Xylanase

About: Xylanase is a research topic. Over the lifetime, 7099 publications have been published within this topic receiving 163793 citations.

Expression of endo-1, 4-beta-xylanase from Trichoderma reesei in Pichia pastoris and functional characterization of the produced enzyme.

Abstract: In recent years, xylanases have attracted considerable research interest because of their potential in various industrial applications. The yeast Pichia pastoris can neither utilize nor degrade xylan, but it possesses many attributes that render it an attractive host for the expression and production of industrial enzymes. The Xyn2 gene, which encodes the main Trichoderma reesei Rut C-30 endo-β-1, 4-xylanase was cloned into the pPICZαA vector and expressed in Pichia pastoris. The selected P. pastoris strains produced as 4,350 nkat/ml β-xylanase under the control of the methanol inducible alcohol oxidase 1 (AOX1) promoter. The secreted recombinant Xyn2 was estimated by SDS-PAGE to be 21 kDa. The activity of the recombinant Xyn2 was highest at 60°C and it was active over a broad range of pH (3.0–8.0) with maximal activity at pH 6.0. The enzyme was quite stable at 50°C and retained more than 94% of its activity after 30 mins incubation at this temperature. Using Birchwood xylan, the determined apparent Km and kcat values were 2.1 mg/ml and 219.2 S-1, respectively. The enzyme was highly specific towards xylan and analysis of xylan hydrolysis products confirmed as expected that the enzyme functions as endo-xylanase with xylotriose as the main hydrolysis products. The produced xylanase was practically free of cellulolytic activity. The P. pastoris expression system allows a high level expression of xylanases. Xylanase was the main protein species in the culture supernatant, and the functional tests indicated that even the non-purified enzyme shows highly specific xylanase activity that is free of cellulolytic side acitivities. Therefore, P pastoris is a very useful expression system when the goal is highly specific and large scale production of glycosyl hydrolases.

Purification and characterization of two extracellular xylanases from Penicillium sclerotiorum: a novel acidophilic xylanase.

Abstract: Two xylanases from the crude culture filtrate of Penicillium sclerotiorum were purified to homogeneity by a rapid and efficient procedure, using ion-exchange and molecular exclusion chromatography. Molecular masses estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 23.9 and 33.1 kDa for xylanase I and II, respectively. The native enzymes’ molecular masses of 23.8 and 30.8 kDa were estimated for xylanase I and II, respectively, by molecular exclusion chromatography. Both enzymes are glycoproteins with optimum temperature and pH of 50 °C and pH 2.5 for xylanase I and 55 °C and pH 4.5 for xylanase II. The reducing agents β-mercaptoethanol and dithio-treitol enhanced xylanase activities, while the ions Hg2+ and Cu2+ as well the detergent SDS were strong inhibitors of both enzymes, but xylanase II was stimulated when incubated with Mn2+. The Km value of xylanase I for birchwood xylan and for oat spelt xylan were 6.5 and 2.6 mg mL−1, respectively, whereas the Km values of xylanase II for these substrates were 26.61 and 23.45 mg mL−1. The hydrolysis of oat spelt xylan by xylanase I released xylobiose and larger xylooligosaccharides while xylooligosaccharides with a decreasing polymerization degree up to xylotriose were observed by the action of xylanase II. The present study is among the first works to examine and describe an extracellular, highly acidophilic xylanase, with an unusual optimum pH at 2.5. Previously, only one work described a xylanase with optimum pH 2.0. This novel xylanase showed interesting characteristics for biotechnological process such as feed and food industries.

Emergence of a subfamily of xylanase inhibitors within glycoside hydrolase family 18.

Abstract: The xylanase inhibitor protein I (XIP-I), recently identified in wheat, inhibits xylanases belonging to glycoside hydrolase families 10 (GH10) and 11 (GH11). Sequence and structural similarities indicate that XIP-I is related to chitinases of family GH18, despite its lack of enzymatic activity. Here we report the identification and biochemical characterization of a XIP-type inhibitor from rice. Despite its initial classification as a chitinase, the rice inhibitor does not exhibit chitinolytic activity but shows specificities towards fungal GH11 xylanases similar to that of its wheat counterpart. This, together, with an analysis of approximately 150 plant members of glycosidase family GH18 provides compelling evidence that xylanase inhibitors are largely represented in this family, and that this novel function has recently emerged based on a common scaffold. The plurifunctionality of GH18 members has major implications for genomic annotations and predicted gene function. This study provides new information which will lead to a better understanding of the biological significance of a number of GH18 'inactivated' chitinases.

Purification and characterization of a type B feruloyl esterase (StFAE-A) from the thermophilic fungus Sporotrichum thermophile

Abstract: A feruloyl esterase (StFAE-A) produced by Sporotrichum thermophile was purified to homogeneity. The purified homogeneous preparation of native StFAE-A exhibited a molecular mass of 57.0±1.5 kDa, with a mass of 33±1 kDa on SDS-PAGE. The pI of the enzyme was estimated by cation-exchange chromatofocusing to be at pH 3.1. The enzyme activity was optimal at pH 6.0 and 55–60 °C. The purified esterase was stable at the pH range 5.0–7.0. The enzyme retained 70% of activity after 7 h at 50 °C and lost 50% of its activity after 45 min at 55 °C and after 12 min at 60 °C. Determination of k cat/K m revealed that the enzyme hydrolyzed methyl p-coumarate 2.5- and 12-fold more efficiently than methyl caffeate and methyl ferulate, respectively. No activity on methyl sinapinate was detected. The enzyme was active on substrates containing ferulic acid ester linked to the C-5 and C-2 linkages of arabinofuranose and it hydrolyzed 4-nitrophenyl 5-O-trans-feruloyl-α-l-arabinofuranoside (NPh-5-Fe-Araf) 2-fold more efficiently than NPh-2-Fe-Araf. Ferulic acid (FA) was efficiently released from destarched wheat bran when the esterase was incubated together with xylanase from S. thermophile (a maximum of 34% total ferulic acid released after 1 h incubation). StFAE-A by itself could release FA, but at a level almost 47-fold lower than that obtained in the presence of xylanase. The potential of StFAE-A for the synthesis of various phenolic acid esters was tested using a ternary water-organic mixture consisting of n-hexane, 1-butanol and water as a reaction system.