Purification, characterization, gene cloning and expression of GH-10 xylanase (Penicillium citrinum isolate HZN13).
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 Kmof 10 mg/ml and 16.7 mg/ml and Vmaxof 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.
Department of Biochemistry, Karnatak University, Dharwad, Karnataka, 580 003, India.
Zabin K
ZK
Bagewadi
Department of Biochemistry, Karnatak University, Dharwad, Karnataka, 580 003, India.
Sikandar I
SI
Mulla
Department of Biochemistry, Karnatak University, Dharwad, Karnataka, 580 003, India. hzninnekar@yahoo.com.
Harichandra Z
http://orcid.org/0000-0002-3435-6457
HZ
Ninnekar
Characterization; Cloning and expression; Penicillium citrinum; Purification; Xylanase