Xylanase Production Research Articles

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

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

Isolation, screening, preliminary optimisation and characterisation of thermostable xylanase production under submerged fermentation by fungi in Durban, South Africa

ABSTRACT Fungi are renowned for their ability to produce extracellular enzymes into their surrounding environment. Xylanases are hydrolytic enzymes capable of xylan degradation. The objectives of this study were to isolate, screen for potential xylanolytic fungi from soil and tree bark samples from three locations in South Africa and to determine their growth conditions for maximum xylanase production. Forty-six isolates were obtained based on clearing zone formation on xylan-enriched agar plates using Congo red indicator. Xylanase activity was quantified during submerged fermentation. Isolate MS5, identified as Trichoderma harzianum with the highest enzyme activity (38.17 U/ml) was selected for further studies based on thermophilic properties (70°C) and pH (5.0). The culture conditions; incubation period (5 days), agitation speed (160 rpm) wheat bran (1%) and ammonium sulphate (1.2%) were optimised further. Biochemical characterisation of the crude enzyme revealed two pH and temperature optima (pH 6.0 at 60°C and 70°C, pH 8.0 at 55°C and 75°C). The enzyme retained >70% activity after 4 h at pH 6.0 at 70°C. SDS-PAGE revealed multiple protein bands with a prominent band at 70 kDa. Substrate Native PAGE revealed multiple isoforms between 55 and 130 kDa. This enzyme will be beneficial for applications in the animal feed and biofuels industries.

Cellulolytic and Xylanolytic Enzymes from Yeasts: Properties and Industrial Applications.

Lignocellulose, the main component of plant cell walls, comprises polyaromatic lignin and fermentable materials, cellulose and hemicellulose. It is a plentiful and renewable feedstock for chemicals and energy. It can serve as a raw material for the production of various value-added products, including cellulase and xylanase. Cellulase is essentially required in lignocellulose-based biorefineries and is applied in many commercial processes. Likewise, xylanases are industrially important enzymes applied in papermaking and in the manufacture of prebiotics and pharmaceuticals. Owing to the widespread application of these enzymes, many prokaryotes and eukaryotes have been exploited to produce cellulase and xylanases in good yields, yet yeasts have rarely been explored for their plant-cell-wall-degrading activities. This review is focused on summarizing reports about cellulolytic and xylanolytic yeasts, their properties, and their biotechnological applications.

Degradative impact of Alternaria multiformis on novel polymeric biocomposites with the fillers of industrial waste materials under different pH and temperature conditions

The impact of Alternaria multiformis on novel polymeric biocomposites formed from epoxidized linseed oil and various types of fillers: pine needles (PN), pine bark (PB), grain mill waste (GW), rapeseed cake (RC) and specimen without filler (WF) under different pH and temperature conditions has been studied. Identification of fungal strain A. multiformis 0065 isolated from synthetic polymer was based on molecular and morphological analysis. Tested fungal strain showed significant production of polyphenol oxidase and weak production of amylase, endoglucanase, xylanase, and lipase. Polymeric biocomposites PB, GW, RC, and WF exposed to A. multiformis for four weeks showed the greatest weight loss at pH 4 or pH 5 and at a temperature of 26 °C, suggesting the optimal conditions for biodegradation of polymeric biocomposites studied. The attenuated total reflectance infrared spectroscopy (ATR-IR) and scanning electron microscopy (SEM) analysis confirmed biodegradation of polymeric biocomposites after fungal attack. ATR-IR technique revealed changes in the intensities of the signals, demonstrating dependence of degradation on the type of the filler. SEM analysis showed significant alterations of the specimen surfaces, indicating degradative impact of A. multiformis. Identification of the most biodegraded constituents could help in the creation of more environmentally friendly biocomposites.

Downstream Process of Xylanase Production from Oil Palm Empty Fruit Bunches: A Review

As an enzyme that can hydrolyze the glycosidic bond of hemicellulose, xylanase is widely used in pulp and paper, feed, food, and beverage industries. The vast availability of lignocellulosic biomass waste offers the potential of xylanase production by microorganisms via submerged and solid-state fermentation. However, the specific activity of the produced crude enzyme is still too low to be directly used in a commercial application. Downstream processing of the enzyme, that is purification and concentration, is necessary. Centrifugation or filtration can be used as a pre-treatment process for biomass separation before the next purification technologies. Precipitation, aqueous two-phase system (ATPS), chromatography, and membrane ultrafiltration (UF) are technologies for enzyme purification. Freeze drying can be used also as a late-stage purification procedure. ATPS and membrane UF have been emerged as a recovery method enabling the purification and concentration of the target biomolecule in a single operation. The downstream process of an enzyme usually accounts for 70-90% of the total production cost. Nonetheless set of purification methods that have high purification factors, cost-effective, high productivity, and environmentally benign strategies are needed such that the xylanase production process can be conducted economically at an industrial/commercial scale. This paper reviews processes for xylanase purification/concentration produced by using solid-state fermentation configuration. A membrane-based process for downstream processing of xylanase production is suggested.

Optimization and characterization of immobilized E. coli for engineered thermostable xylanase excretion and cell viability

There are many parameters that may have influenced the properties of cell during immobilization process. Particularly, the immobilization methods, carrier materials, and enzyme loading amount that have been proved to be important for immobilization process. The physiological responses of microorganisms are depending on the immobilization technique used. Typical alterations to the micro-environment of the immobilized cell involved the altered water activity, presence of ionic charges, cell confinement and modified surface tension. In this study, the graphene oxide was selected as a suitable carrier for immobilization process of recombinant E.coli and adsorption was chosen as an appropriate method to improve the production of engineered thermostable xylanase . High level production of thermostable xylanase by immobilized recombinant cell in the 5 L bioreactor was studied by using optimum research surface methodology (RSM) conditions was studied. The immobilization of E. coli onto nanoparticle matrix manages to improve the cell performance by improving the protein expression, reduced the occurrences of cell lysis as well as improved the plasmid stability of the host cell. Thus, immobilization contributes a physical support for both whole cells as well as enzymes to develop a better operative achievement system for industrialized fields and give rise to the biological advancement existing enzyme for instance xylanase.

Press water from the mechanical drying of Douglas-fir wood chips has multiple beneficial effects on lignocellulolytic fungi

BackgroundThe mechanical drying of wood chips is an innovative method that improves the heating value of sawmill by-products in an energy-efficient continuous process. The liquid that comes out of the wood chips as press water (PW), however, contains a variety of undissolved as well as dissolved organic substances. The disposal of the PW as wastewater would generate additional costs due to its high organic load, offsetting the benefits in energy costs associated with the enhanced heating value of the wood chips. Our research explored if the organic load in PW could be utilized as a substrate by cellulolytic filamentous fungi. Hence, using the industrially relevant Ascomycete Trichoderma reesei RUT-C30 as well as several Basidiomycete wood-rotting fungi, we examined the potential of press water obtained from Douglas-fir wood chips to be used in the growth and enzyme production media.ResultsThe addition of PW supernatant to liquid cultures of T. reesei RUT-C30 resulted in a significant enhancement of the endoglucanase and endoxylanase activities with a substantially shortened lag-phase. A partial replacement of Ca2+, Mg2+, K+, as well as a complete replacement of Fe2+, Mn2+, Zn2+ by supplementing PW of the liquid media was achieved without negative effects on enzyme production. Concentrations of PW above 50% showed no adverse effects regarding the achievable endoglucanase activity but affected the endoxylanase activity to some extent. Exploring the enhancing potential of several individual PW components after chemical analysis revealed that the observed lag-phase reduction of T. reesei RUT-C30 was not caused by the dissolved sugars and ions, nor the wood particles in the PW sediment, suggesting that other, so far non-identified, compounds are responsible. However, also the growth rate of several basidiomycetes was significantly enhanced by the supplementation of raw PW to the agar medium. Moreover, their cultivation in liquid cultures reduced the turbidity of the PW substantially.ConclusionsPW was identified as a suitable media supplement for lignocellulolytic fungi, including the cellulase and xylanase producer T. reesei RUT-C30 and several wood-degrading basidiomycetes. The possibility to replace several minerals, trace elements and an equal volume of fresh water in liquid media with PW and the ability of fungal mycelia to filter out the suspended solids is a promising way to combine biological wastewater treatment with value-adding biotechnological applications.

Fusarium graminearum as a producer of xylanases with low cellulases when grown on wheat bran

The xylanolytic potential of endophytic fungi isolated from leaves of Theobroma cacao was explored for the first time. Four fungal strains showed significant amounts of xylanase activity and low cellulase levels when grown on wheat bran as the sole carbon source. Strain Ec220 of Fusarium graminearum had the highest xylanase production (1.79 U/ml), whereas its cellulase activity was minimal (0.24 U/ml). Optimal conditions for xylanase production were: 154 h of incubation time, pH 5.79 and 29.8 °C. Furthermore, two protein spots detected by two-dimensional gel electrophoresis showed molecular weights (26.05 and 27.70 kDa) and isoelectric points (6.18 and 9.20) corresponding to previously reported F. graminearum xylanases, Xyl A and Xyl B, respectively. Therefore, endophytic fungi of T. cacao can be an important source of xylanolytic activities when cultured on wheat bran, and xylanases with low cellulases found in strain Ec220 require further characterization as they show promise for possible industrial applications.

Xylanase Production by Talaromyces amestolkiae Valuing Agroindustrial Byproducts.

In general, agroindustrial byproducts can be easily assimilated by several microorganisms due to their composition, which is rich in carbohydrates. Therefore, they could be appropriate for use as raw materials in a sustainable refinery concept, including the production of hydrolytic enzymes with industrial applicability. In this work, xylanase production by the filamentous fungi Talaromyces amestolkiae in submerged culture was evaluated using five agroindustrial byproducts, namely, wheat bran, citrus pulp, rice bran, peanut skin, and peanut shell. Firstly, the aforementioned byproducts were characterized in terms of cellulose, xylan, lignin, and extractives. Next, production studies were performed, and wheat bran generated the highest enzymatic activity (5.4 U·mL−1), probably because of its large amount of xylan. Subsequently, a factorial design was performed to evaluate the independent variables yeast extract, wheat bran, K2HPO4, and pH, aiming to improve the variable response, xylanase activity. The condition that promoted the highest production, 13.02 U·mL−1 (141% higher than the initial condition), was 20 g·L−1 wheat bran, 2.5 g·L−1 yeast extract, 3 g·L−1 K2HPO4, and pH 7. Thus, industrial byproducts with a high content of xylan can be used as a culture medium to produce xylanase enzymes with a Talaromyces strain through an economical and sustainable approach.

Utilization of Agroresidues for the Production of Xylanase by Bacillus safensis XPS7 and Optimization of Production Parameters

The main objective of this study was to investigate the utilization of various agro-industrial wastes such as wheat bran, wheat husk, wheat straw, peanut powder, pomace, corn cobs, rice straw, sawdust and sugarcane bagasse for the cost-effective production of xylanase by Bacillus safensis XPS7 using the one-variable-at-a-time approach. A number of bacterial strains were isolated from different locations in the cold desert region of Himachal Pradesh, India. Among these, the hyperproducing strain designated as XPS7 was selected for optimized production of xylanase and identified as B. safensis based on 16S rDNA gene analysis. B. safensis XPS7 produced the maximum xylanase (141.28 U/mL) at 45 °C, pH 9, 24 h incubation time, 2% (w/v) wheat straw and wheat bran mixture as carbon source and 1.5% (w/v) ammonium nitrate as the nitrogen source in modified Riviere’s medium. The results showed that the combination of wheat straw and wheat bran proved to be a cheap and abundant source for the hyper production of xylanase and can be used as an inexpensive base (carbon source) for large scale industrial production of enzymes. In addition, the use of waste for the economic production of enzymes will also help to minimize the environmental problems associated with the disposal of agro-industrial waste.

Valorization of Brewer's Spent Grain Using Biological Treatments and its Application in Feeds for European Seabass (Dicentrarchus labrax).

Brewer’s spent grain (BSG) is the main brewery industry by-product, with potential applications in the feed and food industries due to its carbohydrate composition. In addition, the lignocellulosic nature of BSG makes it an adequate substrate for carbohydrases production. In this work, solid-state fermentation (SSF) of BSG was performed with Aspergillus ibericus, a non-mycotoxin producer fungus with a high capacity to hydrolyze the lignocellulosic matrix of the agro-industrial by-products. SSF was performed at different scales to produce a crude extract rich in cellulase and xylanase. The potential of the crude extract was tested in two different applications: -(1) - the enzymatic hydrolysis of the fermented BSG and (2) - as a supplement in aquafeeds. SSF of BSG increased the protein content from 25% to 29% (w/w), while the fiber content was reduced to 43%, and cellulose and hemicellulose contents were markedly reduced to around 15%. The scale-up of SSF from 10 g of dry BSG in flasks to 50 g or 400 g in tray-type bioreactors increased 55% and 25% production of cellulase and xylanase, up to 323 and 1073 U g−1 BSG, respectively. The optimum temperature and pH of maximal activities were found to be 55°C and pH 4.4 for xylanase and 50°C and pH 3.9 for cellulase, cellulase being more thermostable than xylanase when exposed at temperatures from 45°C to 60°C. A Box–Behnken factorial design was applied to optimize the hydrolysis of the fermented BSG by crude extract. The crude extract load was a significant factor in sugars release, highlighting the role of hydrolytic enzymes, while the load of fermented BSG, and addition of a commercial β-glucosidase were responsible for the highest phenolic compounds and antioxidant activity release. The lyophilized crude extract (12,400 and 1050 U g−1 lyophilized extract of xylanase and cellulase, respectively) was also tested as an enzyme supplement in aquafeed for European seabass (Dicentrarchus labrax) juveniles. The dietary supplementation with the crude extract significantly improved feed and protein utilization. The processing of BSG using biological treatments, such as SSF with A. ibericus, led to the production of a nutritionally enriched BSG and a crude extract with highly efficient carbohydrases capable of hydrolyzing lignocellulosic substrates, such as BSG, and with the potential to be used as feed enzymes with remarkable results in improving feed utilization of an important aquaculture fish species.

Valorization of corn cobs for xylanase production by Aspergillus flavus AW1 and its application in the production of antioxidant oligosaccharides and removal of food stain

Xylanases are hemicellulolytic enzymes specifically catalyze xylan hydrolysis that constitutes the most abundant polysaccharide in the hemicellulose fraction of the lignocellulosic biomasses. The effective conversion of these biomasses to valuable commodities required the use of economic effective biocatalysts . In this study, xylanase production was performed by corn cobs fermentation using Aspergillus flavus AW1 in which the highest productivity was 356.36 U/g dry substrate. The partially purified enzyme was aflatoxin-free as estimated by high performed liquid chromatography . The enzyme was optimally active over a wide pH range from 5.5 to 7.5. Additionally, it was optimally active at temperature of 55 °C and at 60 °C it possessed 95% of its maximum activity. The enzyme half-lives were estimated as 83.81 and 44.69 min at 55 and 60 ° C, respectively. The V max and K m for the purified enzyme fraction were 250 U/mg protein/min and 5.55 mg/mL, respectively. Moreover, beech wood xylan hydrolysis as well as the hydrolysis of the hemicellulosic fraction of corn cobs was examined by applying the produced enzyme. The obtained results indicated hydrolysis percentage values of 39 and 19% for beech wood xylan and the hemicellulosic fraction of corn cobs, respectively with the release of a mixture of xylooligosaccharides confirmed by thin layer and high performed liquid chromatography analysis. The antioxidant activities for the dried oligosaccharide mixtures were estimated by scavenging activity of 1,1-diphenyl-2-picrylhydrazyl free radical. Finally, the efficiency of the purified enzyme fraction in the removal of mango stain was investigated in comparison to the commercial detergent. • Xylanase was produced by solid state fermentation of corn cobs using Aspergillus flavus AW1. • The partially purified enzyme was thermo-active and possessed high affinity toward beech wood xylan. • The enzyme was applied for the removal of food stain and production of xylooligosaccharides. • The produced xylooligosaccharides possessed antioxidant activity.

Spartina argentinensis valorization and process optimization for enhanced production of hydrolytic enzymes by filamentous fungus

Hemicellulose is a major component of plant cell walls and xylan is the most predominant polysaccharide. Xylan degrading enzymes integrate the xylanolytic system. Xylanolytic enzymes were produced by fermentation by Aspergillus niger and Thermomyces lanuginosus grown on grass considered unsuitable for farming called Spartina argentinensis . Significant parameters: type of fermentation used (SmF or SSF), type of leaves of S. argentinensis (green or senescent) and conidia final concentration for xylanase production were screened and optimized. The main results showed that the highest levels of xylanolytic enzyme production were obtained by 1 × 10 5 conidia/mL of A. niger in SmF at 96 h, 30ºC, with a mixture of 20.20% of senescent and 79.80% of green leaves. The xylanase specific activity obtained was 62 U/mg, higher than the activity obtained (23 U/mg) in previous work ( Taddia et al., 2019 ), and the concentration of xylanolytic production over that of glucanase activity was maximized five times. The optimized enzymatic extract obtained was characterized by LC-MS and HPLC of carbohydrates. Six enzymes were identified as constituents of the xylanolytic complex and seven carbohydrates. Moreover, the xylanolytic enzyme extract was stable for 30 days at 20 °C. Thus, S. argentinensis can be used within the framework of a circular economy, rendering a synergistic combination of the xylanolytic enzymes with industrial applications. • Lignocellulosic biomass; Spartina argentinensis was utilized in the fermentation. • Enzyme statistical design production was optimized. • Maximum xylanase specific activity of 61 U/mL was obtained. • The enzymatic extract maintained 100% its activity after 30 days at 20°C.

Enhanced Production, Cloning, and Expression of a Xylanase Gene from Endophytic Fungal Strain Trichoderma harzianum kj831197.1: Unveiling the In Vitro Anti-Fungal Activity against Phytopathogenic Fungi.

Trichoderma sp. is extensively applied as a beneficial fungus for the management of plant diseases, plant growth promotion, induced resistance, and plays an important role in global sustainable agriculture. This study aimed to enhance the production of microbial xylanase in high titer from the endophytic fungus Trichoderma harzianum kj831197.1, and the cloning of xylanase genes in E. coli DH5α using a pUC19 vector. A combination of glucose, 0.1 mM, Tween 80 with lactose, and 2 mM galactose combined with malt extract boostedthe enzyme production. Xylanase production was maximized at a pH of 5.0, temp. of 30 °C, and agitation of 150 rpm in the presence of malt extract and bagasse as the best nitrogen source and waste, respectively, using submerged fermentation. The molecular weight of highly purified xylanase was 32 KDa, identified using SDS-PAGE. The xylanase gene of T. harzianum kj831197.1 was screened in fungal DNA using definite primers specified in the gene bank database. The identified region was excised using restriction enzymes HindIII and EcoRI and cloned into a pUC19 plasmid vector. Optimization of fermentation conditions improved xylanase production about 23.9-fold.The antifungal efficacy of xylanase toward different phytopathogenic fungi was determined. The highest inhibition was against Corynespora cassiicola, Alternaria sp., Fusarium oxysporum, and Botrytis fabae. This study offered an economical, simple, and efficient method using Trichoderma harzianum kj831197.1 for the production of the xylanase enzyme via the submerged fermentation method.

Evaluation of endoglucanase and xylanase production by Aspergillus tamarii cultivated in agro-industrial lignocellulosic biomasses.

To better understand the production of enzymes of industrial interest from microorganisms with biotechnological potential using lignocellulosic biomass, we evaluated the production of endoglucanase and xylanase from Aspergillus tamarii. CAZymes domains were evaluated in the genome, and a screening of the enzymatic potential of A. tamarii in various agricultural biomasses was done. The enzymatic profile could be associated with the biomass complexity, with increased biomass recalcitrance yielding higher activity. A time-course profile defined 48h of cultivation as the best period for cultivating A. tamarii in sugarcane bagasse reached 12.05IU/mg for endoglucanase and 74.86IU/mg for xylanase. Using 0.1% (w/v) tryptone as the only nitrogen source and 12µmol/L CuSO4 addition had an overall positive effect on the enzymatic activity and protein production. A 22 factorial central composite design was used then to investigate the simultaneous influence of tryptone and CuSO4 on enzyme activity. Tryptone strongly affected enzymatic activity, decreasing endoglucanase activity but increasing xylanase activity. CuSO4 supplementation was advantageous for endoglucanases, increasing their activity, and it had a negative effect on xylanases. But overall, the experimental design increased the enzymatic activity of all biomasses used. For the clean cotton residue, the experimental design was able to reach the highest enzyme activity for endoglucanase and xylanase, with 1.195IU/mL and 6.353IU/mL, respectively. More experimental studies are required to investigate how the biomass induction effect impacts enzyme production.

Isolation of Efficient Xylooligosaccharides-Fermenting Probiotic Lactic Acid Bacteria from Ethnic Pickled Bamboo Shoot Products.

Simple SummaryNaw Mai Dong is an ethnic fermented bamboo shoot product that is popular in the upper northern region of Thailand. Xylan is a component of hemicellulose that can be found in bamboo shoots. It is a raw material utilized in xylooligosaccharide production. To produce Naw Mai Dong, bamboo shoots are sliced and pickled in bottles or jars that contain water rinsed from rice crops with or without salt. This would differ from the simple brine comprised of water and salt that is associated with most other pickled products. Naw Mai Dong is sour due to its acidity. Importantly, fermented fruits and vegetables are a potential source of lactic acid bacteria, which have been well-documented to possess a number of probiotic characteristics. This would imply that some of the lactic acid bacteria present during bamboo shoot fermentation could serve as efficient xylooligosaccharides-fermenting probiotic lactic acid bacteria.Xylooligosaccharides (XOSs) are produced from xylan, which is a component of the hemicellulose that can be found in bamboo shoots. Naw Mai Dong, an ethnic pickled bamboo shoot product of northern Thailand, is generally characterized as acidic and has a sour taste. It can be considered a potential source of probiotic lactic acid bacteria (LAB). This study aimed to isolate efficient XOSs-fermenting probiotic LAB from ethnic pickled bamboo shoot products. A total of 51 XOSs-fermenting LAB were recovered from 24 samples of Naw Mai Dong, while 17 strains exhibited luxuriant growth in xylose and XOSs. Among these, seven strains belonging to Levicaseibacillus brevis and Pediococcus acidilactici exhibited similar growth in glucose, xylose, and XOSs, while the rest showed a weaker degree of growth in xylose and XOSs than glucose. Sixteen strains exhibited resistance under gastrointestinal tract conditions and displayed antimicrobial activity against foodborne pathogens. Notably, Lv. brevis FS2.1 possessed the greatest probiotic properties, with the highest %hydrophobicity index and %auto-aggregation. Effective degradation and utilization of XOSs by probiotic strains are dependent upon xylanase and β-xylosidase production, as well as xylose metabolism. It can be concluded that pickled bamboo shoot products can be a beneficial source of XOSs-fermenting probiotic LAB.

Harnessing Mucor spp. for Xylanase Production: Statistical Optimization in Submerged Fermentation Using Agro-Industrial Wastes.

Xylan is the primary hemicellulosic polymer found in lignocellulosic agricultural wastes and can be degraded by xylanase. In the current research, Mucor circinelloides and M. hiemalis were tested for their ability to produce xylanase from tangerine peel by submerged fermentation. Experiments on five variables were designed with Box–Behnken design and response surface methodology. Analysis of variance was exercised, the xylanase output was demonstrated with a mathematical equation as a function of the five factors, and the quixotic states for xylanase biosynthesis was secured. In addition, xylanase was partially purified, characterized, and immobilized on calcium alginate beads. The optimum parameters for xylanase production by M. circinelloides and M. hiemalis were consisted of incubation temperature (30 and 20°C), pH value (9 and 7) incubation period (9 and 9 days), inoculum size (3 and 3 mL), and substrate concentration (3 and 3 g/100 mL), respectively. M. circinelloides and M. hiemalis demonstrated the highest xylanase activities after RSM optimization, with 42.23 and 35.88 U/mL, respectively. The influence of single, interchange, and quadratic factors on xylanase output was investigated using nonlinear regression equations with significant R2 and p values. The partial purification of M. circinelloides and M. hiemalis xylanase yielded 1.69- and 1.97-fold purification, and 30.74 and 31.34% recovery with 292.08 and 240.15 U/mg specific activity, respectively. Partially purified xylanase from M. circinelloides and M. hiemalis demonstrated the highest activity at neutral pH and 60 and 50°C, respectively. The immobilized M. circinelloides and M. hiemalis xylanase retained 84.02 and 79.43% activity, respectively. The production of xylanase from M. circinelloides and M. hiemalis utilizing RSM is deemed profitable for the decomposition of the agro-industrial wastes.

Glycosyl hydrolase 11 (xynA) gene with xylanase activity from thermophilic bacteria isolated from thermal springs

BackgroundHemicellulose is one of the copious polymer in lignocellulosic biomass (LCB). It is primarily composed of xylan linked by β-1,4 glycosidic bonds. Xylanase preferentially cleaves the β-1,4-glycosidic bonds in the xylan backbone resulting in complete hydrolysis of the biomass. Thermostable variants of glycoside hydrolases act as robust catalysts, not only in degradation but also during processing, to obtain specific carbohydrate-containing chemicals and materials (Ramasamy et al. in Madras Agric J 107(special):1. https://doi.org/10.29321/MAJ.2020.000382, 2020).ResultsThe xylanase production by two thermophilic bacteria isolated from thermal springs was evaluated. In addition, the gene encoding this industrially vital enzyme was isolated and characterized, and its protein structure was analyzed. The thermophilic bacteria producing xylanases were isolated from augmented sawdust and banana fiber biomass from hot springs of Himachal Pradesh and identified as Bacillus subtilis VSDB5 and Bacillus licheniformis KBFB4 using 16S rRNA gene sequencing. The persistent xylanase activity revealed that the enzyme is secreted extracellularly with the maximum activity of 0.76 IU mL−1 and 1.0 IU mL−1 at 6 h and 12 h of growth by KBFB4 and VSDB5, respectively, under submerged fermentation. Both the strains exhibited the maximum activity at pH 6 and a temperature of 50 °C. The xylanases of KBFB4 and VSDB5 were thermostable and retained 40% of their activity at 60 °C after incubation for 30 min. Xylanase of VSDB5 had wide thermotolerance and retained 20% of its activity from 60 to 80 °C, whereas xylanase of KBFB4 showed wide alkali tolerance and retained 80% of its activity until pH 10. The xylanase (xynA)-encoding gene (650 bp) cloned from both the strains using specific primers showed 98 to 99% homology to β-1,4-endoxylanase gene. Further in silico analysis predicted that the xylanase protein, with a molecular weight of 23 kDa, had a high pI (9.44–9.65), which explained the alkaline nature of the enzyme and greater aliphatic index (56.29). This finding suggested that the protein is thermostable. Multiple sequence alignment and homology modeling of the protein sequence revealed that the gene product belonged to the GH11 family, indicating its possible application in bioconversion.ConclusionThe strains B. subtilis VSDB5 and B. licheniformis KBFB4 obtained from hot springs of Himachal Pradesh produced potent and alkali-tolerant thermostable xylanases, which belong to the GH11 family. The enzyme can be supplemented in industrial applications for biomass conversion at high temperatures and pH (or in processes involving alkali treatment).Graphical

Mycoremediation of lignocellulosic biorefinery sludge: A reinvigorating approach for organic contaminants remediation with simultaneous production of lignocellulolytic enzyme cocktail

This research work aims to valorize lignocellulosic biorefinery sludge with genetically engineered Trichoderma atroviride for simultaneous removal of organic contaminants, fermentation inhibitors, and lignocellulolytic enzyme cocktail production. Upon analysis, three phenolic compounds (42.6 ± 3.6 μg/g), two polycyclic aromatic hydrocarbons (0.42 ± 0.06 μg/g) and five fermentation inhibitors (2.5 ± 0.3 mg/g) were detected in the sludge. Bioaugmentation of sludge with 72 h-old T. atroviride (5%) results in the production of cellulase (21 U/g), xylanase (84 U/g), laccase (20 U/g), lignin peroxidase (14 U/g) and aryl alcohol oxidase (116 U/g), along with the concomitant removal of organic contaminants (phenol, 2, 4-dinitrophenol, pentchlorophenol, phenanthrene, benzo(a)pyrene) and fermentation inhibitors (furfural, 5-hydroxymethylfurfural, levulinic acid, ferulic acid, and catechol). Subsequently, the enrichment of sludge with nutrients and rhamnolipids enhanced the enzyme production by 5–6-fold and resulted in the removal of 85–95% of organic contaminants and fermentation inhibitors, which constitutes an eco-friendly process.

Xylanase production by Thermobacillus xylanilyticus is impaired by population diversification but can be mitigated based on the management of cheating behavior

BackgroundThe microbial production of hemicellulasic cocktails is still a challenge for the biorefineries sector and agro-waste valorization. In this work, the production of hemicellulolytic enzymes by Thermobacillus xylanilyticus has been considered. This microorganism is of interest since it is able to produce an original set of thermostable hemicellulolytic enzymes, notably a xylanase GH11, Tx-xyn11. However, cell-to-cell heterogeneity impairs the production capability of the whole microbial population.ResultsSequential cultivations of the strain on xylan as a carbon source has been considered in order to highlight and better understand this cell-to-cell heterogeneity. Successive cultivations pointed out a fast decrease of xylanase activity (loss of ~ 75%) and Tx-xyn11 gene expression after 23.5 generations. During serial cultivations on xylan, flow cytometry analyses pointed out that two subpopulations, differing at their light-scattering properties, were present. An increase of the recurrence of the subpopulation exhibiting low forward scatter (FSC) signal was correlated with a progressive loss of xylanase activity over several generations. Cell sorting and direct observation of the sorted subpopulations revealed that the low-FSC subpopulation was not sporulating, whereas the high-FSC subpopulation contained cells at the onset of the sporulation stage. The subpopulation differences (growth and xylanase activity) were assessed during independent growth. The low-FSC subpopulation exhibited a lag phase of 10 h of cultivation (and xylanase activities from 0.15 ± 0.21 to 3.89 ± 0.14 IU/mL along the cultivation) and the high-FSC subpopulation exhibited a lag phase of 5 h (and xylanase activities from 0.52 ± 0.00 to 4.43 ± 0.61 over subcultivations). Serial cultivations on glucose, followed by a switch to xylan led to a ~ 1.5-fold to ~ 15-fold improvement of xylanase activity, suggesting that alternating cultivation conditions could lead to an efficient population management strategy for the production of xylanase.ConclusionsTaken altogether, the data from this study point out that a cheating behavior is responsible for the progressive reduction in xylanase activity during serial cultivations of T. xylanilyticus. Alternating cultivation conditions between glucose and xylan could be used as an efficient strategy for promoting population stability and higher enzymatic productivity from this bacterium.