Improving the thermostability of Trichoderma reesei xylanase 2 by introducing disulfide bonds

Background: Xylanases are considered one of the most important enzymes in many industries. However, their low thermostability hampers their applications in feed pelleting, pulp bleaching, and so on. The main aim of this work was to improve the thermostability of Trichoderma ressei xylanase 2 (Xyn2) by introducing disulfide bonds between the N-terminal and α-helix and the ß-sheet core. Results: In this work, two disulfide bonds were separately introduced in the Xyn2 to connect the N-terminal and α-helix to the ß-sheet core of Xyn2. The two disulfide bonds were introduced by site-directed mutagenesis of the corresponding residues. The half-life of the mutants Xyn2C14­52 (disulfide bond between ß-sheets B2 and B3) and Xyn2C59­149 (disulfide bond between ß-sheets A5 and A6) at 60°C was improved by approximately 2.5- and 1.8-fold compared to that of the wild type Xyn2. In addition, the enzyme's resistance to alkali and acid was enhanced. Conclusion: Our results indicated that the connection of the N-terminal and α-helix to the ß-sheet core is due to the stable structure of the entire protein.

Xylanase and beta-xylosidase from Penicillium janczewskii: Purification, characterization and hydrolysis of substrates

Background: Xylanases and β-D-xylosidases are the most important enzymes responsible for the degradation of xylan, the second main constituent of plant cell walls. Results: In this study, the main extracellular xylanase (XYL I) and p-xylosidase (BXYL I) from the fungus Penicillium janczewskii were purified, characterized and applied for the hydrolysis of different substrates. Their molecular weights under denaturing and non-denaturing conditions were, respectively, 30.4 and 23.6 kDa for XYL I, and 100 and 200 kDa for BXYL I, indicating that the latter is homodimeric. XYL I is highly glycosylated (78%) with optimal activity in pH 6.0 at 65°C, while BXYL I presented lower sugar content (10.5%) and optimal activity in pH 5.0 at 75°C. The half-lives of XYL I at 55, 60 and 65°C were 125,16 and 6 min, respectively. At 60°C, BXYL I retained almost 100% of the activity after 6 h. NH4+,Na+, DTT and β-mercaptoethanol stimulated XYL I, while activation of BXYL I was not observed. Interestingly, XYL I was only partially inhibited by Hg2+, while BXYL I was completely inhibited. Xylobiose, xylotriose and larger xylooligosaccharides were the main products from xylan hydrolysis by XYL I. BXYL I hydrolyzed xylobiose and larger xylooligosaccharides with no activity against xylans. Conclusion: The enzymes act synergistically in the degradation of xylans, and present industrial characteristics especially in relation to optimal activity at high temperatures, prolonged stability of BXYL I at 60°C, and stability of XYL I in wide pH range.

Value addition of corn husks through enzymatic production of xylooligosaccharides

ABSTRACT Corn husks are the major wastes of corn industries with meagre economic significance. The present study was planned for value addition of corn husk through extraction of xylan, followed by its enzymatic hydrolysis into xylooligosaccharides, a pentose based prebiotic. Compositional analysis of corn husks revealed neutral detergent fibre 68.87%, acid detergent fibre 31.48%, hemicelluloses 37.39%, cellulose 29.07% and crude protein 2.68%. Irrespective of the extraction conditions, sodium hydroxide was found to be more effective in maximizing the yield of xylan from corn husks than potassium hydroxide (84% vs. 66%). Application of xylanase over the xylan of corn husks resulted into production of xylooligosaccharides with different degree of polymerization namely, xylobiose and xylotriose in addition to xylose monomer. On the basis of response surface model analysis, the maximum yield of xylobiose (1.9 mg/ml) was achieved with the enzymatic hydrolysis conditions of pH 5.8, temperature 44°C, enzyme dose 5.7U/ml and hydrolysis time of 17.5h. Therefore, the corn husks could be used as raw material for xylan extraction vis a vis its translation into prebiotic xylooligosaccharides.

Production of a cellulase-free alkaline xylanase from Bacillus pumilus MTCC 5015 by submerged fermentation and its application in biobleaching.

Here, we described the production of a cellulase-free alkaline xylanase from Bacillus pumilus MTCC 5015 by submerged fermentation and its application in biobleaching. Various process parameters affecting xylanase production by B. pumilus were optimized by adopting a Plackett-Burman design (PBD) as well as Response surface methodology (RSM). These statistical methods aid in improving the enzyme yield by analysing the individual crucial components of the medium. Maximum production was obtained with 4% yeast extract, 0.08% magnesium sulphate, 30 h of inoculum age, incubation temperature of 33.5 °C and pH 9.0. Under optimized conditions, the xylanase activity was 372 IU/ml. Media engineering improved a 5-fold increase in the enzyme production. Scanning electron microscopy (SEM) showed significant changes on the surface of xylanase treated pulps as a result of xylan hydrolysis. Increased roughness of paper carton fibres was apparent in scanning electron micrograph due to opening of the micro fibrils present on the surface by xylanase action. The untreated pulp did not show any such change. These results demonstrated that the B. pumilus MTCC 5015 xylanase was effective in bio-bleaching of paper carton.

Xylooligosaccharides: an economical prebiotic from agroresidues and their health benefits.

Oligosaccharides and dietary fibres are non-digestible food ingredients that preferentially stimulate the growth of prebiotic Bifidobacterium and other lactic acid bacteria in the gastro-intestinal tract. Xylooligosaccharides (XOS) provide a plethora of health benefits and can be incorporated into several functional foods. In the recent times, there has been an over emphasis on the microbial conversion of agroresidues into various value added products. Xylan, the major hemicellulosic component of lignocellulosic materials (LCMs), represents an important structural component of plant biomass in agricultural residues and could be a potent bioresource for XOS. On an industrial scale, XOS can be produced by chemical, enzymatic or chemo-enzymatic hydrolysis of LCMs. Chemical methods generate XOS with a broad degree of polymerization (DP), while enzymatic processes will be beneficial for the manufacture of food grade and pharmaceutically important XOS. Xylooligomers exert several health benefits, and therefore, have been considered to provide relief from several ailments. This review provides a brief on production, purification and structural characterization of XOS and their health benefits.

Microbial xylanases and their biomedical applications: a review .

Xylanases have a great potential, mainly known for industrial applications. They can hydrolyze the xylose (Hemicellulose of plant cell wall) and can be used for bio-bleaching the kraft pulp. As it reduces the requirement of harsh chemicals in the process, it can be used further to a number of bio-products with a great aggregate value. Microbial-origin xylanases can also be used in improving the nutritional quality of animal feed (e.g. food additives to poultry, piggery or fishery) and indirectly affect the humans. Additionally they can be used directly in human food in bakery, clarification of juices and in xenobiotics like tobacco processing. The great value of xylanase as a bio-bleaching agent has now a new dimension of fiber digesting agent having relevance to food, drugs and cosmetics act. This review presents some important applications of Xylanases extended up to biomedical sciences.

Xylanases: An Overview.

Endo-1, 4-β-xylanase (Endo-β-1, 4-xylan, xylanohydrolase; EC. 3.2.1.8, commonly called xylanase) is an industrially important enzyme which degrades xylan randomly and produces xylooligosaccharides, xylobiose and xylose. It is mainly present in microbes and plants but not in animals. Xylanases from fungal and bacterial sources have been extensively studied and produced commercially. Its potential use in paper industries has been discussed which is directly related to reduction in environmental pollution. It has role in bio-bleaching paper pulp and increasing pulp brightness. Besides, it can be exploited for ethanol production and as an additive in animal feedstock to improve its nutritional value. Endo-1, 4-β-xylanase can also be exploited in baking and fruit juice industries. Here, we reviewed its distribution, structural aspects and industrial/ biotechnological applications. Besides, we also discussed studies related to cloning of the gene encoding endo-1, 4-β-xylanase with the objectives of overproducing the enzyme and altering its properties to suit commercial applications.

Characterization of Xylanase of Cladosporium cladosporioides H1 Isolated from Janggyeong Panjeon in Haeinsa Temple

Cladosporium cladosporioides H1 was found to be the most abundant microbe in Janggyeong Panjeon. C. cladosporioides H1 produced a 20 kDa xylanase, which was generally stable below 60degrees C and had specialized activity in an acidic condition. Our results may lead to the development of a strategy for preservation of organic cultural heritage environments.

Heterologous Expression of Endo-1,4-beta-xylanaseA from Phanerochaete chrysosporium in Pichia pastoris

The cDNA of endo-1,4-beta-xylanaseA, isolated from Phaenerocheate chrysosporium was expressed in Pichia pastoris. Using either the intrinsic leader peptide of XynA or the alpha-factor signal peptide of Saccharomyces cerevisiae, xylanaseA is efficiently secreted into the medium at maximum concentrations of 1,946 U/L and 2,496 U/L, respectively.

Purification and characterization of a new Xylanase from Humicola grisea var. Thermoidea

The thermophilic fungus Humicola grisea var. thermoidea secretes extracellular xylanase when grown on solid and in liquid media containing wheat bran and banana plant residue as substrates, respectively. At 55ºC, xylanase from the culture filtrate of H. grisea var. thermoidea grown on banana stalk retained 50% of its activity after 28 h of incubation. A xylanase (X2) was isolated from solid state cultures with wheat bran as the carbon source. It was purified to apparent homogeneity by ultrafiltration followed by ion-exchange and hydrophobic interaction chromatography on DEAE-Sepharose and Phenyl-Sepharose resins, respectively. The enzyme had an apparent molecular weight of 29 kDa, as determined by SDS-PAGE. The purified enzyme was most active at pH and temperature ranges of 4.5-6.5 and 55-60ºC, respectively. In addition, X2 showed thermostability at 60ºC with a half-life of approx. 5.5 h. The apparent Km values, using soluble and insoluble arabinoxylans as substrates, were 10.87 and 11.20 mg/ml, respectively.

O fungo termofílico Humicola grisea var. secreta xilanase extracelular quando cultivado em meios sólidos e líquidos contendo farelo de trigo e engaço de bananeira como substratos, respectivamente. À temperatura de 55ºC, xilanase do filtrado de meio de cultura de H. grisea var. thermoidea cultivado em engaço de bananeira reteve 50% de sua atividade após 28 de incubação. Uma xilanase (X2) foi isolada de culturas de estado sólido contendo farelo de trigo como fonte de carbono. X2 foi purificada por ultrafiltração, seguido por cromatografias de interação hidrofóbica e troca iônica em resinas de Phenyl-Sepharose e DEAE-Sepharose, respectivamente. A enzima apresentou peso molecular de 29 kDa, como determinado por SDS-PAGE. A enzima purificada foi mais ativa em intervalos de pH e temperatura de 4,5-6,5 and 55-60ºC, respectivamente. Além disso, X2 mostrou termoestabilidade a 60ºC com meia vida de aproximadamente 5,5 h. Os valores de Km aparente, utilizando arabinoxilanas solúveis e insolúveis, foram 10,87 and 11,20 mg/ml, respectivamente.

INSOLUBLE DYE XYLAN OF SUGAR CANE BAGASSE AND ITS APPLICATION FOR MEASUREMENT OF XYLANASE ACTIVITY / 微生物学通报

Xylan from sugar cane bagasse can be cross linked by 1,4 butanedioldiglycidylether with dye Cibacron blue 3GA to form an insoluble dye solid This substrate was stable from pH4 to pH8 at 50℃ for 24h or 100℃ for 30min It is easy and sensitive to use this dyed xylan from sugar cane bagasse in measuring xylanase activity and screening xylanase producing microorganisms on agar plate