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The Heterologous Expression and Characterization of Lytic Polysaccharide Monooxygenase from Actinosynnema mirum DSM 43827 |
SHI Xian-wei, ZHANG Wei-tao, ZHANG Xiao-fei, YANG Guang-yu, FENG Yan |
State Key Laboratory of Microbial Metabolism, School of Life Science and Biotechnology, Shanghai Jiaotong University, Shanghai 200240, China |
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Abstract Lytic polysaccharide monooxygenases (LPMO) is a recently discovered new type of oxidase that catalyzes oxidative cleavage of recalcitrant polysaccharides (such as cellulose and chitin), andits oxidative cleavage are advantages for degradation byglycoside hydrolasesystem to obtain soluble oligosaccharides. To get more LPMOs, the Amir_5334 gene coding LPMO was cloned successfully from Actinosynnema mirum DSM 43827 by PCR. The gene was inserted into the pET28a(+) vector that contained maltose binding protein (MBP-tag) and transformed into E.coli BL21(DE3)cell for induced expression. The MBP-Am5 fusion protein was purified by Ni-column affinity chromatography and cleavaged by Factor Xa, and obtained the nature Am5 finally. The predicted molecular weight of nativeAm5 is about 26 kDa, and its isoelectric point is 8.3.The analysis of Am5 sequence shows that the sequence identityis very low, compared with other LPMOs in AA10 family.The characterization of Am5 shows that Am5 is a LPMO that could act on chitin but not cellulose. On the other hand, Am5 can degrade chitin with chitinase synergistically, and the hydrolysis efficiency increased by 60%. The affinity of Am5 demonstrated Am5 combined with chitin more strongly than cellulose. All of above indicates that Am5 may be a LPMO acted on chitin with high efficiency and selectivity.
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Received: 16 May 2014
Published: 25 July 2014
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