<em>Actinosynne mamirum</em> DSM43827溶解性多糖单加氧酶的异源表达和酶学性质表征

doi:10.13523/j.cb.20140703

中国生物工程杂志

2014, Vol. 34

Issue (7): 17-23 DOI: 10.13523/j.cb.20140703

研究报告

Actinosynne mamirum DSM43827溶解性多糖单加氧酶的异源表达和酶学性质表征

施贤卫, 张伟涛, 张小飞, 杨广宇, 冯雁

上海交通大学生命科学技术学院微生物代谢国家重点实验室上海 200240

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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摘要：

溶解性多糖单加氧酶（lytic polysaccharide monooxygenases，LPMO）是近年来新发现的一类作用于结晶多糖（如纤维素和几丁质）的氧化酶；LPMO通过氧化的方式来裂解底物从而有利于水解酶系进一步作用，获得可溶性寡糖。为获得更多新酶资源，通过PCR方法从Actinosynnema mirum DSM 43827菌株中成功地克隆了编码LPMO的基因Amir_5334；将该基因构建到含麦芽糖结合蛋白（maltose binding protein，MBP）标签的pET-28a（+）载体（pET-28a-MBP）上，并转化至E. coli BL21（DE3）中进行诱导表达。利用镍柱亲和层析进行纯化，获得融合表达蛋白后，使用Factor Xa蛋白酶切除MBP标签，最终得到成熟的LPMO（Am5）。成熟Am5的预测分子量约为26 kDa，等电点为8.3。分析Am5序列发现，Am5与同家族中LPMO的序列一致性较低，具有良好的序列新颖性。酶学性质分析表明Am5是对几丁质有氧化活性而对纤维素无氧化活性的LPMO；与几丁质酶协同降解几丁质时可提高几丁质酶60%的水解效率；吸附实验显示，Am5对几丁质具有较强的吸附作用，而对纤维素吸附能力较弱。以上研究表明，Am5是一种针对几丁质底物具有高效选择性的新型氧化酶。

关键词： 溶解性多糖单加氧酶; 融合表达; 酶学性质; 几丁质

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.

Key words: Lytic polysaccharide monooxygenases(LPMO) Fusion expression Enzymatic property Chitin

收稿日期: 2014-05-16 出版日期: 2014-07-25

ZTFLH:

Q78

基金资助:

国家“863”计划资助项目（2013AA102801）

通讯作者: 冯雁 E-mail: yfeng2009@sjtu.edu.cn

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引用本文:

施贤卫, 张伟涛, 张小飞, 杨广宇, 冯雁. Actinosynne mamirum DSM43827溶解性多糖单加氧酶的异源表达和酶学性质表征[J]. 中国生物工程杂志, 2014, 34(7): 17-23.

SHI Xian-wei, ZHANG Wei-tao, ZHANG Xiao-fei, YANG Guang-yu, FENG Yan. The Heterologous Expression and Characterization of Lytic Polysaccharide Monooxygenase from Actinosynnema mirum DSM 43827. China Biotechnology, 2014, 34(7): 17-23.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20140703 或 https://manu60.magtech.com.cn/biotech/CN/Y2014/V34/I7/17

[1] Svein J H, Vaaje-Kolstad G, Westereng B,et, al.Novel enzymes for the degradation of cellulose. Biotechnology for Biofuels, 2012, 5:45.

[2] Hemsworth G R, Davies G J, Walton P H. Recalcitrant polysaccharide degradation by noveloxidative biocatalysts. Appl Microbiol Biotechnol, 2013, 97(19):8455-8465.

[3] Davies G, Henrissat B. Structures and mechanisms of glycosyl hydrolases. Structure, 1995, 3(9):853-859.

[4] Horn S J, Sikorski P, Cederkvist J B, et, al. Costs and benefits of processivity in enzymaticdegradation of recalcitrant polysaccharides. Proc Natl Acad Sci USA, 2006,103(48):18089-18094.

[5] Eijsink VGH, Vaaje-Kolstad G, Vrum K M, et, al. Towards new enzymes forbiofuels: lessons from chitinase research. Trends Biotechnol, 2008,26(5):228-235.

[6] Hemsworth G R, Davies G J, Walton P H.Recent insights into copper-containing lytic polysaccharidemono-oxygenases. Current Opinion in Structural Biology, 2013, 23(5):660-668.

[7] Quinlan R J, Sweeney M D, Lo Leggio L, et al. Insights into theoxidative degradation of cellulose by a copper metalloenzyme thatexploits biomass components. ProcNatlAcad Sci USA, 2011,108(37):15079-15084.

[8] Westereng B, Ishida T, Vaaje-Kolstad G,et al. The putativeendoglucanase PcGH61D from Phanerochaete chrysosporium is a metal-dependentoxidative enzyme that cleaves cellulose. PLoS One, 2011,6(11):e27807.

[9] Vaaje-Kolstad G, Westereng B, Horn S J, et al. An oxidative enzyme boosting the enzymatic conversion of recalcitrantpolysaccharides. Science, 2010, 330(6001):219-222.

[10] Vaaje-Kolstad G, Liv A B, Sigrid G, et al. Characterization of the Chitinolytic Machinery of Enterococcus faecalis V583 and High-Resolution Structure of Its Oxidative CBM33 Enzyme. Journal of Molecular Biology, 2011, 416(2): 239-254.

[11] Forsberg Z, Vaaje-Kolstad G, Westereng B, et al. Cleavage of cellulose by aCBM33 protein. Prot Science, 2011, 20(9):1479-1483.

[12] Miller GL. Use of dinitrosalicyclic acid regent for determination ofreducing sugar. Anal Chem, 1951, 31: 426-4281

[13] 苏畅,夏文水,姚惠源.氨基葡萄糖和乙酰氨基葡萄糖的测定方法.食品工业技术,2003.(6):74-75. Su C, Xia W S, Yao H Y. Determination methods of glucosamine and acetyl glucosamine. Science and technology of food industry, 2003.(6):74-75.

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