Xylanase Production Research Articles

Valorization of agro-wastes by Bacillus altitudinis XYL17 through simultaneous production of xylanase, xylooligosaccharides, and antioxidant compounds

Bacterial strains were isolated from undisturbed regions of Sikkim Himalaya for the production of highly active xylanase and xylooligosaccharides (XOS). Seven bacterial isolates showed xylanase production while fermenting beechwood xylan (BX) and the agrowastes corncob (CC), rice straw (RS), and sugarcane bagasse (SB). Bacillus altitudinis XYL17, isolated from soil at an elevation of 4083 m, produced the highest amount of xylanase. Maximum xylanase production was observed during BX fermentation after 24 h with the an observed activity of 6576.96 ± 82.75 U/mg. CC was observed to be the best agrowaste substrate for xylanase production with an observed activity of 4150.25 ± 94.64 U/mg. Xylopentaose was the most prominent XOS, with a highest observed content of 11.206 ± 0.331 mg/mL in RS hydrolysate. Significant increase of total phenolic content was observed for hydrolysates of B. altitudinis XYL17 fermented agro-wastes as compared to the control, while there was no significant difference (p < 0.05) in phenolic content between the control and fermented BX. The biotechnological potential of the XOS-rich hydrolysate was demonstrated by the observed radical scavenging, HOCl reducing, and myeloperoxidase (MPO) inhibitory activity. MPO inhibitory IC50 of 6.88 mg/mL and 9.4 mg/mL was observed for hydrolysates of SB and CC, respectively. We conclude that B. altitudinis XYL17 can be used for the valorization of agricultural wastes for the simultaneous production of highly efficient xylanase along with XOS and other antioxidant compounds.

Optimization of hydrolases production by Penicillium crustosum in submerged fermentation using agro-waste residues as cosubstrate

This study aimed to enhance the production of exo-polygalacturonases (exo-PG), xylanases, and endoglucanases using Penicillium crustosum through submerged fermentation. Lemon peel (LP) and wheat bran (WB) were examined as potential cosubstrate for hydrolase production. The highest exo-PG and endoglucanase activities were observed with LP, whereas xylanase activity showed improvement with the addition of WB. To optimize exo-PG and endoglucanase production, a 23 Face-Centered Central Composite Design (FCCCD) was employed, varying crucial physicochemical factors. Similarly, a 24 FCCCD was utilized to optimize xylanase production by varying temperature, pH, MnSO4 concentration as a cofactor, and the PDB culture medium. Under optimized conditions, exo-PG production reached 1427.99 ± 27.41 U/mL, marking a 45% increase compared to initial fermentation conditions. Endoglucanase production peaked at 62.68 ± 9.65 U/mL under conditions of 45 °C, pH 5.5, and 2.5 g/L MnSO4. Xylanase production reached 271.77 ± 8.50 U/mL under optimal conditions (37.5 °C, pH 5.5, 2.5 g/L MnSO4, and 62.5% PDB). The optimized simultaneous production of the hydrolytic cocktail occurred at 45 °C, pH 5.5, and 2.5 g/L MnSO4 with LP, yielding maximum activities of 959.74 U/mL for exo-PG, 61.98 U/mL for endoglucanases, and 65.60 U/mL for xylanases as determined by FCCCD. In conclusion, Penicillium crustosum shows promise as a hydrolase producer, and the utilization of agro-wastes as cosubstrate enhances the yield of enzymes with industrial relevance.

Production of Phytase, Protease and Xylanase by Aspergillus niveus with Rice Husk as a Carbon Source and Application of the Enzymes in Animal Feed

Production of Phytase, Protease and Xylanase by Aspergillus niveus with Rice Husk as a Carbon Source and Application of the Enzymes in Animal Feed

Optimization of xylanase production by Bacillus sp. MCC2212 under solid-state fermentation using response surface methodology

Xylanases have become increasingly significant due to their versatile applications in the food, paper, and pharmaceutical industries. Nevertheless, the current production of these enzymes relies on costly substrates, with estimates indicating that over 30% of the production expenses are attributed to these substrates. The objective of this study is to optimize the physico-chemical parameters for obtaining the maximum production of xylanase enzyme from Bacillus sp. MCC2212. The production conditions were statistically optimized using Plackett-Burman design (PBD) and Central Composite design (CCD). The significant variables identified through PB design including temperature, substrate to moisture ratio, MgSO4, peptone, tween 20, inoculum size, inoculum age, incubation time, and extracted xylan, were further optimized using the CCD approach. This optimization process revealed the most influential factors affecting xylanase production, with optimal conditions observed at a temperature, 35 °C; a substrate to moisture ratio, 1:1.75 g:mL; MgSO4, 1.1%; peptone, 1.5%; tween 20, 1.25%; inoculum size, 10 % (v/w); inoculum age, 20 h; an incubation time, 50 h; and crude xylan extracted from wheat bran at a concentration of 6% v/w. The R2 value of the model was found to be 0.9901, indicating its effectiveness. The optimized CCD model displayed a 1.84-fold greater xylanase production than the PB design approach. These findings suggest that wheat bran, a readily available agro-waste, could be a feasible alternative to the conventional substrate, beechwood xylan, for the production of xylanase enzyme with the possibility of achieving higher production levels optimized by using statistical design approach.

Exploring the Hot Springs of Golan: A Source of Thermophilic Bacteria and Enzymes with Industrial Promise.

In recent years, there has been a surge in research on extremophiles due to their remarkable ability to survive in harsh environments. Extremophile thermophilic bacteria provide thermostable enzymes for biotechnology and industry. Thermophilic bacteria live in extreme environments like hot springs at 45-80°C. This study screens and isolates thermophilic bacteria and thermozymes from the Golan hot springs in Karakocan, Elazig, Turkey. The study also characterizes thermophilic bacteria and their thermozymes to understand their features and applications better. Golan hot spring water samples at 50°C yielded 12 isolates. GKE 02, 07, 08, and 10 produce amylase, GKE 04, 08, and 11 cellulase, and GKE 06 xylanase. One isolate (GKE 08) displayed both amylolytic and cellulolytic activity on agar plates. GKE 02 had the highest plate assay amylolytic index (2.3) and amylase activity (67.87 U/ml). Plate assay indicates GKE 08 has 1.5 amylolytic index, 1.1 cellulolytic index, 38.57 U/ml amylase, and 6.81 U/ml cellulase. GKE 04 had the greatest cellulolytic index (1.7) and cellulase activity (27.46). GKE 06, the only xylanase producer, has 19.67 U/ml activity and 1.4 plate assay index. The investigation also included determining the optimal pH and temperature conditions for each enzyme. 16S rDNA gene sequencing revealed seven thermozyme-producing bacteria Bacillus, Geobacillus, and Thermomonas. Thermomonas hydrothermalis genome annotation showed glycosyl hydrolase genes for amylolytic and cellulolytic activity. The findings of this study on thermophilic bacteria and thermostable enzyme synthesis in the Golan hot springs are promising, particularly for T. hydrothermalis, which has limited research on its potential as a thermozyme producer.

An efficient CRISPR/Cas9 genome editing system based on a multiple sgRNA processing platform in Trichoderma reesei for strain improvement and enzyme production.

The CRISPR/Cas9 technology is being employed as a convenient tool for genetic engineering of the industrially important filamentous fungus Trichoderma reesei. However, multiplex gene editing is still constrained by the sgRNA processing capability, hindering strain improvement of T. reesei for the production of lignocellulose-degrading enzymes and recombinant proteins. Here, a CRISPR/Cas9 system based on a multiple sgRNA processing platform was established for genome editing in T. reesei. The platform contains the arrayed tRNA-sgRNA architecture directed by a 5S rRNA promoter to generate multiple sgRNAs from a single transcript by the endogenous tRNA processing system. With this system, two sgRNAs targeting cre1 (encoding the carbon catabolite repressor 1) were designed and the precise deletion of cre1 was obtained, demonstrating the efficiency of sgRNAs processing in the tRNA-sgRNA architecture. Moreover, overexpression of xyr1-A824V (encoding a key activator for cellulase/xylanase expression) at the ace1 (encoding a repressor for cellulase/xylanase expression) locus was achieved by designing two sgRNAs targeting ace1 in the system, resulting in the significantly enhanced production of cellulase (up to 1- and 18-fold on the Avicel and glucose, respectively) and xylanase (up to 11- and 41-fold on the Avicel and glucose, respectively). Furthermore, heterologous expression of the glucose oxidase gene from Aspergillus niger ATCC 9029 at the cbh1 locus with the simultaneous deletion of cbh1 and cbh2 (two cellobiohydrolase coding genes) by designing four sgRNAs targeting cbh1 and cbh2 in the system was acquired, and the glucose oxidase produced by T. reesei reached 43.77U/mL. Besides, it was found the ER-associated protein degradation (ERAD) level was decreased in the glucose oxidase-producing strain, which was likely due to the reduction of secretion pressure by deletion of the major endogenous cellulase-encoding genes. The tRNA-gRNA array-based CRISPR-Cas9 editing system was successfully developed in T. reesei. This system would accelerate engineering of T. reesei for high-level production of enzymes including lignocellulose-degrading enzymes and other recombinant enzymes. Furthermore, it would expand the CRISPR toolbox for fungal genome editing and synthetic biology.

Porcine in vitro digestion and solubilization of non-starch polysaccharides in corn and wheat supplemented with xylanase and feruloyl esterase

A study evaluated porcine in vitro digestion and non-starch polysaccharide (NSP) solubilization of corn and wheat without or with xylanase alone or xylanase plus feruloyl esterase (FE). The enzymes supplied 4000 U of xylanase and 35 U of FE per kilogram of sample. Samples were digested with pepsin at a pH of 2.0 and then with pancreatin at a pH of 6.8. The digested samples were filtered to obtain unhydrolyzed residue, which was then washed using alcohol and acetone. The cereal grains (CGs) before in vitro digestion and the washed unhydrolyzed residues were analyzed for dry matter and NSP. Xylanase improved ( P &lt; 0.05) in vitro digestibility of dry matter (IVDDM) for wheat (85% vs. 89%), but not for corn (78% vs. 79%). Addition of FE to the xylanase-supplemented CGs increased ( P &lt; 0.05) IVDDM for both CGs, and reduced ( P &lt; 0.05) the arabinoxylans in the unhydrolyzed residue for corn, but not for wheat. In conclusion, xylanase product used in the current study is more effective in improving the digestibility of nutrients in wheat than in corn. The FE product used in the current study can improve the efficacy of xylanase in improving the nutrient digestibility of wheat and corn.

Optimization of the extracellular secretion of black goat rumen metagenome-derived KG42 xylanase by Bacillus subtilis

Xylanase (E.C. 3.2.1.8) is the enzyme that breaks down β-1,4 xylan by cleaving β-1,4 glycosidic linkages. Production of xylanases is important for various industrial applications. Here, we aimed to determine the optimal incubation conditions for expression and secretion of KG42 xylanase in Bacillus subtilis using response surface methodology based on Box-Behnken design in preparation for industrial applications. Among nine broth media tested in this study, Power Broth was chosen as a basal medium. In addition to the basal medium, the four other independent variables of extra carbon sources (glucose, lactose, mannose, fructose, and sucrose), extra nitrogen sources (beef extract, yeast extract, tryptone, urea, NaNO3, and (NH4)2SO4), isopropyl β-D-1-thiogalactopyranoside concentrations, and induction times were individually tested using one factor at a time in an optimization experiment. Next, a Box-Behnken design-based response surface methodology approach was used to identify and validate the optimized incubation conditions with the four variables in batch culture. The statistically optimized incubation conditions obtained from this study yielded a maximum of approximately 3- to 4-fold increases in the expression and secretion of KG42 xylanase by B. subtilis in comparison with unoptimized medium and incubation conditions.

Enhancing the efficiency and functionality of xylanase from Bacillus sp. RTS11: Optimization, purification, characterization, and prospects in kraft pulp bleaching.

Bacillus sp. RTS11, a xylanolytic strain, was isolated from the Algerian desert rocks. Genetic analysis revealed a remarkable 98.69% similarity to Bacillus pumilus. We harnessed optimization techniques, including Plackett-Burman screening and Box-Behnken optimization design, to amplify xylanase production and activity. The outcome of these efforts was an optimized medium that yielded an impressive xylanase production titer of 448.89 U, a threefold increase compared to the non-optimized medium (146 U). The Purification of xylanase was achieved through the three-phase partitioning technique, employing t-butanol and various chromatographic methods. Notably, anion exchange chromatography led to isolating a highly pure enzyme with a molecular weight of 60 kDa. The xylanase exhibited its peak activity at a temperature of 60°C and a pH of 9.0. When applied to pulp pretreatment, 20 U/g of xylanase demonstrated a substantial increase in the release of phenolic and chromophore compounds while reducing sugar content in the pulp. Furthermore, this versatile xylanase shows its ability to efficiently hydrolyze a variety of agro-industrial residues, including wheat bran, corn and grape waste, wheat straw, and sugarcane bagasse. These findings underscore the significant potential of this xylanase enzyme in biobleaching processes and the utilization of agro-industrial waste, opening up exciting avenues for sustainable and environmentally friendly industrial applications.

Optimization of culture conditions for xylanase production by mixed fungal fermentation: Effects on pretreated maize cobs

The use of microorganisms for xylanase production plays important role in the bioconversion of lignocelluloses and also required in huge amount for industrial level application. This necessitates the need to select potent microorganisms for xylanase production, followed by optimization of media components for enhanced production. The effects of altering cultural fermentation conditions on the xylanase production ability in maize cobs were investigated. A consortium of four fungi; Lenzites betulina, Trichoderma reesei, Lachnocladium specie and Aspergillus niger were used to carry out single and mixed solid-state fermentation on NaOH pretreated maize cobs. Optimization of fermentation factors were carried out from ten groups of individual and co-fermented fungal combinations. L. flavidum was found to be the most effective xylanase producer with optimal conditions at pH of 5.5, moisture 75%, inoculum concentration at 5-6 x 103 spores/ml, incubation period of 7-9 days and 1% peptone as the best nitrogen media supplement. Variation to different degrees in the degradation of the maize cobs were observed. A 10% decrease in cellulose was observed with co- cultures of T. reesei and A. niger and a 15% decrease in the hemicellulose fraction. The biotechnological potential of corn cobs has been enhanced by the screening and optimizing the culture conditions.

Endogenous Enzyme Activities, Digestibility of Nutrients and Digestive Physiology as Affected by High Inclusion of Cottonseed Meal in Broiler Chicken Diets

A 3 × 3 factorial study examined the performance, endogenous enzyme activities and apparent ileal digestibility of nutrients, including energy, protein, starch, dry matter, amino acids and minerals of birds fed diets containing graded levels of cottonseed meal (CSM) with or without microbial enzymes. Nine iso-caloric and iso-nitrogenous diets were formulated mainly from wheat/sorghum/soybean meal. Three levels of CSM none (0%), low (5%, 10%, and 15%) or high (6%, 12%, and 18%) in the starter, grower, and finisher diets, respectively, were included. The diets were supplemented with three levels of a composite xylanase and beta-glucanase product at 0, 100 or 150 mg/kg diet. Each of the nine dietary treatments was randomly assigned to 6 replicates, with 10 birds per replicate. Feed intake (FI) and weight gain (WG) were recorded on day 10, 24 and 35, and feed conversion ratio (FCR) was obtained from the data, while enzyme activities and nutrient digestibility were measured on samples collected on day 10 and 24, and day 24 only, respectively. Feed intake up to day 35 decreased (P &lt; 0.05) significantly with increasing enzyme supplementation. On the CSM-containing diets, enzyme supplementation at 100 or 150 mg/kg improved WG up to day 24, with the heaviest birds (1514 g/b) observed in the low CSM with 100 mg/kg enzyme group. Enzyme supplementation improved (P &lt; 0.01) FCR all through the growth phases and also improved (P &lt; 0.05) ileal crude protein and starch digestibility. High levels of CSM decreased the digestibility of starch, but this was improved (P &lt; 0.05) by enzyme supplementation, showing an interaction between CSM and microbial enzyme on starch digestibility. The digestibility of arginine, glutamic acid and threonine improved with increased CSM inclusion, and that of methionine improved with increased enzyme supplementation. There was an interaction effect (P &lt; 0.01) between CSM and enzyme on magnesium digestibility. Cottonseed meal inclusion improved the digestibility of copper and potassium and reduced the digestibility of phosphorus, while that of calcium and manganese was increased (P &lt; 0.01) with enzyme supplementation. At day 10, lipase activity was increased (P &lt; 0.05) by higher CSM levels, while on day 24, general proteolytic activity was highest (P &lt; 0.05) when 100 mg enzyme was supplemented in the diet. These results indicate that relatively high levels of CSM in diet have no negative effects on the growth and digestive physiology of broiler chickens when supplemented with xylanase and β–glucanase.

Production of Xylanase by Trichoderma Species Growing on Olive Mill Pomace and Barley Bran in a Packed-Bed Bioreactor.

Xylanases are hydrolytic enzymes that have tremendous applications in different sectors of life, but the high cost of their production has limited their use. One solution to reduce costs and enhance xylanase production is the use of agro-wastes as a substrate in fungal cultures. In this study, olive mill pomace (OMP) and barley bran (BB) were used as carbon sources and possible inducers of xylanase production by three species of Trichoderma (atroviride, harzianum, and longibrachiatum), one major xylanase producer. The experiments were conducted under a solid-state fermentation system (SSF) in flask cultures and a packed-bed bioreactor. Cultures of OMP and BB were optimized by examining different ratios of OMP and BB, varied particle sizes, and inoculum size for the three species of Trichoderma. The ratio of 8:2 OMP and BB yielded the highest xylanase activity, with a particle size of 1 mm at 29 °C and an inoculum size of 1 × 107 spores/mL. Studying the time profile of the process revealed that xylanase activity was highest after seven days of incubation in flask SSF cultures (1.779 U/mL) and after three days in a packed-bed bioreactor (1.828 U/mL). The maximum percentage of OMP degradation recorded was about 15% in the cultures of T. harzianum flask SSF cultures, compared to about 11% in T. longibrachiatum bioreactor cultures. Ammonium sulfate precipitation and dialysis experiments showed that Xylane enzyme activity ranged from 0.274 U/mL in T. harzianum to 0.837 U/mL in T. atroviride when crude extract was used, with the highest activity (0.628 U/mL) at 60% saturation. Xylose was the main sugar released in all purified fractions, with the G-50 and G-75 fractions showing the maximum units of xylanase.

Isolation and screening of potential lignocellulolytic microbes from Phra Nakhon Si Ayutthaya Province

Phra Nakhon Si Ayutthaya Province is an important area for rice cultivation in Thailand. However, large amounts of rice straw generated at harvest time are often burnt in the fields, causing environmental degradation and air pollution. To address this, rice straw composting by degrading microorganisms has been identified as an effective alternative for sustainable waste management, with potential applications in environmental biotechnology. For this study, 28 microorganism samples were collected from various sources in Phra Nakhon Si Ayutthaya Province, including a corrugated paper manufacturer, plant-cultivated soil, compost from dung, black soldier flies, and vermicompost. Lignocellulolytic microorganisms were screened for cellulase, xylanase, and manganese peroxidase production on agar plates and broth media using commercial and rice straw substrates. The lignocellulolytic activities were compared among the isolated and non-pathogenic microorganisms. A microbial consortium with high potential to degrade rice straw was identified, consisting of two thermophilic bacteria, Bacillus licheniformis BKT1 and BOT4, two mesophilic actinomycetes, Streptomyces ardesiacus AQ4 and An6, two thermophilic actinomycetes, Streptomyces thermoalcalitolerans APT3 and Streptomyces thermoviolaceus subsp. thermoviolaceus AQT2, and two fungi, Penicillium sp. Fh11 and Aspergillus sp. Fj6. This microbial consortium induced rapid composting of rice straw, thereby supporting sustainable agricultural waste management and reducing PM25 air pollution.

Insights into Endophytic and Rhizospheric Bacteria of Five Sugar Beet Hybrids in Terms of Their Diversity, Plant-Growth Promoting, and Biocontrol Properties

Sugar beet is the most important crop for sugar production in temperate zones. The plant microbiome is considered an important factor in crop productivity and health. Here, we investigated the bacterial diversity of seeds, roots, and rhizosphere of five sugar beet hybrids named Eduarda (ED), Koala (KO), Tibor (T), Tajfun (TF), and Cercospora-resistant (C). A culture-independent next-generation sequencing approach was used for the further investigation of seed-borne endophytes. Hybrid-associated bacteria were evaluated for their plant growth–promoting (PGP) characteristics, antagonistic activity towards Cercospora beticola and several Fusarium strains in dual culture assays, and drought and salinity tolerance. High-throughput sequencing revealed that the Proteobacteria phylum was most dominant in the seeds of all hybrids, followed by Cyanobacteria and Actinobacteriota. The predominant genus in all hybrids was Pantoea, followed by Pseudomonas, Acinetobacter, Chalicogloea, Corynebacterium, Enterobacter, Enterococcus, Glutamicibacter, Kosakonia, and Marinilactibacillus. Unique genera in the hybrids were Pleurocapsa and Arthrobacter (T), Klebsiella (TF), Apibacter (ED), and Alloscardovia (KO). The genera that were most represented in one hybrid were Weissella and Staphylococcus (TF); Streptococcus (T); Gardnerella, Prevotella, and Rothia (KO); and Gilliamella, Lactobacillus, and Snodgrassella (ED). Thirty-two bacteria out of 156 isolates from the rhizosphere, roots, and seeds were selected with respect to various plant growth–promoting activities in vitro, i.e., nitrogen fixation, phosphate solubilization, siderophore production, indole-3-acetic acid production, 1-aminocyclopropane-1-carboxylic acid deaminase activity, hydrogen cyanide production, exoenzymatic activity (amylase, protease, lipase, cellulase, xylanase, mannanases, gelatinase, and pectinase), mitigation of environmental stresses, and antifungal activity. Mixta theicola KO3-44, Providencia vermicola ED3-10, Curtobacterium pusillum ED2-6, and Bacillus subtilis KO3-18 had the highest potential to promote plant growth due to their multiple abilities (nitrogen fixation, phosphate solubilization, production of siderophores, and IAA). The best antagonistic activity towards phytopathogenic fungi was found for Bacillus velezensis C3-19, Paenibacillus polymyxa C3-36 and Bacillus halotolerans C3-16/2.1. Only four isolates B. velezensis T2-23, B. subtilis T3-4, B. velezensis ED2-2, and Bacillus halotolerans C3-16/2.1 all showed enzymatic activity, with the exception of xylanase production. B. halotolerans C3-16/2.1 exhibited the greatest tolerance to salinity, while two B. subtilis strains (C3-62 and TF2-1) grew successfully at the maximum concentration of PEG. The current study demonstrates that sugar beet–associated bacteria have a wide range of beneficial traits and are therefore highly promising for the formulation of biological control and PGP agents.

Recent developments in heterologous production of cellulases and xylanases using the Pichia pastoris expression system

AbstractThe yeast expression system, Pichia pastoris, is one of the most robust and versatile expression systems in biotechnology and molecular biology, generally considered as a safe host for heterologous protein expression, especially for producing cellulases and xylanases at the industrial level. Despite the high recombinant protein expression rate, the potential of the P. pastoris expression system has still not been fully explored. Cultivation of P. pastoris under optimized conditions greatly relies on the strain and is associated with certain problems such as promoter strength, sensitivity to methanol, and oxygen demand. To address these issues, different genetic engineering strategies have been employed. Advancements in promoter engineering, optimization of gene dosage and codon usage, recombinant plasmid engineering using CRISPR/Cas9 system, and directed evolution strategies have proven beneficial to the yield of cellulase expression levels. This study will systemically review recent progress in various genetic engineering strategies to enhance cellulase and xylanase expression in the P. pastoris expression system. The utilization of alcohol oxidase 1 promoter (pAOX1), methanol‐free system, and recombinant plasmid engineering for improved production of these enzymes are highlighted. Additionally, we discuss the recent advancements in the P. pastoris expression system toolbox for improved cellulase and xylanase production, thus providing a deep insight into how P. pastoris is becoming the indispensable platform for heterologous protein production. © 2023 Society of Chemical Industry (SCI).

Coffee cherry processing waste to produce value-added products through clean production biorefineries

Indonesia is the 4th largest coffee-producing country in the world. Coffee cherry can produce 60% CCPW, more than the core product of coffee beans as the main product produced. CCPW contains lignocellulosic material that can be converted into value-added products. This research optimizes the production of xylose and xylanase enzymes as an integrated process with a biorefinery model to increase the added value of CCPW using the Research Surface Methodology (RSM). Enzyme production is carried out using a bioprocess using Trichoderma viride. Xylose production is carried out using the dregs of xylanase production as raw material and xylanase enzymes which are produced in the integrated process. The xylanase and xylose produced are in the liquid phase. The treatment design for xylanase production is substrate concentration and incubation time, while for xylose production it is substrate concentration and xylanase concentration. Based on the optimization conducted, the optimum condition of xylanase production was 75% substrate concentration with an incubation time of 72 hours, which will result in xylanase an enzyme with an activity of 29.275 U/ml with desirability of 99.55%. On the other hand, the optimum condition of xylose production was 12.54% substrate concentration with a xylanase concentration of 4.01%, which will result in 0.53 g/L xylose content with desirability of 94.06%. These findings show that xylose production efficiency can be achieved through integrated two-stage biorefinery production for efficient and sustainable utilization of CCPW.

Enhanced production of a recombinant xylanase (XT6): optimization of production and purification, and scaled-up batch fermentation in a stirred tank bioreactor

The endoxylanase XT6 produced by Geobacillus stearothermophilus is a desirable candidate for industrial applications. In this study, the gene encoding XT6 was cloned using the pET-28a expression vector and expressed in Escherichia coli BL21 (DE3) cells. Recombinant XT6 production was improved by optimizing cell lysis (sonication, chemical, and enzymatic lysis) and expression conditions. Sonication in a 0.05 M sodium phosphate (pH 6.0) buffer resulted in the highest xylanase activity (16.48 U/ml). Screening and optimization of induction conditions using the Plackett–Burman Design and Box-Behnken Design (BBD) approaches revealed that cell density pre-induction (OD600 nm), post-induction incubation time, and IPTG concentration significantly (p < 0.05) influenced the expression levels of XT6 (16.48 U/ml to 40.06 U/ml) representing a 3.60-fold increase. BBD resulted in a further 8.74-fold increase in activity to 144.02 U/ml. Batch fermentation in a 5-l stirred tank bioreactor at 1 vvm aeration boosted recombinant xylanase production levels to 165 U/ml suggesting that heterologous expression of the XT6 enzyme is suitable for scaled-up production. The pure enzyme with a molecular weight of 43 kDa and a 15.69-fold increase in purity was obtained using affinity chromatography and a cobalt column. Future studies will include application of the purified recombinant xylanase to animal feed.

Sustainable and Eco-Friendly Production of Xylanase and Other Enzymes from Tomato Paste through Solid-State Fermentation Using Bacillus safensis MABS6

Sustainable and Eco-Friendly Production of Xylanase and Other Enzymes from Tomato Paste through Solid-State Fermentation Using Bacillus safensis MABS6

Molecular and Biochemical Characterization of Xylanase Produced by Streptomyces viridodiastaticus MS9, a Newly Isolated Soil Bacterium.

A xylan-degrading bacterial strain, MS9, was recently isolated from soil samples collected in Namhae, Gyeongsangnam-do, Republic of Korea. This strain was identified as a variant of Streptomyces viridodiastaticus NBRC13106T based on 16S rRNA gene sequencing, DNA-DNA hybridization analysis, and other chemotaxonomic characteristics, and was named S. viridodiastaticus MS9 (=KCTC29014= DSM42055). In this study, we aimed to investigate the molecular and biochemical characteristics of a xylanase (XynCvir) identified from S. viridodiastaticus MS9. XynCvir (molecular weight ≍ 21 kDa) was purified from a modified Luria-Bertani medium, in which cell growth and xylanase production considerably increased after addition of xylan. Thin layer chromatography of xylan-hydrolysate showed that XynCvir is an endo-(1,4)-β-xylanase that degrades xylan into a series of xylooligosaccharides, ultimately converting it to xylobiose. The Km and Vmax values of XynCvir for beechwood xylan were 1.13 mg/ml and 270.3 U/mg, respectively. Only one protein (GHF93985.1, 242 amino acids) containing an amino acid sequence identical to the amino-terminal sequence of XynCvir was identified in the genome of S. viridodiastaticus. GHF93985.1 with the twin-arginine translocation signal peptide is cleaved between Ala-50 and Ala-51 to form the mature protein (21.1 kDa; 192 amino acids), which has the same amino-terminal sequence (ATTITTNQT) and molecular weight as XynCvir, indicating GHF93985.1 corresponds to XynCvir. Since none of the 100 open reading frames most homologous to GHF93985.1 listed in GenBank have been identified for their biochemical functions, our findings greatly contribute to the understanding of their biochemical characteristics.

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.