. The results confirm the significant positive effect of dilute acid treatment in making DDGS a feasible feedstock for hydrolytic enzyme production.

Bioprocessing of oilseed cakes by fungi consortia: Impact of enzymes produced on antioxidants release

Oilseed cakes (OC) present high potential as feedstock for the biobased industry. Biotechnological processes allow OC valorization by the production of diverse value-added products and simultaneously altering OC structure, improving their nutritional value, and boosting OC utilization in animal feed. This work explored the use of fungi consortium of two different species as a bioprocessing approach to improve the nutritional quality of OC, obtain enzymes and antioxidants by solid-state fermentation (SSF) of sunflower cake (SFC) and rapeseed cake (RSC). Rhyzopus oryzae and Aspergillus ibericus consortium led to the highest production of cellulase (135 U/g) and β-glucosidase (265 U/g) while maximum protease (228 U/g) was obtained with A. niger and R. oryzae consortium. Maximum xylanase production (886 U/g) was observed in SSF of RSC resulting in high hemicellulose reduction. The synergistic action of lignocellulosic enzymes resulted in extracts with increased antioxidant potential with possible application as food additives against oxidative stress.

Application of baby corn husk as a biological sustainable feedstock for the production of cellulase and xylanase by Lentinus squarrosulus Mont.

In an effort to use baby corn husk (BCH) as a sustainable feedstock for cellulase and xylanase production by the Lentinus squarrosulus Mont. isolate LS-YA (LSM-LS-YA), a suitable pretreatment method and fermentation strategies were developed. BCH pretreated with 1 M sodium hydroxide for 90 min, an alkaline pretreatment, exemplified an appropriate pretreatment method. In a 10-L external Venturi injector bioreactor, the highest cellulase and xylanase production was 4.12 ± 0.36 unit/mL and 6.15 ± 0.36 unit/mL, respectively, when 1 g/L diammonium hydrogen phosphate was used as the nitrogen source and the aeration rate was controlled at 0.2 vvm. This study provides an informative perspective on the production of cellulase and xylanase from agricultural lignocellulosic materials, which could reduce agricultural waste while supporting a zero-waste circular economy, and this fermentation process would be applicable to larger-scale production.

Biomass-Degrading Enzyme(s) Production and Biomass Degradation by a Novel Streptomyces thermocarboxydus.

Modern society has a great challenge to decrease waste and minimize the adverse effects of wastes on the economy, environment, and individual health. Thus, this study focuses on the use of eight agro-wastes (banana peel, barley straw, canola straw, pomegranate peel, orange peel, pumpkin pulp+seeds, maple leaf, and brewer's spent grains) by a novel bacterium (Streptomyces thermocarboxydus) for enzymes production. Further, the study explored the subsequent degradation of those wastes by the bacterium. This bacterium was isolated from forest soil and identified as Streptomyces thermocarboxydus by 16S rRNA sequence analysis. The biodegrading capability of S. thermocarboxydus was determined by observing the clear zone around the colony cultured on the agar plate containing the different biomasses as sole carbon sources and calculating the substrate degradation ratios. Furthermore, scanning electron microscopy images of eight agro-wastes before and after bacterial treatment and weight loss of agro-wastes revealed the bacterium degraded the biomasses. The different trends of enzyme activities were observed for various wastes, and the maximum activity depended on the type of agro-wastes. Overall, S. thermocarboxydus was found to be a potential candidate for pectinase and xylanase production. The enzyme production varies with the concentration of the biomasses.

Breeding of a thermostable xylanase-producing strain of Myceliophthora thermophila by atmospheric room temperature plasma (ARTP) mutagenesis.

Introduction: Hemicellulose is an important component in lignocellulose materials, which is second only to cellulose, accounting for 15%-35% of the dry weight of plants. In the current situation of energy shortage, making full use of lignocellulose materials to produce fuel ethanol has become an important way to solve the energy problem. Xylanase plays a crucial role in the utilization of hemicellulose. It is a necessary means to reduce the cost of hemicellulose utilization by improving the activity of xylanase. Moreover, most naturally xylanases are mesophilic enzymes, which limits their industrial application. Methods:In this study, Myceliophthora thermophila was used to produce xylanases and a thermostable mutant M 2103 was obtained by atmospheric room temperature plasma (ARTP) mutagenesis. The research work started with exploring the effects of ARTP mutagenesis on the antioxidase system [superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), polyphenol oxidase (PPO), and antioxidant capacity (AOC)] of M. thermophile, and found that superoxide dismutase activity increased by 221.13%, and polyphenol oxidase activity increased by 486.04% as compared with the original strain when the implantation time was 300s. So as to determine the conditions for subsequent mutagenesis. Results and Discussion:For the mutant M 2103, the reaction temperature for xylanase production remained stable in the range of 70°C-85°C. Its optimum temperature was 75°C, which was 15°C higher than that of the original strain. And its xylanase activity increased by 21.71% as compared with the original strain. M 2103 displayed a significantly higher relative xylanase activity than the original strain in the acidic (pH 4.0-7.0) range, and the xylanase activity was relatively stable in the pH range of 6.0-8.5. These results provide an alternative biocatalyst for the production of xylooligosaccharide, and a potential usage of ARTP in the mutagenesis of thermostable mutant.

Efecto del tamaño de partícula, aireación y altura del sustrato sobre la producción de enzimas lignocelulolíticas producidas por Trametes polyzona HHM001 crecido sobre residuos de hojas de maíz

The objective of the work was to evaluate the production of lignocllulolytic enzymes produced by Trametes polyzona HHM001 during its growth on corn leaf residues. Two particle sizes (PS) (PS8 and PS12), two levels of aeration (1 vvm and 0 vvm), as well as the height of the substrate in the production of lignocellulolytic enzymes were tested. The enzymatic activities of Laccase (Lcc), Lignin peroxidase (LiP), and Manganese peroxidase (MnP) were favored under aerated conditions (1 vvm). The enzymatic activity of Lcc was the most favored, with 80 Activity Units (AU)/mL, compared to the culture without aeration, in which 40 AU/mL was obtained. The production of xylanases (Xyl) and cellulases (Cel) were not influenced by aeration under the tested conditions. The results indicated that the particle size has more effect on enzyme production than the presence or absence of air, with a particle size of 8 where the best levels of enzymatic activity were observed. It was observed that the height of the substrate in the fermentation column strongly affects the activity of ligninolytic enzymes and not of the hydrolytic; at 5 cm, the highest ligninolytic activity was detected, where aeration favors oxidative conditions.

Modulating the production of xylanase by Bacillus pumilus BS131 through optimization using waste fiber sludge.

In recent days, cheapest alternative carbon source for fermentation purpose is desirable to minimize production cost. Xylanases have become attractive enzymes as their potential in bio-bleaching of pulp and paper industry. The objective of the present study was to identify the potential ability on the xylanase production by locally isolated Bacillus pumilus BS131 by using waste fiber sludge and wheat bran media under submerged fermentation. Culture growth conditions were optimized to obtain significant amount of xylanase. Maximum xylanase production was recorded after 72 hours of incubation at 30 °C and 7 pH with 4.0% substrate concentration. In the nutshell, the production of xylanase using inexpensive waste fiber sludge and wheat-bran as an alternative in place of expensive xylan substrate was more cost effective and environment friendly.

INDUSTRIALLY IMPORTANT XYLANASE FROM MICROBIAL SOURCES AND THEIR APPLICATIONS

One of the major constituents of hemicellulose is xylan. Among different natural polymers, xylan can be found more abundantly and is ranked second among them. There are four types of xylans, and the physical conformation and solubility of each are determined by the side chains. The phenomenon of reactions with other components of hemicelluloses can be attributed to their side chains. Xylanase is an enzyme from the hydrolase family that can catalyze the hydrolysis of xylan polysaccharides. Aside from plant and animal sources, the enzyme xylanase is produced from a different microorganism. Some fungi as Trichoderma sp., Aspergillus sp., yeast like Cryptococcus sp., and bacteria such as Streptomyces sp., are very well-known for the production of microbial xylanase. Microbial enzyme production will boost employment and revenue, allowing for the exploration of certain alternate techniques to preserve foreign exchange reserves. Because industrial enzymes are imported with precious foreign exchange, this endeavor can address national demand while simultaneously benefiting industry and the economy. Xylanase has several applications; it is used in the beverage industry, the bakery industry, the paper industry, etc. They can also be used in poultry as food additives.

Optimization of xylanase production from Aspergillus Spp. hau-f-6 using plackett burman screening design and path of steepest ascent in submerged fermentation

Optimization of xylanase production from Aspergillus Spp. hau-f-6 using plackett burman screening design and path of steepest ascent in submerged fermentation

Xylanase production by Trichoderma virens MLT2J2 under solid-state fermentation using corn cob as a substrate

Abstract. Istiqomah L, Cahyanto MN, Zuprizal. 2022. Xylanase production by Trichoderma virens MLT2J2 under solid-state fermentation using corn cob as a substrate. Biodiversitas 23: 6530-6538. Xylanase is one of non-starch polysaccharide (NSP) degrading enzyme encompass in industrial application like animal feed, food, biofuel, and textile. The objective of this study was to produce and characterize the crude xylanase from MLT2J2 isolate that isolated from coconut husk. Molecular identification revealed that MLT2J2 isolate was identified as Trichoderma virens. The xylanase was produced from Trichoderma virens MLT2J2 (108 spores/g of corncob) using corn cob as substrate with 80% initial moisture content using under solid-state fermentation (SSF) at 30°C for 7 d of incubation. Changes in surface morphology and structure of fermented corn cob were monitored by Scanning Electron Microscope (SEM). Statistical analysis was performed using One-way of analysis of variance (ANOVA) followed Tukey Kramer post hoc test to compare treatment means. Under fermentation parameters the maximum xylanase activity was 181.22 U/g-IDW, loss of dry matter 11.43%, and pH was decreased (4.11) at 5 d of incubation compared to initial pH (5.47). Xylanase was produced between a broad range pH 3-8 and showed acidophilic and mesophilic characteristics (optimum at pH 5,0 and 40°C), and also conserved more than 50% of activity before 4 h of incubation at 30°C and after 5 h at 40°C. The micrographs surface of corn cob before SSF appeared intact and smoother, while turned coarser after SSF, suggestive of a disrupted and more porous surface. These findings reveled that T. virens MLT2J2 could produce extracellular xylanase and potential to disrupt the cell wall of corn cob as a cheap substrate for enzyme production thereby increased the reactive surface area of corn cob for enzymatic in situ hydrolysis.

A novel fusion transcription factor drives high cellulase and xylanase production on glucose in Trichoderma reesei

To reduce the high cost of (hemi)cellulase production in lignocellulose biorefining, it is important to develop strategies to enhance enzyme productivity from economic and also readily manipulatable carbon sources. In this study, an artificial transcription factor XT was designed by fusing the DNA binding domain of Xyr1 to the transactivation domain of Tmac1. When overexpressed in Trichoderma reesei QM9414 Δxyr1, the XT recombinant strain (OEXT) greatly improved (hemi)cellulase production on repressing glucose compared with QM9414 on Avicel with 1.7- and 8.2-fold increases in pNPCase and xylanase activity, respectively. Both activities were even higher (0.9- and 33.8-fold higher, respectively) than the recombinant strain similarly overexpressing Xyr1. The dramatically enhanced xylanase activities in OEXT resulted from the elevated expression of various hemicellulases in the secretome. Moreover, the enzyme cocktail from OEXT improved the saccharification efficiency toward corn stover by 60% compared with enzymes from QM9414 with equal volume loading.

Solid-State Fermentation of Steam-Exploded Opuntia ficus-indica Cladodes Using Trichoderma reesei CCT-2768 for the Production of Cellulolytic Enzymes.

The sustainable development of the drylands, i.e., regions with limited availability of water, depends on the exploitation of the few biomass types that can thrive in such conditions, such as the Opuntia ficus-indica, a plant of the Cactaceae family. In the present study, the cladodes of O. ficus-indica were used as a substrate by the fungus Trichoderma reesei CCT-2768 for the production of cellulolytic enzymes through solid-state fermentation. Firstly, the extraction of the mucilage, soluble components of industrial interest, was evaluated. Temperature, water-to-biomass ratio, and time of mixture were varied using an experimental design and impacted, especially, the pectin removal. Then, the lignocellulosic residue was used for the production of enzymes; the effect of the water activity, biomass pretreatment, mineral supplementation, temperature, and inoculum size on the enzymatic production were investigated using two sets of experimental designs. The steam explosion pretreatment exposed the fiber to the microbial action and boosted the enzyme production, provided that the medium was supplemented with salts. This combination has improved the production of xylanase, CMCase, FPase, and polygalacturonase by 27, 62, 98, and 185%, respectively. The temperature of 35°C was determined as the optimal for the production of FPase, xylanase, and polygalacturonase, while no effect was observed on the production of CMCase and β-glucosidase. The optimization of the enzymatic production performed in this study can potentially provide a new application for the Opuntia biomass and improve the sustainable development of the drylands.

Solid-state fermentation of Saba banana peel for pigment production by Monascus purpureus.

Eco-friendly natural pigment demand has ever-increasing popularity due to health and environmental concerns. In this context, the aim of this study was to evaluate the feasibility use of Saba banana peel as low-cost fermentable substrate for the production of pigments, xylanase and cellulase enzymes by Monascus purpureus. Among the strains tested, M. purpureus TISTR 3385 produced pigments better and had higher enzyme activities. Under the optimal pigment-producing conditions at the initial moisture content of 40% and initial pH of 6.0, the pigments comprising yellow, orange, and red produced by the fungi were achieved in the range of 0.40-0.93 UA/g/day. The maximum xylanase and cellulase activities of 8.92 ± 0.46 U/g and 4.72 ± 0.04 U/g were also obtained, respectively. More importantly, solid-state fermentation of non-sterile peel could be achieved without sacrificing the production of the pigments and both enzymes. These indicated the potential use of the peel as fermentable feedstock for pigment production by the fungi and an environmental-friendly approach for sustainable waste management and industrial pigment and enzyme application.

Impact of structure and composition of different sorghum xylans as substrates on production of xylanase enzyme by Aspergillus fumigatus RSP-8

Xylan composition from six different bmr sorghum (Sorghum bicolor L.) biomasses (Atlas 12, BN 612, Atlas 3986, IS 27333, Atlas N598 & IS 27206) with variable lignin content were analyzed for their structure and evaluated as a substrate for xylanase complex production using Aspergillus fumigatus RSP-8. Chemical compositional analysis of xylans revealed that all the xylans belong to arabinoxylan (AX) type and are composed of xylose, glucose, and arabinose in varying concentrations. The relative content of xylose varied from 90 % (of total sugars) in the variety IS 27206 to 72 % in Atlas N598. Atlas N598 xylan contained the highest relative glucose content of 22 % and the least content was observed in IS 27206 xylan (2.9 %). The relative content of L-arabinose was constant with fewer variations (5.1–6.7 %). FT-IR analysis showed that the xylans extracted from different sorghum substrates varied in structure. The spectra of all the xylans exhibited absorption maxima of ∼895 cm-1 which is characteristic for the β-(1−4) xylans. The strongest absorption bands in the IR spectra were observed at 3700–3000 and 1200–1000 cm-1 with varying intensities. Xylanase production revealed that the source of xylan significantly affected the fungal growth and xylanase productivity. The highest xylanase production (1248 U/mL) and fungal biomass dry weight (5.94 g/L) was observed with IS 27206 xylan and the least productivity of 964 U/mL and fungal biomass dry weight (3.74 g/L) with Atlas N598 xylan noticed as substrate.

Enhancing xylanase expression by Komagataella phaffii by formate as carbon source and inducer

Komagataella phaffii (syn. Pichia pastoris), a methylotrophic yeast, has many advantages as a protein expression system, but has the disadvantage of hazardous methanol as an inducer and carbon source. To enable substitution of formate for methanol, a formate assimilation pathway was constructed by the co-expression of acetyl-CoA synthase, acetaldehyde dehydrogenase, and transcription factor Mit1, resulting in a 103.5 ± 12.5% increase in xylanase production. Recombinant K. phaffii was able to use formate as a carbon source, indicating successful substitution of formate for methanol. Xylanase production, using the safe and sustainable formate as an inducer and carbon source, is a major advance in the field of industrial enzyme production. KEY POINTS: • Change to formate assimilation by recombinant K. phaffii instead of methanol • K. phaffii expressed xylanase by formate induction instead of methanol induction • Increased xylanase expression by transcription factor co-expression.

Optimization, purification, and characterization of xylanase production by a newly isolated Trichoderma harzianum strain by a two-step statistical experimental design strategy

Xylanases are hydrolytic enzymes with a wide range of applications in several industries such as biofuels, paper and pulp, food, and feed. The objective of this study was to optimize the culture conditions and medium components for maximal xylanase production from a newly isolated Trichoderma harzianum strain using the Plackett–Burman Design (PBD) and Box Behnken Design (BBD) experimental strategies. Xylanase production was enhanced 4.16-fold to 153.80 U/ml by BBD compared to a preliminary one-factor-at-a-time (OFAT) activity of 37.01 U/ml and 2.24-fold compared to the PBD (68.70 U/ml). The optimal conditions for xylanase production were: 6 days of fermentation, incubation temperature of 70 °C, pH 5.0, agitation of 160 rpm, and 1.2% wheat bran and ammonium sulphate. The experimental design effectively provided conditions for the production of an acidic-thermostable enzyme with exciting potential for application in animal feed improvement. The acidic-thermostable xylanase was purified from the submerged culture and SDS-PAGE analysis revealed a molecular weight of 72 kDa. This protein had maximum xylanolytic activity at pH 6.0 and 65 °C and was stable for 4 h retaining > 70% activity and exhibited substrate specificity for beechwood xylan with a Km of 5.56 mg/ml and Vmax of 1052.63 µmol/min/mg. Enzyme activity was enhanced by Fe2+, Mg2+, and Zn2+. There was an absence of strong inhibitors of xylanase activity. Overall, these characteristics indicate the potential for at least two industrial applications.

Fluorescent labeling of dicysteine‐tagged peptide for monitoring and optimization of protein bio‐production in bacteria

AbstractA rapid and convenient strategy to monitor the productivity of biomanufacturing is essential for the research in optimizing relevant bioprocesses. In this work, we have developed a fluorescein‐derived probe (FL‐DT) that reacts rapidly with thiol groups via 1,4‐Michael addition reaction of the sulfhydryl to unsaturated ketone and releases fluorescence. FL‐DT specifically forms fluorescent adduct with two adjacent thiols in a protein of interest (POI), making the probe a reliable tool for protein quantification. The production of xylanase fused with a short di‐Cys tag was then successfully monitored and quantified with FL‐DT in Escherichia coli system under different protein expression conditions, providing useful information for optimizing the bioprocess. Our work provides a convenient and efficient strategy for POI labeling and monitoring bioproduction.

Optimization of the fermentation parameters to maximize the production of cellulases and xylanases using DDGS as the main feedstock in stirred tank bioreactors

Lignocellulolytic enzymes such as cellulase and xylanases are needed on the industrial scales for low-cost production of biofuels and the other value-added products from lignocellulosic biomass such as distillers' dried grains with solubles (DDGS). Optimization of fermentation variables such as agitation, aeration, and inoculum size for fungal enzyme production by submerged fermentation can enhance the enzyme production levels. Therefore, this research focused on the statistical optimization of fungal inoculum size (1–10%), aeration (0.5-2vvm) and agitation rates (100–500rpm) through response surface methodology (RSM) to obtain highest amounts of cellulases and xylanases. Aspergillus niger (NRRL 330) was grown in dilute acid treated DDGS supplemented with peptone, yeast extract, and ammonium sulfate in benchtop bioreactors to produce these enzymes. The optimal fermentation results were determined to be 6.5% inoculum size, 310 rpm agitation rate and 1.4 vvm aeration, which revealed increases of cellulase activity from 0.6 to 0.82 IU/ml and xylanase activity from 3.99 to 52.76 IU/ml after optimization. The results prove the need for optimization of culture conditions such as aeration, agitation and inoculum size for higher production of hydrolytic enzymes by A. niger using DDGS as the main feedstock.

Safety and efficacy of a feed additive consisting of endo-1,4-β-xylanase produced by Komagataella phaffiiDSM 33574, and viable spores of Bacillus velezensisDSM 21836 and Bacillus licheniformisATCC 53757 (EnzaPro) for chickens for fattening, chickens reared for laying/breeding, turkeys for fattening, turkeys reared for breeding and growing minor poultry species (BioResource International (BRI), Inc.).

Following a request from the European Commission, the EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the safety and efficacy of the product EnzaPro containing viable cells/spores of strains of Bacillus velezensis (DSM 21836) and Bacillus licheniformis (ATCC 53757) and an endo‐1,4‐β‐xylanase produced by a genetically modified strain of Komagataella phaffii (DSM 33574) as a zootechnical additive in chickens for fattening, chickens reared for laying/breeding, turkeys for fattening, turkeys reared for breeding and minor poultry species for fattening or raised to the point of lay. The strains B. velezensis DSM 21836 and B. licheniformis ATCC 53757 were considered to meet the qualified presumption of safety (QPS) requirements. The K. phaffii xylanase production strain is genetically modified. No viable cells and no recombinant DNA of the genetically modified production strain were detected in the final product. Therefore, the Panel concluded that the additive does not pose any safety concern regarding the xylanase production strain. EnzaPro is safe for all poultry species for fattening or reared to the point of lay at the proposed conditions of use. The FEEDAP Panel concluded that the use of EnzaPro in animal nutrition is safe for the consumers and the environment. EnzaPro is not a skin irritant but should be considered an eye irritant and a respiratory sensitiser. No conclusions could be drawn on the potential of the additive to cause skin sensitisation. Due to the lack of data, the FEEDAP Panel could not conclude on the efficacy of EnzaPro for the target species. EnzaPro is compatible with diclazuril, halofuginone and nicarbazin.

AKTIVITAS XILANASE DARI Bacillus altitudinis YANG DIPRODUKSI DENGAN VARIASI WAKTU INKUBASI DAN KONDISI PENGUJIAN pH NETRAL DAN ALKALIN

Xylanases are hydrolytic enzymes which randomly cleave the β-1,4 backbone of the complex plant cell wall polysaccharide xylan. In this study, we investigate the extracellular enzyme activity of three strains of Bacillus altitudinis which produced with various production times in neutral (pH 7) and alkaline (pH 9) conditions. Crude enzyme was obtained from inoculated production medium containing 2% w/v xylan after six different time of incubations, including 0, 24, 48, 72, 96 and 120 hours. The stationary phase of those three strains was observed after 72 hours of incubation, in accordance with significant increase of xylanase activity both in neutral and alkaline assay conditions. However, the highest of xylanase activity was obtained after 92 hours of incubation, in all three bacterial strains tested. A thin layer chromatography (TLC) was performed, and it confirmed that the crudes enzymes was able to breakdown the xylan into oligosaccharides. A higher activity of xylanase was obtained in alkaline condition but not significantly different between those two conditions. B. altitudinis KBX08 has the highest xylanase activity (46.9U/ml) which produced after 96 hours of incubation, indicating their potential for further development as xylanase producer.