Purification of the alkaliphilic xylanases from Myceliophthora sp. IMI 387099 using cellulose-binding domain as an affinity tag |
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Authors: | A K Badhan B S Chadha H S Saini |
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Institution: | (1) Department of Microbiology, Guru Nanak Dev University, Amritsar, 143 005, India |
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Abstract: | Ten xylanase isoforms produced by Myceliophthora sp. were characterized for their ability to bind to avicel. Three of the xylanases showing differential affinity for avicel
were purified by column chromatography. The purified xylanase Xyl IIa, IIb and IIc showed molecular mass of 47, 41 and 30 kDa
and pI of ∼3.5, 4.8 and 5.2, respectively. Xyl IIa was optimally active at pH 8.0 and temperature 70 °C, while Xyl IIb and
IIc were optimally active at pH 9.0 and 60 °C and 7.0 and 80 °C, respectively. Xyl IIa and Xyl IIb showed higher stability
under alkaline conditions (pH 9.0) and retained 80% of the original activity upto 1 h and 3 h respectively, at 50 °C. All
three purified iso-xylanases showed enhanced activities in presence of Na+, Mg2+, Mn2+ and K+ ions, whereas, Zn2+ and Cu2+ showed negative effect on Xyl IIa. The activity of Xyl IIa increased in presence of reducing agents DTT and mercaptoethanol,
however, SDS showed inhibitory effect. Kinetic studies showed that Xyl IIb and IIc degrade rye arabinoxylan, much more efficiently
than oat spelt xylan, whereas, Xyl IIa showed much higher Kcat/Km value for birch wood xylan as compared to oat spelt xylan. The purified xylanases were apparently classified in family 10. |
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Keywords: | Myceliophthora sp Cellulose binding domains Affinity tag Purification of xylanase isoforms |
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