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Purification and Properties of a Psychrotrophic Trichoderma sp. Xylanase and its Gene Sequence

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Abstract

A psychrotrophic fungus identified as Trichoderma sp. SC9 produced 36.7 U/ml of xylanase when grown on a medium containing corncob xylan at 20 °C for 6 days. The xylanase was purified 37-fold with a recovery yield of 8.2%. The purified xylanase appeared as a single protein band on SDS-PAGE with a molecular mass of approximately 20.5 kDa. The enzyme had an optimal pH of 6.0, and was stable over pH 3.5–9.0. The optimal temperature of the xylanase was 42.5 °C and it was stable up to 35 °C at pH 6.0 for 30 min. The xylanase was thermolabile with a half-life of 23.9 min at 45 °C. The apparent K m values of the xylanase for birchwood, beechwood, and oat-spelt xylans were found to be 3, 2.1, and 16 mg/ml respectively. The xylanase hydrolyzed beechwood xylan and birchwood xylan to yield mainly xylobiose as end products. The enzyme-hydrolysed xylotriose, xylotetraose, and xylopentose to produce xylobiose, but it hardly hydrolysed xylobiose. A xylanase gene (xynA) with an open reading frame of 669 nucleotide base pairs (bp), encoding 222 amino acids, from the strain was cloned and sequenced. The deduced amino acid sequence of XynA showed 85% homology with Xyn2 from a mesophilic strain of Trichoderma viride.

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Abbreviations

BSA:

Bovine serum albumin

CMC:

Carboxymethylcellulose

DNS:

Dinitrosalicylic acid

DTT:

Dithiothreitol

EDTA:

Ethylenediaminetetracetic acid

GH:

Glycoside hydrolase

MES:

2-(N-Morpholino)ethane sulfonic acid

MOPS:

3-(N-Morpholino)-propane sulphonic acid

ORF:

Open reading frame

PCR:

Polymerase chain reaction

PDA:

Potato dextrose-agar

pNP:

p-Nitrophenyl

RACE:

Rapid amplification of cDNA ends

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

TLC:

Thin-layer chromatography

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Acknowledgments

This work was financially supported by the Specialized Research Fund for the Doctoral Program of Higher Education and the Foundation for the Author of National Excellent Doctoral Dissertation of China (Project No. 200555).

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Authors and Affiliations

  1. Department of Biotechnology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, 100083, People’s Republic of China

    Peng Zhou, Huifang Zhu, Priti Katrolia & Zhengqiang Jiang

  2. Bioresource Utilization Laboratory, College of Engineering, China Agricultural University, Beijing, 100083, People’s Republic of China

    Qiaojuan Yan

  3. China Agricultural University, PO Box 294, No.17 Qinghua Donglu, Haidian District, Beijing, 100083, China

    Zhengqiang Jiang

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  1. Peng Zhou
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  2. Huifang Zhu
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Correspondence to Zhengqiang Jiang.

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Zhou, P., Zhu, H., Yan, Q. et al. Purification and Properties of a Psychrotrophic Trichoderma sp. Xylanase and its Gene Sequence. Appl Biochem Biotechnol 164, 944–956 (2011). https://doi.org/10.1007/s12010-011-9186-2

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  • DOI: https://doi.org/10.1007/s12010-011-9186-2

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