Extracellular Xylanase Research Articles

Production and Optimization of Xylanase Enzyme from Bacillus cereus by Submerged Fermentation Method

Three strains of Bacillus cereus were isolated from soil samples in this study in an effort to enhance the circumstances for the production of xylanase by the bacteria. The bacteria released extracellular xylanase throughout the submerged fermentation process. In a 250 ml conical flask, the whole fermentation process was conducted at 150 rpm stirring speed. The highest amount of xylanase synthesis was demonstrated by the isolated strains at 45°C, 48 hours of incubation, pH 5, 1.5% oat splet xylan base concentration, lactose carbon source, and beef extract source.

Aspergillus labruscus ITAL 22.223 xylanase - immobilization and application for the obtainment of corncob xylan targeting xylitol production.

Corncob is an agro-residue rich in lignocellulosic material that can be used for the xylitol production, through its enzymatic conversion obtaining fermentable sugars and their subsequent fermentation. In light of the above, this study targeted the immobilization of Aspergillus labruscus xylanase and the use of the derivative to hydrolyze the corncob xylan for the obtainment of xylose, and its subsequent use for the production of xylitol. The extracellular xylanase was immobilized using different supports (sodium alginate, DEAE-Cellulose, DEAE-Sephadex and CM-Sephadex). Among all supports used, the best results were obtained with the DEAE-Cellulose derivative showing an efficiency of immobilization of 97-99%, yield of 93-95% and recovered activity of 81-100%. The sodium alginate derivative showed 3 cycles of reuse, with drop in activity of about 65% in the 3rd cycle using both CaCl2 and MnCl2 as crosslinkers. The best enzymatic activity for the DEAE-Cellulose derivative was observed at 55ºC and pH 5.0. This derivative presented reuse of 10 cycles using commercial xylan as substrate, and 4 cycles using corncob xylan. This derivative was used in an enzymatic reactor to hydrolyze corncob xylan, obtaining 2.7mg/mL of xylose after 48h of operation under optimal condition of temperature and pH. The xylose obtained from the corncob was fermented by Candida tropicalis for 96h with consumption of 60%. The HPLC analyses indicated a production of 1.02mg/mL of xylitol with 48h of fermentation. In conclusion, this is the first report on the immobilization of the A. labrucus xylanase as an alternative for the obtainment of xylose from corncob xylan, and the subsequent production of xylitol.

Aspergillus brasiliensis E_15.1: A Novel Thermophilic Endophyte from a Volcanic Crater Unveiled through Comprehensive Genome-Wide, Phenotypic Analysis, and Plant Growth-Promoting Trails.

Thermophilic fungi have been seldom studied despite the fact that they can contribute to understanding ecological mechanisms of adaptation in diverse environments and have attractive toolboxes with a wide range of biotechnological applications. This work describes for the first time an endophytic and thermophilic strain of Aspergillus brasiliensis that was isolated in the crater of the active volcano "El Chichonal" in Mexico. This strain was capable of surviving in soil with a temperature of 60 °C and a pH of neutral acidity, which preluded a high thermostability and a potential in industrial application. The complete genome of A. brasiliensis E_15.1 was sequenced and assembled in 37 Mb of genomic DNA. We performed a comprehensive phylogenomic analysis for the precise taxonomic identification of this species as a novel strain of Aspergillus brasiliensis. Likewise, the predicted coding sequences were classified according to various functions including Carbohydrate-Active Enzymes (CAZymes), biosynthetic gene clusters of secondary metabolites (BGCs), and metabolic pathways associated with plant growth promotion. A. brasiliensis E_15.1 was found to degrade chitin, chitooligosaccharides, xylan, and cellulose. The genes to biosynthesize clavaric acid (a triterpene with antitumor activity) were found, thus probably having antitumor activity. In addition to the genomic analysis, a set of enzymatic assays confirmed the thermostability of extracellular xylanases and cellulases of A. brasiliensis E_15.1. The enzymatic repertoire of A. brasiliensis E_15.1 suggests that A. brasiliensis E_15.1 has a high potential for industrial application due to its thermostability and can promote plant growth at high temperatures. Finally, this strain constitutes an interesting source of terpenoids with pharmacological activity.

Transcriptional activator UvXlnR is required for conidiation and pathogenicity of rice false smut fungus Ustilaginoidea virens.

Transcription factors play critical roles in diverse biological processes in fungi. XlnR, identified as a transcriptional activator that regulates the expression of the extracellular xylanase genes in fungi, has not been extensively studied for its function in fungal development and pathogenicity in rice false smut fungus Ustilaginoidea virens. In this study, we characterized UvXlnR in U. virens and established that the full-length, N- and C-terminal forms of the UvXlnR have the ability to activate transcription. The study further demonstrated that UvXlnR plays crucial roles in various aspects of U. virens biology. Deletion of UvXlnR affected growth, conidiation, and stress response. UvXlnR mutants also exhibited reduced pathogenicity, which could be partially attributed to the reduced expression of xylanolytic genes and extracellular xylanase activity of U. virens during the infection process. Our results indicate that UvXlnR is involved in regulating growth, conidiation, stress response, and pathogenicity.

Optimization of xylanase production by Pichia kudriavzevii and Candida tropicalis isolated from the wood product workshop.

Enzymatic compounds can be found abundantly and provide numerous advantages in microbial organisms. Xylanases are used in various pharmaceutical, food, livestock, poultry, and paper industries. This study aimed to investigate xylanase-producing yeasts, xylose concentration curve and their enzymatic activity under various factors including carbon and nitrogen sources, temperature, and pH. Enzyme activity was evaluated under different conditions before, during, and after purification. The yeast strains were obtained from the wood product workshop and were subsequently cultivated on YPD (yeast extract peptone dextrose) medium. Additionally, the growth curve of the yeast and its molecular identification were conducted. The optimization and design process of xylan isolated from corn wood involved the use of Taguchi software to test different parameters like carbon and nitrogen sources, temperature, and pH, with the goal of determining the most optimal conditions for enzyme production. In addition, the Taguchi method was utilized to conduct a multifactorial optimization of xylanase enzyme activity. The isolated species were partially purified using ammonium sulfate precipitation and dialysis bag techniques. The results indicated that 3 species (8S, 18S, and 16W) after molecular identification based on 18S rRNA gene sequencing were identified as Candida tropicalis SBN-IAUF-1, Candida tropicalis SBN-IAUF-3, and Pichia kudriavzevii SBN-IAUF-2, respectively. The optimal parameters for wheat carbon source and peptone nitrogen source were found at 50°C and pH 9.0 through single-factor optimization. By using the Taguchi approach, the best combination for highest activity was rice-derived carbon source and peptone nitrogen source at 50°C and pH 6.0. The best conditions for xylanase enzyme production in single-factor optimization of wheat bran were 2135.6 U/mL, peptone 4475.25 U/mL, temperature 50°C 1868 U/mL, and pH 9.0 2002.4 U/mL. Among the tested yeast, Candida tropicalis strain SBN-IAUF-1 to the access number MZ816946.1 in NCBI was found to be the best xylanase product. The highest ratio of enzyme production at the end of the delayed phase and the beginning of the logarithmic phase was concluded by comparing the growth ratio of 8S, 16W, and 18S yeasts with the level of enzymatic activity. This is the first report on the production of xylan polymer with a relative purity of 80% in Iran. The extracellular xylanases purified from the yeast species of C. tropicalis were introduced as a desirable biocatalyst due to their high enzymatic activity for the degradation of xylan polymers.

A novel transcriptional repressor specifically regulates xylanase gene 1 in Trichoderma reesei

BackgroundThe well-known industrial fungus Trichoderma reesei has an excellent capability of secreting a large amount of cellulases and xylanases. The induced expression of cellulase and xylanase genes is tightly controlled at the transcriptional level. However, compared to the intensive studies on the intricate regulatory mechanism of cellulase genes, efforts to understand how xylanase genes are regulated are relatively limited, which impedes the further improvement of xylanase production by T. reesei via rational strain engineering.ResultsTo identify transcription factors involved in regulating xylanase gene expression in T. reesei, yeast one-hybrid screen was performed based on the promoters of two major extracellular xylanase genes xyn1 and xyn2. A putative transcription factor named XTR1 showing significant binding capability to the xyn1 promoter but not that of xyn2, was successfully isolated. Deletion of xtr1 significantly increased the transcriptional level of xyn1, but only exerted a minor promoting effect on that of xyn2. The xylanase activity was increased by ~ 50% with XTR1 elimination but the cellulase activity was hardly affected. Subcellular localization analysis of XTR1 fused to a green fluorescence protein demonstrated that XTR1 is a nuclear protein. Further analyses revealed the precise binding site of XTR1 and nucleotides critical for the binding within the xyn1 promoter. Moreover, competitive EMSAs indicated that XTR1 competes with the essential transactivator XYR1 for binding to the xyn1 promoter.ConclusionsXTR1 represents a new transcriptional repressor specific for controlling xylanase gene expression. Isolation and functional characterization of this new factor not only contribute to further understanding the stringent regulatory network of xylanase genes, but also provide important clues for boosting xylanase biosynthesis in T. reesei.

Xylanase Production by Solid-State Fermentation for the Extraction of Xylooligosaccharides from Soybean Hulls§.

The development of a novel process for the production of xylooligosaccharides (XOS) based on the 4R concept is made possible by the integration of numerous techniques, especially enzymatic modification together with the physical pretreatment of renewable materials. This study aims to integrate the use of agricultural wastes for the production of xylanase by a new strain of Penicillium sp. and value-added products, XOS. For the production of xylanase, a solid-state fermentation was performed using wheat bran as substrate. To obtain the most active crude extract of xylanase, the time frame of cultivation was first adjusted. Then, the downstream process for xylanase purification was developed by combining different membrane separation units with size exclusion chromatography. Further characterisation included determination of the optimal pH and temperature, determination of the molecular mass of the purified xylanase and analysis of kinetic parameters. Subsequently, the hydrolytic ability of the partially purified xylanase in the hydrolysis of alkali-extracted hemicellulose from soybean hulls was investigated. Our results show that Penicillium rubens produced extracellular xylanase at a yield of 21 U/g during solid-state fermentation. Using two ultrafiltration membranes of 10 and 3 kDa in combination with size exclusion chromatography, a yield of 49 % and 13-fold purification of xylanase was achieved. The purified xylanase (35 kDa) cleaved linear bonds β-(1→4) in beechwood xylan at a maximum rate of 0.64 μmol/(min·mg) and a Michaelis constant of 44 mg/mL. At pH=6 and 45 °C, the purified xylanase showed its maximum activity. The xylanase produced showed a high ability to hydrolyse the hemicellulose fraction isolated from soybean hulls, as confirmed by thin-layer chromatography. In the hydrothermally pretreated hemicellulose hydrolysate, the content of XOS with different degrees of polymerisation was detected, while in the non-pretreated hemicellulose hydrolysate, the content of xylotriose and glucose was confirmed. Future research focusing on the creation of new enzymatic pathways for use in processes to convert renewable materials into value-added products can draw on our findings.

Effects of Graded Levels of Xylanase-Treated Rice Husk on Nutrient Digestibility and Growth Performance of Broiler Chickens

This study investigated the feeding value of Aspergillus tubingensis xylanase-treated rice husk in broiler chickens. The objectives of the study are (I) to investigate the effect of xylanase treatment on the utilization of rice husk in broiler chickens and (II) to determine the optimum level of xylanase-treated rice husk that can be tolerated by broiler chickens. Xylanases are hydrolytic enzymes that degrade xylan and hemicelluloses located in plant cell walls, into xylose which is a reducing sugar and enhancing nutrient digestibility in animal feeds. The production of extracellular xylanase by a locally isolated Aspergillus tubingensis was conducted using solid-state fermentation. The selected isolate was identified by cultural techniques and verified by molecular identification. Conventional feed ingredients such as maize is the primary cereal as source of energy in broiler diet. High cost, availability and competition existing among man, industry and livestock has necessitated the need to find cheap and available alternative feedstuffs for maize in poultry diet. Rice husk is one of the alternative feedstuffs but characterized by high fibre content and Non-Starch Polysaccharide (NSP) (Dalibord, 2006). Thus, addition of the xylanase enzyme breaks the NSPs resulting in plant cell wall destruction after releasing the trapped nutrients such as starches and proteins within fibre-rich cell walls (Gade et al., 2017). Crude xylanase produced was used for enzymatic degradation of rice husk to improve its nutritional value. The rice husk was initially subjected to physical pre-treatment by soaking in water for 24 hours or ground to increase the surface area. Pre-treated rice husk was treated by spraying xylanase onto the rice husk at 100g/ 0.2mL representing the concentration level that recorded the best degradation of fibre content in the treated rice husk. Xylanase-treated rice husk (XTRH) was used with other ingredients in formulating a broiler chicken diet at different inclusion levels. The experiment was a complete randomized design with five experimental diets containing xylanase-treated rice husk at different inclusion levels 0, 5, 10, 15 and 20 kg/100kg. The control diet contained no xylanase-treated rice husk (XTRH). There were five (5) treatments each with three (3) replicate cages of eight (8) broiler chicks totalling 120 birds for the feeding trial. One hundred and twenty (120) day-old chicks with an average weight of 54.85g were randomly allotted to five dietary treatments formulated with 0 (control), 5%, 10%, 15% and 20% (XTRH), and fed for eight weeks. There were three replicates of eight (8) birds per treatment. Results showed that the performance of Broiler chickens such as feed intake, and weight gain feed fed diet containing a 20% inclusion level of xylanase-treated rice husk was significantly higher (p<0.05) compared to other treatment diets. This showed an improvement in the utilization of a high-fibre diet, nutrient digestibility, growth performance, and carcass characteristics which can be beneficial to farmers in reducing the cost of feed, and increasing savings and profit margin. Conclusively, the application of xylanase produced by Aspergillus tubingensis on rice husk may have enhanced efficient enzymatic hydrolysis of its fibre fractions and improved its nutritional values. Supplementation of rice husk treated with xylanase at 100g/0.2mL concentration level and included at 20% in broiler chicken may enhance nutrient digestion and utilization and improve the growth performance of broiler chickens.

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.

Isolation, morphological identification, and xylanase characteristics of anaerobic gut fungi Neocallimastix from Anatolian wild goat.

This study shows the morphological identification of anaerobic fungal strains isolated from fecal samples of goats inhabiting Turkey and the effects of various metal ions and chemicals on extracellular xylanase production. Three different anaerobic gut fungi isolated from wild goats in Turkey were identified as Neocallimastix spp. xylanase, cellulase, and lichenase production were tested in culture supernatants, and the maximum-specific activities were found as 560.42 ± 9.39, 159.70 ± 3.88, and 157.36 ± 3.83 (μmol/min/mg protein), respectively. While the optimum temperature range of exo-xylanases was found as 40-50°C, their optimum pH range was determined as 6.0-6.5. Xylanase activity decreased in metal ions and other chemical reactants based on dose. The metal ion that significantly inhibited xylanase activity was Fe+3 . It was found that the ferric ions inhibited xylanase activity in all three anaerobic gut fungi by 30%-90% depending on molarity. On the contrary, the 1 mM concentrations of the Mn+2 , Ba+2 , Co+2 , Cu+2 , Sn+2 , and Mg+2 metal ions and the ethylenediaminetetraacetic acid and β-mercaptoethanol reagents had a positive effect at rates in the range of 3%-92%. In conclusion, these findings demonstrate that anaerobic gut fungus has very stable fibrolytic enzymes that need to be separated, as well and the existence of a unique resource for industrial applications.

An environment-benign approach of bamboo pulp bleaching using extracellular xylanase of strain Bacillus stratosphericus EB-11 isolated from elephant dung.

The use of microbial enzymes is highly encouraged in paper and pulp industries to reduce the excessive use of hazardous chemicals. During the study, xylanase of BacillusstratosphericusEB-11 was characterized for pulp bleaching applications. The extracellular xylanase was produced under submerged fermentation using bamboo wasteas a natural carbon source. There was fast cell division and enzyme production under optimized fermentation conditions in the bioreactor. The highest activity was 91,200Uafter30h of growth with Km and Vmax of 3.52mg/mL and 391.5μmol/min per mgrespectively. The purified enzyme with molecular mass ~ 60kDa had conferred positive activity on native PAGE. The strong inhibition by ethylenediaminetetraacetate and SDS showed the metallo-xylanase nature of the purified enzyme.The bacterial xylanase reduces the use of hydrogen peroxide by 0.4%. Similarly, biological oxygen demand and chemical oxygen demand were reduced by 42.6 and 35.2%. The xylanase-hydrogen peroxide combined treatment and conventional chlorine dioxide-alkaline (CDE1D1D2) bleaching showed almost similar improvement in physicochemical properties of bamboo pulp. Xylanase-peroxide bleaching reduces the lignin content to 4.95% from 13.32% unbleached pulp. This content after CDE1D1D2treatment was 4.21%. The kappa number decreased from 15.2 to 9.46 with increasing the burst factor (15.51),crystallinity index (60.25%), viscosity (20.1 cp), and brightness (65.4%). The overall finding will encourage the development of new cleaner methods of bleaching in the paper and pulp industry.

High cellulase‐free xylanases production by Moesziomyces aphidis using low‐cost carbon and nitrogen sources

AbstractBackgroundEnzymes involved in xylan hydrolysis have several industrial applications. Selection of efficient microbial hosts and scalable bioreaction operations can lower enzyme production costs and contribute to their commercial deployment. This work aims at investigating the Moesziomyces aphidis yeast cultivation conditions that deliver maximal xylanase titres, yields and productivities using low‐cost nitrogen (N) and carbon (C) sources.ResultsNaNO3 and KNO3 supplementation improved xylanase production 2.9‐ and 2.7‐fold (against 67.2 U mL−1), respectively, using xylan as C source. Interestingly, the use of KNO3, instead of NaNO3, results in 2‐ to 3‐fold higher specific activity, highlighting the potassium ion role. In addition, this study investigates synergetic effects on using ionic and organic N sources. A 4.9‐fold increase in xylanase production, with high specific activity, is attained combining KNO3 and corn steep liquor (CSL). Exploring the previous findings, this study reports one of the highest extracellular xylanase production titres (864.7 U mL−1) by yeasts, using a media formulation containing dilute‐acid pre‐treated brewery spent grains (BSG), as C source and inducer, supplemented with KNO3 and CSL. Replacement of dilute‐acid pre‐treatmed BSG by untreated BSG had low impact on xylanase production, of only 6%.ConclusionEfficient production of M. aphidis xylanolytic enzymes, using low‐cost N and C sources, is attractive for deployment of on‐site enzyme production targeting different biotechnological applications under circular economy and biorefinery concepts. Potential xylanases end‐users include industries such as brewing (using BSG as substrate for enzyme production), pulp and paper (benefiting from the cellulase‐free xylanase activity) or lignocellulosic ethanol (for cellulase supplementation). © 2022 Society of Chemical Industry (SCI).

Optimization of procedural factors for advanced xylanase synthesis by Lysinibacillus fusiformis using Kolanut husk as substrate

Xylan is a complex hetero-polysaccharide consisting of different monosaccharides held together by glycosidic and ester bonds. Extracellular xylanase fashioned by numerous microbes principally from bacterial species such as Bacillus species are responsible for cleaving the glycosidic linkages. Microbial xylanases exhibit different substrate specificities and biochemical peculiarities. This study was carried out for optimization of cultivation conditions for xylanase production using the bacterium Lysinibacillus fusiformis and Kolanut husk as a component of cultivation medium. The bacterium was isolated from Kolanut plantation waste soil and screened for the production of xylanase qualitatively on xylan nutrient agar and quantitatively under submerged fermentation. The different conditions optimized included substrate concentration, additional sugars, incubation period, temperature, initial pH, nitrogen supplementation and inoculum mass through one factor at a time approach. Maximum xylanase production was obtained at substrate concentration of (1 % xylan and 1.5 % Kolanut husk), nitrogen source (yeast extract plus peptone), carbon source (sucrose), incubation period (24 h), pH (5.0), temperature (35 oC) and inoculum size (1 %). Lysinibacillus fusiformis has been proven to be a promising bacterium for xylanase production using Kolanut husk as substrate. The use of Kolanut husk as foremost carbon source is predominantly precious as being an agricultural waste, affordable, and locally available compared to expensive commercially sold xylan.

One-step fermentation for producing xylo-oligosaccharides from wheat bran by recombinant Escherichia coli containing an alkaline xylanase

BackgroundOne-step fermentation is a cheap way to produce xylo-oligosaccharides (XOS), where production of xylanases and XOS is integrated into a single process. In spite of cost advantage, one-step fermentation is still short in yield so far due to the limited exploration. To cope with this issue, production of XOS from wheat bran by recombinant Escherichia coli through one-step fermentation was investigated in this study.ResultsAn endo-β-1,4-xylanase gene belonging to glycoside hydrolase family 11 of Bacillus agaradhaerens was employed to construct recombinant E. coli. This xylanase showed maximal activity at 60 °C and pH 8.0–8.5. Its activity retained more than 60% after incubation at 70 °C for 4 h, showing a good stability. The recombinant E. coli BL21(DE3) could secreted xylanases that directly hydrolyzed de-starched wheat bran to XOS in fermentation medium. The XOS generated from hydrolysis consisted of xylose, xylobiose and xylotriose accounting for 23.1%, 37.3% and 39.6%, respectively. Wheat bran concentration was found to be the most crucial factor affecting XOS production. The XOS concentration reached 5.3 mg/mL at 10% loading of wheat bran, which is higher than those of previous researches. Nitrogen source type could also affect production of XOS by changing extracellular xylanase activity, and glycine was found to be the best one for fermentation. Optimal fermentation conditions were finally studied using response surface optimization. The maximal concentration emerged at 44.3 °C, pH 7.98, which is affected by characteristics of the xylanase as well as growth conditions of E. coli.ConclusionsThis work indicates that the integrated fermentation using recombinant E. coli is highly competitive in cost and final concentration for producing XOS. Results can also provide theoretical basis for large-scale production and contribute to the wide adoption of XOS.

Deletion analysis of Pichia pastoris alcohol dehydrogenase 2 (ADH2) promoter and development of synthetic promoters.

Pichia pastoris (Komagataella phaffii) is a non-conventional Crabtree-negative yeast with the capability of reaching very high cell densities in a fed-batch fermentation process. The alcohol dehydrogenase (ADH) genes of P. pastoris involved in ethanol metabolism were identified and were previously characterized. This work aimed to extend current knowledge of the regulation of the ADH2 promoter. To this end, we first determined the upstream activator (UAS) and repressor (URS) sequences of the promoter by deletion assays. Two upstream activator sites have been identified, positioned between -900 and -801bp,and -284 and -108bp upstream of the ADH2 transcription start site. The sequences positioned between -361 and -262bp had a negative effect on the promoter activity and designated a repressor sequence (URS). We then demonstrated that Mxr1 (methanol expression regulator 1) transcription factor activates the ADH2 promoter through the direct interaction with UAS regions in response to ethanol. Furthermore, five different synthetic promoters were constructed by adding or deleting the regulatory sites. These synthetic promoters were tested for extracellular xylanase production at shake flask level by inducing with ethanol. These promoter variants improved the xylanase production ranging between 165% and 200% of the native promoter. The synthetic promoter 5 (SNT5) that displayed the highest activity was further evaluated at the fermenter scale. The modification in the promoter features might have several implications for industrial processes where decoupling the cell growth and product formation is advantageous.

Purification, Identification, and Characterization of a Glycoside Hydrolase Family 11-Xylanase with High Activity from Aspergillus niger VTCC 017

Xylanases (EC 3.2.1.8) have been considered as a potential green solution for the sustainable development of a wide range of industries including pulp and paper, food and beverages, animal feed, pharmaceuticals, and biofuels because they are the key enzymes that degrade the xylosidic linkages of xylan, the major component of the second most abundant raw material worldwide. Therefore, there is a critical need for the industrialized xylanases which must have high specific activity, be tolerant to organic solvent or detergent and be active during a wide range of conditions, such as high temperature and pH. In this study, an extracellular xylanase was purified from the culture broth of Aspergillus niger VTCC 017 for primary structure determination and properties characterization. The successive steps of purification comprised centrifugation, Sephadex G-100 filtration, and DEAE-Sephadex chromatography. The purified xylanase (specific activity reached 6596.79 UI/mg protein) was a monomer with a molecular weight of 37kDa estimating from SDS electrophoresis. The results of LC/MS suggested that the purified protein is indeed an endo-1,4-β-D-xylanase. The purified xylanase showed the optimal temperature of 55°C, and pH 6.5 with a stable xylanolytic activity within the temperature range of 45-50°C, and within the pH range of 5.0-8.0. Most divalent metal cations including Zn2+, Fe2+, Mg2+, Cu2+, Mn2+ showed some inhibition of xylanase activity while the monovalent metal cations such as K+ and Ag+ exhibited slight stimulating effects on the enzyme activity. The introduction of 10-30% different organic solvents (n-butanol, acetone, isopropanol) and several detergents (Triton X-100, Tween 20, and SDS) slightly reduced the enzyme activity. Moreover, the purified xylanase seemed to be tolerant to methanol and ethanol and was even stimulated by Tween 80. Overall, with these distinctive properties, the putative xylanase could be a successful candidate for numerous industrial uses.

Multi-efficient thermostable endoxylanase from Bacillus velezensis AG20 and its production of xylooligosaccharides as efficient prebiotics with anticancer activity

In this study, we have unveiled the novel potential of xylanase production by Bacillus velezensis, previously known only for its PGPR traits. According to our investigations, Bacillus velezensis AG20 produced extracellular xylanase with a fold purification of 5.3 and a yield of 21 %. The purified xylanase (45 kDa) aptly cleaved linear β-(1→4)-xylan with a maximum velocity of 21.0 ± 3.0 U/mL, a turnover of 1.75/s, and a catalytic cycle of 0.571 at optimum pH 7. The temperature of 50 °C was found optimum for enzyme activity. Purified endoxylanase was thermostable and retained its significant residual activities at 50 °C and 60 °C. Metal cations such as Ca2+ and Mg2+ enhanced the substrate catalysis up to 1.5 fold. The purified endoxylanase also demonstrated multi-substrate hydrolyzing properties. However, the enzymatic hydrolysis of sugarcane bagasse produced xylooligosaccharides (XOS), including xylobiose, xylotriose, and xylotetrose. The mixture of released XOS has profound stability in gastric juice, intestinal fluid, and α-amylase and facilitates probiotic bacteria Bifidobacterium. The mixed XOS (300 μg/mL) significantly inhibited the HT-29 and Caco-2 cell proliferation by 90 % and 75 %, respectively, at 48 h. The possible mechanism of XOS uptake through the BIAXP receptor of Bifidobacterium has been contemplated and established using the caver analysis, Molecular Dynamics (MD) simulation, and Density Functional Theory (DFT) studies. Thus, here we reveal the utility of this novel bacterial strain in industries as high-temperature catalysts.

Purification and characterization of two thermostable xylanases from a halotolerant Bacillus sp. Asc6BA isolated from Salar de Ascotán, Atacama Desert.

Two extracellular xylanases, denominated X2 and X3, were purified and characterized from the halotolerant bacterium Bacillus sp. Asc6BA isolated from "Salar de Ascotán" in the Atacama Desert. Xylanases were purified by anion exchange, cation exchange and size exclusion liquid chromatography. Xylanase X2 and X3 were purified ~ 690-fold and ~ 629-fold, respectively, compared to the concentrated extracellular fraction with a final specific activity of 169 and 154umg-1, respectively. Optimal conditions of pH and temperature of xylanolytic activity were 6.0 and 60°C for X2 and 7.0 and 60°C for X3. Half-life of X2 xylanase was 30min at 50°C, while X3 xylanase was remarkably more thermostable, retaining more than 70% of its activity after 32h of incubation at 50°C. X2 exhibited Km, Vmax and kcat values of 7.17mgmL-1, 1.28mMmin-1mg-1 and 425.33s-1, respectively. X3 exhibited Km, Vmax and kcat values of 6.00mgmL-1, 19.25mMmin-1mg-1 and 82,515s-1, respectively. In addition to their thermal stabilities, these enzymes were shown to be resistant to freeze-drying. These stability properties, in addition to the ability of these enzymes to be active in a wide range of temperatures and pHs, make these xylanases good candidates for industrial applications.

Immobilization of the extracellular recombinant Lucky9 xylanase from Bacillussubtilis enhances activity at high temperature and pH.

In the paper industry, chlorine is often used to treat the pulp for bleaching. After pulping, a large amount of xylan is present in the fiber. Xylanase can be used to degrade xylan in an eco-friendly process called biobleaching, which can help minimize the usage of chlorine in the delignification process. However, a bottleneck in the adoption of biobleaching is the cost of xylanase and the requirement that xylanase be active and stable at extreme conditions. Here, we investigated whether using sodium alginate beads to immobilize an extracellular xylanase from Bacillussubtilis (Lucky9) can reduce the potential cost of enzyme usage. The optimal pH and the activity of the immobilized enzyme were increased at optimal temperature compared with the free enzyme. In addition, immobilized xylanase was shown to be more stable than free xylanase. The results of this study suggest that the immobilized xylanase has potential applications in the biobleaching industry.

Efficient production of acetylated xylooligosaccharides from Hawthorn kernels by a xylanase from Paecilomyces aerugineus

Hawthorn (Crataegus pinnatifida) kernels can be utilized as a good source for production of acetylated XOS. A GH family 10 xylanase gene (PaXyn10A) from Paecilomyces aerugineus was cloned and expressed in Pichia pastoris. PaXyn10A shared the highest identity of 77 % with a xylanase from Aspergillus niger. The highest extracellular xylanase activity of 20,100 U/mL with protein concentration of 19 mg/mL was obtained in a 5-L fermentor. PaXyn10A was most active at pH 5.5 and 55 °C, respectively. It hydrolyzed different xylans to produce mainly xylooligosaccharidess (XOS) with degree of polymerization 2–6. To produce XOS, hawthorn kernels (HK) were pretreated by steam explosion at 185 °C with 25 min, and then hydrolyzed by PaXyn10A. The highest XOS yield of 18.7 g/100 g HK with hydrolysis ratio of 66.8 % was achieved. Xylooligosaccharides from HK were heavily acetylated at positions of 2-, 3-, and 2, 3-. This is a promising strategy for utilization of HK to produce acetylated XOS in an industrial point of view.