Purification, characterization, gene cloning and expression of GH-10 xylanase (Penicillium citrinum isolate HZN13)
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- 作者：Zabin K. Bagewadi ; Sikandar I. Mulla ; Harichandra Z. Ninnekar
- 刊名：3 Biotech
- 出版年：2016
- 出版时间：December 2016
- 年：2016
- 卷：6
- 期：2
- 全文大小：784 KB
- 刊物主题：Biotechnology; Agriculture; Cancer Research; Bioinformatics; Stem Cells; Biomaterials;
- 出版者：Springer Berlin Heidelberg
- ISSN：2190-5738
- 卷排序：6
文摘
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 Km of 10 mg/ml and 16.7 mg/ml and Vmax 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.KeywordsXylanasePenicillium citrinumPurificationCharacterizationCloning and expression