Studies on the extracellular xylanase activity of some thermophilic actinomycetes

AbstractXylanases are widely used enzymes in the food, textile, and paper industries. Most efficient xylanases have been identified from lignocellulose-degrading microbiota, such as the microbiota of the cow rumen and the termite hindgut. Xylanase genes from efficient pulp and paper wastewater treatment (PPWT) microbiota have been previously recovered by metagenomics, assigning most of the xylanase genes to the GH10 family. In this study, a total of 40 GH10 family xylanase genes derived from a certain PPWT microbiota were cloned and expressed in Escherichia coli BL21 (DE3). Among these xylanase genes, 14 showed xylanase activity on beechwood substrate. Two of these, PW-xyl9 and PW-xyl37, showed high activities, and were purified to evaluate their xylanase properties. Values of optimal pH and temperature for PW-xyl9 were pH 7 and 60 ℃, respectively, while those for PW-xyl37 were pH 7 and 55 ℃, respectively; their specific xylanase activities under optimal conditions were 470.1 U/mg protein and 113.7 U/mg protein, respectively. Furthermore, the Km values of PW-xyl9 and PW-xyl37 were determined as 8.02 and 18.8 g/L, respectively. The characterization of these two xylanases paves the way for potential application in future pulp and paper production and other industries, indicating that PPWT microbiota has been an undiscovered reservoir of efficient lignocellulase genes. This study demonstrates that a metagenomic approach has the potential to screen efficient xylanases of uncultured microorganisms from lignocellulose-degrading microbiota. In a similar way, other efficient lignocellulase genes might be identified from PPWT treatment microbiota in the future.

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Method for the Production and Purification of Plant Immuno-Active Xylanase from Trichoderma

International Journal of Molecular Sciences ◽

10.3390/ijms22084214 ◽

2021 ◽

Vol 22 (8) ◽

pp. 4214

Author(s):

Gautam Anand ◽

Meirav Leibman-Markus ◽

Dorin Elkabetz ◽

Maya Bar

Keyword(s):

Immune System ◽

Plant Defense ◽

Plant Immunity ◽

Xylanase Activity ◽

Hydrolytic Enzymes ◽

Adaptive Immune System ◽

Defense Responses ◽

Plant Defense Responses ◽

Xylanase Production ◽

Ideal System

Plants lack a circulating adaptive immune system to protect themselves against pathogens. Therefore, they have evolved an innate immune system based upon complicated and efficient defense mechanisms, either constitutive or inducible. Plant defense responses are triggered by elicitors such as microbe-associated molecular patterns (MAMPs). These components are recognized by pattern recognition receptors (PRRs) which include plant cell surface receptors. Upon recognition, PRRs trigger pattern-triggered immunity (PTI). Ethylene Inducing Xylanase (EIX) is a fungal MAMP protein from the plant-growth-promoting fungi (PGPF)–Trichoderma. It elicits plant defense responses in tobacco (Nicotiana tabacum) and tomato (Solanum lycopersicum), making it an excellent tool in the studies of plant immunity. Xylanases such as EIX are hydrolytic enzymes that act on xylan in hemicellulose. There are two types of xylanases: the endo-1, 4-β-xylanases that hydrolyze within the xylan structure, and the β-d-xylosidases that hydrolyze the ends of the xylan chain. Xylanases are mainly synthesized by fungi and bacteria. Filamentous fungi produce xylanases in high amounts and secrete them in liquid cultures, making them an ideal system for xylanase purification. Here, we describe a method for cost- and yield-effective xylanase production from Trichoderma using wheat bran as a growth substrate. Xylanase produced by this method possessed xylanase activity and immunogenic activity, effectively inducing a hypersensitive response, ethylene biosynthesis, and ROS burst.

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Optimisation of xylanases production by two Cellulomonas strains and their use for biomass deconstruction

Applied Microbiology and Biotechnology ◽

10.1007/s00253-021-11305-y ◽

2021 ◽

Author(s):

Ornella M Ontañon ◽

Soma Bedő ◽

Silvina Ghio ◽

Mercedes M Garrido ◽

Juliana Topalian ◽

...

Keyword(s):

Extracellular Enzymes ◽

Culture Supernatant ◽

Xylanase Activity ◽

Barley Straw ◽

Proteomic Profiling ◽

Biomass Hydrolysis ◽

Biomass Deconstruction ◽

Xylanolytic Activity ◽

Key Points ◽

Distinguishing Features

Abstract One of the main distinguishing features of bacteria belonging to the Cellulomonas genus is their ability to secrete multiple polysaccharide degrading enzymes. However, their application in biomass deconstruction still constitutes a challenge. We addressed the optimisation of the xylanolytic activities in extracellular enzymatic extracts of Cellulomonas sp. B6 and Cellulomonas fimi B-402 for their subsequent application in lignocellulosic biomass hydrolysis by culture in several substrates. As demonstrated by secretomic profiling, wheat bran and waste paper resulted to be suitable inducers for the secretion of xylanases of Cellulomonas sp. B6 and C. fimi B-402, respectively. Both strains showed high xylanolytic activity in culture supernatant although Cellulomonas sp. B6 was the most efficient xylanolytic strain. Upscaling from flasks to fermentation in a bench scale bioreactor resulted in equivalent production of extracellular xylanolytic enzymatic extracts and freeze drying was a successful method for concentration and conservation of the extracellular enzymes, retaining 80% activity. Moreover, enzymatic cocktails composed of combined extra and intracellular extracts effectively hydrolysed the hemicellulose fraction of extruded barley straw into xylose and xylooligosaccharides. Key points • Secreted xylanase activity of Cellulomonas sp. B6 and C. fimi was maximised. • Biomass-induced extracellular enzymes were identified by proteomic profiling. • Combinations of extra and intracellular extracts were used for barley straw hydrolysis.

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Phospholipid fatty acid profiles and xylanase activity in particle size fractions of forest soil and casts of Lumbricus terrestris L. (Oligochaeta, Lumbricidae)

Applied Soil Ecology ◽

10.1016/j.apsoil.2006.06.003 ◽

2007 ◽

Vol 35 (2) ◽

pp. 412-422 ◽

Cited By ~ 32

Author(s):

Sven Marhan ◽

Ellen Kandeler ◽

Stefan Scheu

Keyword(s):

Particle Size ◽

Fatty Acid ◽

Forest Soil ◽

Phospholipid Fatty Acid ◽

Xylanase Activity ◽

Lumbricus Terrestris ◽

Fatty Acid Profiles ◽

Size Fractions ◽

Particle Size Fractions

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Synergistic Effect of Different Plant Cell Wall–Degrading Enzymes Is Important for Virulence of Fusarium graminearum

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-07-17-0179-r ◽

2017 ◽

Vol 30 (11) ◽

pp. 886-895 ◽

Cited By ~ 26

Author(s):

Maria Chiara Paccanaro ◽

Luca Sella ◽

Carla Castiglioni ◽

Francesca Giacomello ◽

Ana Lilia Martínez-Rocha ◽

...

Keyword(s):

Cell Wall ◽

Fusarium Graminearum ◽

Plant Cell ◽

Plant Cell Wall ◽

Xylanase Activity ◽

Cellulase Activity ◽

Wild Type ◽

Cell Wall Degrading Enzymes ◽

Xylanase Production ◽

Significant Difference

Endo-polygalacturonases (PGs) and xylanases have been shown to play an important role during pathogenesis of some fungal pathogens of dicot plants, while their role in monocot pathogens is less defined. Pg1 and xyr1 genes of the wheat pathogen Fusarium graminearum encode the main PG and the major regulator of xylanase production, respectively. Single- and double-disrupted mutants for these genes were obtained to assess their contribution to fungal infection. Compared with wild-type strain, the ∆pg mutant showed a nearly abolished PG activity, slight reduced virulence on soybean seedlings, but no significant difference in disease symptoms on wheat spikes; the ∆xyr mutant was strongly reduced in xylanase activity and moderately reduced in cellulase activity but was as virulent as wild type on both soybean and wheat plants. Consequently, the ΔpgΔxyr double mutant was impaired in xylanase, PG, and cellulase activities but, differently from single mutants, was significantly reduced in virulence on both plants. These findings demonstrate that the concurrent presence of PG, xylanase, and cellulase activities is necessary for full virulence. The observation that the uronides released from wheat cell wall after a F. graminearum PG treatment were largely increased by the fungal xylanases suggests that these enzymes act synergistically in deconstructing the plant cell wall.

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Identification of Poly(cis-1,4-Isoprene) Degradation Intermediates during Growth of Moderately Thermophilic Actinomycetes on Rubber and Cloning of a Functional lcp Homologue from Nocardia farcinica Strain E1

Applied and Environmental Microbiology ◽

10.1128/aem.72.5.3375-3382.2006 ◽

2006 ◽

Vol 72 (5) ◽

pp. 3375-3382 ◽

Cited By ~ 58

Author(s):

Ebaid M. A. Ibrahim ◽

Matthias Arenskï¿½tter ◽

Heinrich Luftmann ◽

Alexander Steinbï¿½chel

Keyword(s):

Thermophilic Bacteria ◽

Natural Rubber Latex ◽

Gel Permeation Chromatography ◽

Mycolic Acid ◽

Rrna Gene ◽

Ionization Mass ◽

Thermophilic Actinomycetes ◽

Moderately Thermophilic ◽

Nocardia Farcinica ◽

Different Strains

ABSTRACT The enrichment and isolation of thermophilic bacteria capable of rubber [poly(cis-1,4-isoprene)] degradation revealed eight different strains exhibiting both currently known strategies used by rubber-degrading mesophilic bacteria. Taxonomic characterization of these isolates by 16S rRNA gene sequence analysis demonstrated closest relationships to Actinomadura nitritigenes, Nocardia farcinica, and Thermomonospora curvata. While strains related to N. farcinica exhibited adhesive growth as described for mycolic acid-containing actinomycetes belonging to the genus Gordonia, strains related to A. nitritigenes and T. curvata formed translucent halos on natural rubber latex agar as described for several mycelium-forming actinomycetes. For all strains, optimum growth rates were observed at 50ï¿½C. The capability of rubber degradation was confirmed by mineralization experiments and by gel permeation chromatography (GPC). Intermediates resulting from early degradation steps were purified by preparative GPC, and their analysis by infrared spectroscopy revealed the occurrence of carbonyl carbon atoms. Staining with Schiff's reagent also revealed the presence of aldehyde groups in the intermediates. Bifunctional isoprenoid species terminated with a keto and aldehyde function were found by matrix-assisted laser desorption ionization-time-of-flight and electrospray ionization mass spectrometry analyses. Evidence was obtained that biodegradation of poly(cis-1,4-isoprene) is initiated by endocleavage, rather than by exocleavage. A gene (lcp) coding for a protein with high homology to Lcp (latex-clearing protein) from Streptomyces sp. strain K30 was identified in Nocardia farcinica E1. Streptomyces lividans TK23 expressing this Lcp homologue was able to cleave synthetic poly(cis-1,4-isoprene), confirming its involvement in initial polymer cleavage.

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Improved Production ofAspergillus usamiiendo-β-1,4-Xylanase inPichia pastorisvia Combined Strategies

BioMed Research International ◽

10.1155/2016/3265895 ◽

2016 ◽

Vol 2016 ◽

pp. 1-9 ◽

Cited By ~ 6

Author(s):

Jianrong Wang ◽

Yangyuan Li ◽

Danni Liu

Keyword(s):

Pichia Pastoris ◽

Cell Viability ◽

Recombinant Proteins ◽

Recombinant Strain ◽

Xylanase Activity ◽

Vitreoscilla Hemoglobin ◽

Wild Type ◽

Methanol Induction ◽

Aspergillus Usamii ◽

Induction Temperature

A series of strategies were applied to improve expression level of recombinant endo-β-1,4-xylanase fromAspergillus usamii(A. usamii) inPichia pastoris(P. pastoris). Firstly, the endo-β-1,4-xylanase (xynB) gene fromA. usamiiwas optimized forP. pastorisand expressed inP. pastoris. The maximum xylanase activity of optimized (xynB-opt) gene was 33500 U/mL after methanol induction for 144 h in 50 L bioreactor, which was 59% higher than that by wild-type (xynB) gene. To further increase the expression ofxynB-opt, theVitreoscilla hemoglobin(VHb) gene was transformed to the recombinant strain containingxynB-opt. The results showed that recombinant strain harboring thexynB-optandVHb(named X33/xynB-opt-VHb) displayed higher biomass, cell viability, and xylanase activity. The maximum xylanase activity of X33/xynB-opt-VHbin 50 L bioreactor was 45225 U/mL, which was 35% and 115% higher than that by optimized (xynB-opt) gene and wild-type (xynB) gene. Finally, the induction temperature of X33/xynB-opt-VHbwas optimized in 50 L bioreactor. The maximum xylanase activity of X33/xynB-opt-VHbreached 58792 U/mL when the induction temperature was 22°C. The results presented here will greatly contribute to improving the production of recombinant proteins inP. pastoris.

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