Abstract
An extracellular thermostable xylanase (Xyl-IIb) produced by Penicillium citrinum isolate HZN13 was purified to homogeneity using DEAE-Sepharose, Sephadex G-100 and Bio-Gel P-60 chromatography with specific activity of 6272.7 U/mg and 19.6-fold purification. The purification revealed the occurrence of multiple forms of xylanases (Xyl-I, Xyl-IIa, Xyl-IIb and Xyl-III). The molecular mass of highly purified Xyl-IIb was ~31 kDa with SDS-PAGE. The enzyme was cellulase-free, thermostable (55–75 °C) and acidophilic (3.5–5.0). It was activated by Ca2+, Ba2+, DTT and β-mercaptoethanol, whereas inhibited by Hg2+, Pb2+, Ni2+ and p-CMB. Purified Xyl-IIb exhibited highest specificity toward birchwood and oat spelts xylan. Kinetics of Xyl-IIb revealed a K m of 10 mg/ml and 16.7 mg/ml and V max of 9523g and 15,873 U/mg with birchwood and oat spelts xylan, respectively, indicating high affinity toward birchwood xylan. The xylanase (Xyl-IIb) belongs to glycosyl hydrolase (GH) family 10 based on conserved regions. Xylanase-encoding gene (xynB) consists of 1501 bp with an open reading frame of 264 bp which was predicted to encode a protein having 87 amino acids and shared homology with endo-1,4-beta-xylanase (xynB) gene from Penicillium citrinum. Cloned xynB gene was expressed in E. coli BL21 (DE3) with xylanase activity (80 U/mg) and confirmed to be GH-10 Xyl-IIa based on molecular mass (~40 kDa). These properties of xylanase make it promising for their applications in biofuel industries.Electronic supplementary materialThe online version of this article (doi:10.1007/s13205-016-0489-4) contains supplementary material, which is available to authorized users.
Highlights
Xylan, the structural polysaccharide, is a heteroglycan composed of a backbone of b-(1,4)-linked D-xylopyranosyl residues
Cloned xynB gene was expressed in E. coli BL21 (DE3) with xylanase activity (80 U/mg) and confirmed to be glycosyl hydrolase (GH)-10 Xyl-IIa based on molecular mass
We report the occurrence of multiple xylanases as well as purification, properties, gene cloning and sequencing and expression of GH10 xylanase from Penicillium citrinum isolate HZN13
Summary
The structural polysaccharide, is a heteroglycan composed of a backbone of b-(1,4)-linked D-xylopyranosyl residues. The complete hydrolysis of xylan requires the synergistic action of different xylanolytic enzymes (xylanase, b-xylosidase, a-L-arabinofuranosidase, a-D-glucuronidase and acetyl xylan esterase) (Beg et al 2001). Xylanases (EC 3.2.1.8) deserve special attention as they degrade major hemicellulosic polysaccharides by catalyzing the hydrolysis of xylopyranosyl linkages of b-1,4-xylan (Zhang et al 2011). Xylanases are produced from a variety of agricultural wastes. Sweet sorghum bagasse is cheap, widely distributed, available in plenty and serves as a better feedstock for xylanase production as it is non-food based agricultural residue having high carbohydrate content. Sweet sorghum is unique among the other crops as feedstock for renewable energy due to high photosynthetic activity, thereby generating high biomass yields and survives water deficit conditions, abiotic stresses, heat and dry environments, 169 Page 2 of 9
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