Xylanase Research Articles

Secretome analysis of thermophilic mould Myceliophthora thermophila cultivated on rice straw and hydrolysis of lignocellulosic biomass for bioethanol production

Myceliophthora thermophila encodes for large number of carbohydrate-active enzymes (CAZymes) involved in lignocellulosic biomass degradation. The mould was grown on rice straw in solid state fermentation at pH 5.0 and 45 °C that produced high levels of cellulolytic and xylanolytic enzymes i.e. 2218.12, 515.23, 478.23, 13.34 U/g DMR for xylanase, CMCase, FPase and β-glucosidase, respectively. The secretome analysis of M. thermophila BJAMDU5 by mass spectroscopy, described 124 different proteins with majority of CAZymes consisting of glycosyl hydrolases (GH), lytic polysaccharide mono-oxygenases (LPMO), carbohydrate esterases (CE) and polysaccharide lyases (PL). Furthermore, the enzyme cocktail of the mould was evaluated for hydrolysis of steam treated rice straw that produced 184.59 mg/g substrate reducing sugars after 24 h, which was used for production of bioethanol by using fast fermenting yeast Saccharomyces cerevisiae resulting in high production of bioethanol.

Streptomyces suşlarında farklı genetik manipulasyonların ksilinaz enzim üretimi üzerine etkisi

Çukurova Üniversitesi’nin 5 farklı bölgesinden alınan toprak örneklerinden 3 Streptomyces suşu izole edildi. S40, S47, S48 suşlarının 7 gün sonundaki ksilanaz aktivitesi sırasıyla 50,92 U/mL, 49,51 U/mL, 47,79 U/mL tespit edilmiştir. Yüksek ksilanaz aktivitesi gösteren bu suşlar arasında gerçekleştirilen protoplast füzyon çalışması sonucu farklı koloni morfolojsi gösteren 9 suş seçilmiştir. (40-48)R2 ve (47-48)R3 suşlarının ksilanaz aktivitesi ebeveyn suşlara göre sırasıyla %25,9 ve % 31,9 oranında arttığı tespit edilmiştir. Protoplast füzyon tekniğiyle enzim aktivitesinin geliştirilebileceği gösterilmiştir.

An integrated approach to the sustainable production of xylanolytic enzymes from Aspergillus niger using agro-industrial by-products

Xylanolytic enzymes were produced by Aspergillus niger NRRL3 grown on agro-industrial by-products obtained from the processing of wheat flour without pretreatment. Significant parameters for xylanase production were screened and optimized. The xylanolytic activity obtained in the optimized extract was 138.3 ± 2.6 U/mL, higher than the activity obtained in an unoptimized medium (14.5 ± 0.3 U/mL) in previous work. The optimized fermentation process was performed in a successful 40-fold scale-up. The optimized enzymatic extract obtained was characterized by LC-MS. Nine enzymes were identified as constituents of the xylanolytic complex. Moreover, the xylanolytic enzymes were stable until 60 °C and over a broad range of pH. Sodium, calcium, cobalt and manganese had no inhibitory effect, meanwhile 1% w/v polyvinylpyrrolidone and 1% w/v dextran increased the xylanolytic activity. The saccharification efficiency was evaluated and the surface morphology of the lignocellulosic substrate was monitored by using scanning electron microscopy (SEM). The synergistic combination of the extracted (o purified) xylanolytic enzymes permitted a higher xylan conversion beneficial for diverse applications, such as bioethanol production. Thus, these agroindustrial by-products can be used within the framework of a circular economy, rendering an added value bioproduct, which is reused in the industry.

Microbial cellulolytic enzymes: diversity and biotechnology with reference to lignocellulosic biomass degradation

Lignocellulosic biomass is the earth’s most abundant renewable feedstock alternative that comprises of cellulose, hemi-cellulose and lignin. The synergistic action of cellulolytic/xylanolytic enzymes produced by lignocellulolytic micro-organisms such as bacteria, algae and fungi are capable of robust cellulosic biomass deconstruction. Most of the microorganisms dwelling in extreme environmental habitats such as rumen environment, hot/cold springs, deep ocean trenches, acidic/alkaline pH environment have been considered as an attractive producers of hemi/cellulolytic lignocellulolytic and other biotechnological enzymes with enhanced bio-chemical properties essential for industrial bioconversion processes. However, the potential microbial sources of cellulolytic enzymes and the underlying mechanism to achieve this is not fully elucidated. In this review article, first we detail the composition of lignocellulosic biomass. Next, we describe the structure and functions of divergent hydrolytic enzymes (cellulolytic and xylanolytic enzymes) involved in cellulosic biomass degradation. Third, we analyze, outline and unveil the prospective source of microbes encoding exceptionally diverse set of biotechnologically relevant cellulolytic enzymes which are critical to answer the specific ecological question of by whom, where and how cellulosic biomass is degraded in the environment. Finally, this review article features the snapshot about the future developments and perspectives on microbial enzymes, high-throughput techniques and molecular tools that could be exploited to derive those enzymes from the potential sources.

Composition and foam properties of whole wheat dough liquor as affected by xylanase and glucose oxidase

Xylanase (XYL) and glucose oxidase (GOX) have been applied to improve the crumb structure of whole wheat buns. However, their effects on the constituents dissolved in the dough liquor (DL), which supposedly contributed to gas cell stabilization and crumb structure, have not been elucidated. In this study, we aimed to unravel the effects of these enzymes on the protein and arabinoxylan contents, DL yields, bulk viscosity, as well as foaming properties. DL was first separated through ultracentrifugation from whole wheat dough prior treated by XYL and GOX. The results indicated that XYL and GOX treatment increased the DL yields. Moreover, while XYL increased the water extractable arabinoxylan content, GOX increased the protein level in the DL. The free sulfhydryl groups of soluble proteins declined significantly in the presence of GOX. Moreover, GOX might have catalyzed the formation of large protein aggregates and protein-polysaccharide complexes. Furthermore, while the foaming capacity of DL increased only in the presence of XYL, the foam stability was increased by both XYL and GOX, especially under heat treatment. The present research might help to understand the mechanism of crumb structure improvement of whole wheat buns by XYL/GOX treatment from the view of their effects on the DL.

XynDZ5: A New Thermostable GH10 Xylanase.

Xylanolytic enzymes have a broad range of applications in industrial biotechnology as biocatalytic components of various processes and products, such as food additives, bakery products, coffee extraction, agricultural silage and functional foods. An increasing market demand has driven the growing interest for the discovery of xylanases with specific industrially relevant characteristics, such as stability at elevated temperatures and in the presence of other denaturing factors, which will facilitate their incorporation into industrial processes. In this work, we report the discovery and biochemical characterization of a new thermostable GH10 xylanase, termed XynDZ5, exhibiting only 26% amino acid sequence identity to the closest characterized xylanolytic enzyme. This new enzyme was discovered in an Icelandic hot spring enrichment culture of a Thermoanaerobacterium species using a recently developed bioinformatic analysis platform. XynDZ5 was produced recombinantly in Escherichia coli, purified and characterized biochemically. This analysis revealed that it acts as an endo-1,4-β-xylanase that performs optimally at 65–75°C and pH 7.5. The enzyme is capable of retaining high levels of catalytic efficiency after several hours of incubation at high temperatures, as well as in the presence of significant concentrations of a range of metal ions and denaturing agents. Interestingly, the XynDZ5 biochemical profile was found to be atypical, as it also exhibits significant exo-activity. Computational modeling of its three-dimensional structure predicted a (β/α)8 TIM barrel fold, which is very frequently encountered among family GH10 enzymes. This modeled structure has provided clues about structural features that may explain aspects of its catalytic performance. Our results suggest that XynDZ5 represents a promising new candidate biocatalyst appropriate for several high-temperature biotechnological applications in the pulp, paper, baking, animal-feed and biofuel industries.

Rapid Screening of Additive Formulations for Enhancing Xylanase Stability in Pulp Bleaching and Storage Conditions

Enzyme deactivation is a significant parameter to restrain the industrial commercialization of enzymes. In this study, a high-throughput screening technique using differential scanning fluorimetry (DSF) incorporated with enzyme activity analysis was thus developed in order to identify a suitable additive formulation for promoting enzyme stability in terms of conformational stability and functional activity. To demonstrate the concept feasibility, endo-xylanase obtained from metagenome of termite gut-inhabiting bacteria (XYN12) was used as a study model and several group of additives including salts, polymers, polyols and sugars at various concentrations and different combination patterns (single, double and triple pairing) were applied for investigating their effects on enzyme stability. As a result, a total of 80 reaction mixtures were retrieved including a native control (enzyme without any additives). DSF assay was performed using real-time PCR instrument to detect unfolding of protein in the presence of a florescence dye. Accurate melting temperature (Tm) values of each enzyme formulation were calculated using Boltzmann equation and compared to the Tm of the native control in order to find ?Tm. After that, the residual activity of xylanase in all reaction mixtures were measured using pHBAH method in automated liquid handling system. For single additives, glycerol had a stabilizing effect to the target enzyme, as observed by a significant increase in the ?Tm, while ?(NH4)2SO4, CaCl2, NaCl destabilized it. In addition, cocktail additives included glycerol, PEG4000 and trehalose dramatically enhanced the enzyme stability by positive shifting of Tm from 54 to 56 (C and increasing of relative activity by more than 4 folds compared to single additive. Storage stability of the enzyme was improved when mixed with this cocktail additive as indicated by remaining of the enzyme activity (86 %) after kept at 30 (C, pH 7.4 for 262 days, whereas native control lost almost of its activity within 40 days when stored at the same condition.

Production of ethanol and xylanolytic enzymes by yeasts inhabiting rotting wood isolated in sugarcane bagasse hydrolysate

Yeasts associated with rotting wood from four Atlantic Rain forest sites in Brazil were investigated using a culture medium based on sugarcane bagasse hydrolysate. A total of 330 yeast strains were isolated. Pichia manshurica, Candida pseudolambica, and Wickerhamomyces sp. 3 were the most frequently isolated species. Fourteen novel species were obtained in this study. All isolates were tested for their ability to ferment d-xylose and to produce xylanases. In the fermentation assays using d-xylose (30 g L−1), the main ethanol producers were Scheffersomyces stipitis (14.08 g L−1), Scheffersomyces sp. (7.94 g L−1) and Spathaspora boniae (7.16 g L−1). Sc. stipitis showed the highest ethanol yield (0.42 g g−1) and the highest productivity (0.39 g L−1h−1). The fermentation results using hemicellulosic hydrolysate showed that Sc. stipitis was the best ethanol producer, achieving a yield of 0.32 g g−1, while Sp. boniae and Scheffersomyces sp. were excellent xylitol producers. The best xylanase-producing yeasts at 50 °C belonged to the species Su. xylanicola (0.487 U mg−1) and Saitozyma podzolica (0.384 U mg−1). The results showed that rotting wood collected from the Atlantic Rainforest is a valuable source of yeasts able to grow in sugarcane bagasse hydrolysate, including species with promising biotechnological properties.

Enzymatic cross-linking to improve the handling properties of dough prepared within a normal and reduced NaCl environment.

This research examines the use of three enzymes [glucose oxidase (GOX), hexose oxidase (HOX), and xylanase (XYL)] and their combinations [GOX-XYL and HOX-XYL] on the dough handling properties of CDC Plentiful and Stettler wheat cultivars prepared at reduced (1.0% wt. by flour) and normal (2.0% wt. by flour) NaCl levels. Properties investigated include dough rheology, stickiness, and ratio of resistance to extension and extensibility. The inclusion of XYL and its combinations with GOX and HOX increased the stickiness, yielded lower dough strength indicated by rheology, and reduced the ratio of resistance to extension and extensibility. The inclusion of oxidative enzymes yielded a stronger dough, where HOX addition to dough had the lowest stickiness values and highest |G*| values, whereas GOX addition led to the highest ratio of resistance to extension-extensibility. NaCl only had minor effects overall on dough strength and stickiness for the cultivars studied. Overall, superior dough handling properties were observed with oxidative enzyme addition (GOX and HOX) suggesting that the increased crosslinking that occurs could aid in improving low sodium bread dough properties.

Optimization of concurrent production of xylanolytic and pectinolytic enzymes by Bacillus safensis M35 and Bacillus altitudinis J208 using agro-industrial biomass through Response Surface Methodology

Application of crude xylanolytic and pectinolytic enzymes in diverse industrial processes make these enzymes commercially valuable and demand their production process to be cost-effective. Out of four different agrowaste biomass, wheat bran (WB) and citrus peel (CP), when amended as fermentation substrates, respectively induced the highest xylanolytic enzymes and pectinolytic enzymes from both, B. safensis M35 and B. altitudinis J208. Further, the simultaneous amendment of WB and CP yielded concurrent production of these cellulase free xylanolytic and pectinolytic enzymes. Hence, the quadratic model was developed using the Central Composite Design of Response Surface Method (CCD-RSM). The model gave the concentration values for WB and CP substrates to be amended in one single production medium for obtaining two optimized predicted response values of xylanase activity and pectinase activity units, which were further practically validated for the xylanase and pectinase production responses from the optimized production medium (OPM). These practically obtained response values from OPM were found to be in accordance with a range of 95% predicted intervals (PI) values. These observations verified the validity of the predicted quadratic model from RSM and suggested that both xylanase and pectinase enzymes can be induced concurrently from both of the bacterial strains.

PREBIOTIC EFFECTS OF XYLANASE MODIFICATION OF ?-GLUCAN FROM OAT BRAN ON BIFIDOBACTERIUM BIFIDUM

Oat ?-glucan (BG) was isolated from oat bran, and the xylanase treatment was conducted to obtain modification of ?-glucan (MBG). The relative molecular weight (Mw) of BG and MBG was determined by gel permeation chromatography (GPC). Results demonstrated that the Mw of BG was reduced from 1.66×104 to 5.43×103. We assessed the prebiotic effect of BG and MBG on human colon Bifidobacterium bifidum?B. bifidum). Our findings suggest that the addition of BG and MBG resulted in a lower pH of the fermentation broths. Both lactic and acetic acid production increased in the fermentation broths. While BG was found to significantly promote the proliferation of B. bifidum, MBG had a greater effect on B. bifidum.

Physicochemical Characteristics of Fungal Xylanases and their Potential for Biobleaching of Kraft and Non-wood Pulps

Xylanases are enzymes with a wide variety of biotechnological applications, such as in the bioconversion of lignocellulosic materials, improvement of feed digestibility, and bleaching kraft pulps to increase pulp brightness. Many studies have been conducted and published over the years on cellulose pulp bleaching due to the need to search for more sustainable tools and thus reduce environmental pollution. Thus, in this review, we focus on analyzing the biochemical properties of xylanases produced by mesophilic and thermophilic fungi that have been used in the bleaching processes of kraft and non-wood pulps. Eucalyptus kraft pulp is still one of the most used raw materials in the production of pulp and paper, while straw and bagasse are alternative sources of non-wood pulps. Thermophilic fungal xylanases show optimum enzymatic activity at high temperatures and a shorter treatment period when compared to mesophilic xylanases in the bleaching step. However, mesophilic fungal xylanases exhibit a longer period of enzymatic treatment but achieve a satisfactory brightness and kappa number. Hence, these approaches will contribute to future applications of these xylanolytic enzymes in pulp and paper industries.

Bioinformatics Analyses of Endo-1,4-beta-xylanase from Bacillus sp. SS3.4

Various types of microorganisms particularly bacteria and fungi are capable of producing xylanolytic enzymes that degrade xylan into simple sugars to be used as their carbon sources. Xylanolytic enzymes, particularly endo-1,4-β-xylanase, has been extensively used in various industrial sectors, including biofuel, livestock, paper, and food. Previously, Bacillus sp. SS3.4 was observed to have xylanolytic activities on wheat bran, suggesting its capability of producing xylanolytic enzymes, endo-1,4-β-xylanase included. With great numbers of data available in nucleotide and protein sequence databases, bioinformatics analyses of protein structure for function prediction have become attractive to supplement wet laboratory experiments to reduce time and money consumption. Therefore, in this study, bioinformatics analyses were performed on Bacillus sp. endo-1,4-β-xylanase sequence in order to further evaluate the enzyme’s function by exploring its protein structure in comparison to other known bacterial endo-1,4-β-xylanase. In this study, Bacillus sp. endo-1,4-β-xylanase sequence was identified from its whole genome sequence. The sequence was then analyzed by using BLAST to perform a homology search for identifying the protein, MUSCLE to perform multiple sequence alignment for comparing with other enzyme sequences, PHYLOGENY-FR to display its relatedness among Bacillus, Aspergillus niger and Trichoderma reesei, and SWISS-MODEL to generate its three-dimensional structure. Results from BLAST confirmed that the identified sequence was endo-1,4-β-xylanase, with greater relatedness to Bacillus velezensis and Bacillus amyloliquefaciens endo- 1,4-β-xylanase. While results from MUSCLE and SWISS-MODEL suggested that the endo-1,4-β-xylanase belongs to the Glycosyl Hydrolase 11 (GH11) family bearing the distinct shape of a jelly roll with well-conserved binding and catalytic residues. These results strongly suggest that Bacillus sp. endo-1,4-β-xylanase is potentially capable of degrading xylan with highly similar mechanism as with other endo-1,4-β-xylanase.
 Keywords: Bacillus, bioinformatics analysis, endo-1,4-

Factors affecting the hydrolytic action of xylanase during pennisetum saccharification: role of lignin

Inhibition of cellulose hydrolysis has been reported extensively, however, there is a paucity of information describing the effect of lignin on xylan hydrolysis by endo-xylanase and β-xylosidase. In this report, the effects of two different lignins on enzymatic hydrolysis of isolated xylan and NaOH-pretreated pennisetum by endo-xylanase and β-xylosidase were assessed. Both acid insoluble lignin (AIL) and enzymatic hydrolysis lignin (EHL) were found to inhibit hydrolysis of endo-xylanase and β-xylosidase, and AIL had a stronger negative effect on enzymatic hydrolysis when compared with that of EHL. Results from inhibitory kinetics experiments showed that the inhibition of AIL and EHL on xylanase did not follow Michaelis–Menten kinetics. The higher adsorption capacity of AIL toward xylanase arose from its higher hydrophobicity and lower absolute zeta potential, and this likely explains the higher inhibitory effect of AIL than that of EHL. The results aid our understanding of the role of lignin in xylan hydrolysis by xylanolytic enzymes.

Lignin degradation and detoxification of eucalyptus wastes by on-site manufacturing fungal enzymes to enhance second-generation ethanol yield

Novel laccases have promising and valuable applications in biorefineries. This investigation documents, for the first time, the potential of depolymerising and repolymerising lignin by the secretome, rich in laccases, from a newly isolated white-rot basidiomycete Marasmiellus palmivorus VE111, for further saccharification and ethanolic fermentation steps. Proteomic analyses of the secretome of M. palmivorus show that laccases are the most predominant enzyme released by this fungus. The whole crude enzymatic broth is used for the delignification of lignin in Eucalyptus globulus wood, with the aim of enhancing the saccharification by cellulolytic and xylanolytic enzymes from Penicillium echinulatum S1M29. In addition, two different strategies, namely, laccase treatment before and after enzymatic hydrolysis, are employed to detoxify steam-exploded E. globulus wood. The objective is to increase the fermentative performance by removing substances formed during the feedstock pretreatment that can inhibit microbial fermentation. The E. globulus wood delignification results in a 31% decrease in the lignin content and a 10% increase in the glucose yield after hydrolysis. An important finding of the present work is the successful wood delignification in the absence of laccase mediators. This laccase-rich preparation also demonstrates its potential in removing the phenolic inhibitors present in steam-exploded E. globulus wood, increasing the ethanol yield by an additional 10%. Furthermore, it is important to highlight that these findings are achieved in the absence of commercial enzymes, making M. palmivorus laccases a potential candidate not only for the production of biofuels but also for the generation of lignin-derived aromatic compounds for different applications in the biotechnology industry.

Effect of xylanase, cellulase and natural maguey extract on the chemical composition of corn silage and in vitro rumen gas production

This study considered the application of two exogenous enzymes, xylanase (XYL) and cellulase (CELL), and maguey extract (ME), (applying 1 ml per kg as fresh matter) and a control (without additive) in four maize silage varieties (San Diego, Cacahuacintle, P-1832 and Victoria), to investigate their effect on the chemical composition (CC), gas production (GP) and in vitro ruminal fermentation. The GP was measured at 0, 3, 6, 9, 12, 24, 36, 48, 72 and 96 hours of incubation. Dry matter disappearance (DMD), organic matter disappearance (OMD), metabolizable energy (ME), and short chain fatty acids (SCFAs) were determined after 96 h of incubation. Data were analyzed using a completely randomized design with a 4×4 factorial arrangement with three replications. The CC showed a significant effect (P<0.05) for varieties with the exception of organic matter (OM), and the inclusion of additives increased the dry matter (DM) and crude protein (CP). A significant effect (P<0.01) was observed for the varieties in the GP parameters and ruminal fermentation. The addition of XYL, CEL and ME promoted dry matter degradation and increased energy availability, with increased in vitro gas production.

Enhancement and bioavailability of phenolic content in Kappaphycus alvarezii through solid substrate fermentation

Known as carrageenan, Kappaphycus alvarezii is a popular species of red edible seaweed which is mainly cultivated for its hydrocolloids. K. alvarezii is a good source of phenolics which are considered to be natural antioxidants that promote myriads of health benefits. Extraction of phenolic compounds using conventional solvent extraction may result in hazards and toxicological effect and low yield of bound phenolics. Therefore, this study was conducted to increase the release of bound phenolics through solid substrate fermentation. In the present study, three different varieties of K. alvarezii were fermented with Aspergillus oryzae through solid substrate fermentation (SSF) at 30 ℃ for 2 to 6 days. The changes of total phenolic content and antioxidant activity due to the effects of SSF were investigated. Results obtained revealed that the highest total phenolic content for green (10.022 mg GAE/g) and purple (4.037 mg GAE/g) varieties of K.alvarezii were on the fourth day of fermentation while for yellow (4.479 mg GAE/g) variety was on the sixth day of fermentation. A remarkable enhancement of antioxidant activity was discovered through DPPH radical scavenging activity on the fourth day of fermentation for all varieties of K. alvarezii tested. Total phenolic content also showed a significant correlation with antioxidant activity of fermented seaweed. Cellulase, xylanase, and β-glucosidase enzymes produced by A.oryzae during SSF were also investigated to see the relationship with the release of phenolic compounds after fermentation. The highest cellulase activity was observed on the fourth day of fermentation. The similar observation was also found for xylanase enzyme in yellow and purple varieties of K. alvarezii , except for green variety where the highest xylanase activity was on the sixth day of fermentation. The maximum β-glucosidase activity in yellow and green varieties was observed on the fourth day of fermentation while purple variety exhibited the maximum β-glucosidase activity on the sixth day. There are positive correlation between the enzymes studied and the polyphenols content in all varieties of K. alvarezii . This study demonstrated that fermented K. alvarezii contained more phenolic content compared to non-fermented seaweed.

Salt-tolerant α-arabinofuranosidase from a new specie Aspergillus hortai CRM1919: Production in acid conditions, purification, characterization and application on xylan hydrolysis

Abstract Hemicelluloses are mainly branched heteropolysaccharides composed by xylose, arabinose, mannose, galactose, rhamnose and glucose. The main hemicellulose is xylan, a structure composed by xylose main chain branched with other monosaccharides or glucuronic and galacturonic acids. Among various hydrolytic enzymes acting on the degradation of xylan, α-L-arabinofuranosidases catalyze the hydrolysis of linkages between α-L-arabinofuranosides and the polysaccharide main chain. In this study, Aspergillus hortai strain CRM1919, isolated from soil surrounding saline and alkaline lagoons of the Nhecolândia region in Pantanal Matogrossense was investigated regarding the production of α-L-arabinofuranosidases. A. hortai produced α- l -arabinofuranosidase at high levels when cultivated in medium containing citrus pulp and orange peel at 1% (w/v) under static submerged cultures for 4 days and at pH 2.5 and 30 °C. The optimization steps increased 15-fold the enzymatic activity. The purified α- l -arabinofuranosidase was optimally active at pH of 4.0 and 60 °C, presenting half-lives of 265 and 230 min at 30 and 40 °C, respectively. High enzyme stability was observed after 24 h incubation at pH 5.0 and in presence of high concentration of NaCl in the reaction medium. The purified enzyme was mainly active against ρ-nitrophenyl-α- l -arabinofuranoside presenting the following kinetic parameters Km of 8.73 mmol/L, Vmax of 7.91 μmol/min.mg of protein and Kcat of 0.59/min. Hydrolysis of oat xylan performed in the presence of the purified α-arabinofuranosidase individually and in association with a purified xylanase made possible to verify the cooperation between these xylanolytic enzymes and the sequential action of each enzyme in the xylan structure hydrolysis.

Screening of fungal contaminants in banana tissue cultures in Jkuat, Kenya

Tissue culture is prone to high costs of production arising from losses incurred from fungal contamination. The aim of the study was to characterise fungal contaminants and elucidate the exhibited mode of resistance to most preferred sterilants. Twenty nine fungal samples were collected at the different stages of tissue culture growth, using purposive sampling technique. Morphology results were confirmed by molecular characterization using fungal 18S rRNA sequences. Biochemical and antibiosis tests, identification of genes for capsulation and ATP binding Cassete (ABC) transporters, were performed to show the relationship between the fungi and sterilants resistance. Amylases and proteases were highly expressed by all isolates while xylanases and lipases were moderately expressed and esterases were lowly expressed. Only fourteen isolates had antagonistic activity for Candida albicans while nine of them had antagonistic activity for Pseudomonas aeruginosa. Three isolates were both antagonistic for Staphylococcus aureus and for Escherichia coli. Cunninghamella bainieri (10R) recorded a unique antibiosis and extra cellular enzymatic activity (p<0.05). All the isolates were positive for mdr1 gene and three isolates had CAP64 capsule genes. Aspergillus sp., Penincillium sp., Cladosporium sp., Cunninghamella sp. and Fusarium sp. were identified to be the major fungal contaminants of tissue culture banana cultures in JKUAT laboratories. Key words: Tissue culture, 18S rRNA, fungal contaminants, banana cultures.

Recombinant expression of Aspergillus niger GH10 endo‐xylanase in Pichia pastoris by constructing a double‐plasmid co‐expression system

AbstractBACKGROUNDPrevious work has shown that GH10 endo‐xylanase presented better synergistic cooperation with cellulase to achieve effective hydrolysis. However, most currently reported commercial xylanolytic enzymes from Aspergillus niger belong to GH11 and there have been few studies regarding GH10 xylanase. In addition, to increase overall expression of heterologous endo‐xylanase in Pichia pastoris, a double‐plasmid co‐expression method, including the construction of recombinant P. pastoris using one expression vector followed by inserting another vector which targeted to different locus, was introduced.RESULTSA GH10 xylanase gene from A. niger BE‐2 (XynC) was optimized and constitutively expressed in P. pastoris GS115. Compared with the conventional single‐plasmid method, it appeared that the double‐plasmid strategy could considerably increase the XynC yield by ∼33%. To further improve enzyme production, cultivation conditions were optimized at shake‐flask level and then scaled up to a 5‐L bioreactor. Through high cell‐density fermentation, the expression level of GH10 XynC reached 1650 U mL−1. The XynC showed maximum activity at 55 °C and pH 5.0, and exhibited an excellent stability over a wide range of pH from 4.5 to 7.0. The kinetic parameters Km and Vmax values for beechwood xylan were 3.5 mg mL−1 and 2327 U mg−1, respectively.CONCLUSIONThe double‐plasmid co‐expression strategy introduced herein could greatly improve the XynC yield in P. pastoris. The GH10 xylanase from A. niger BE‐2 shared similar reaction conditions with the existing enzymatic hydrolysis, which might be a good candidate to assist cellulases for industrial application. © 2019 Society of Chemical Industry