核盘菌角质酶的克隆、表达及活性分析

Cloning, expression and activity analysis of cutinase from Sclerotinia sclerotiorum

摘要:

角质酶可降解脂肪族或芳香族聚酯，对聚对苯二甲酸乙二醇酯也具有较好的降解作用，但目前市面上角质酶产品非常稀缺，因此寻求一种高效表达的角质酶用于工程酶的开发非常有必要。本研究从核盘菌中克隆得到8个角质酶基因，利用PCR结合RT-PCR技术筛选出主效基因SsCut-52，利用大肠杆菌Escherichia coli BL21将SsCut-52进行异源表达并通过镍柱亲和层析的方法分离纯化，研究重组酶的特性及其致病性。SsCut-52全长为768 bp，N端有17个信号肽，进化树分析显示其氨基酸序列和葡萄孢属(Botrytis) 角质酶同源性最高，和蜡盘菌属(Rutstroemia) 角质酶亲缘关系也较近。SsCut-52在核盘菌侵染植物的过程中高效表达，在菌丝形成菌核的过程中表达量也高于其他角质酶基因。异源表达检测结果显示，角质酶以包涵体的形式存在，复性的SsCut-52在pH 6.0时活性最高，比活力达到3.45 U/mg，在pH为4.0–10.0时活性均较高。最适温度为20–30 ℃，当温度高于50 ℃时该酶的残余活性低于60%。植物叶片侵染结果表明，SsCut-52对核盘菌侵染板蓝根叶片具有一定的促进作用。

Abstract:

Cutinase can degrade aliphatic and aromatic polyesters, as well as polyethylene terephthalate. Lack of commercially available cutinase calls for development of cost-effective production of efficient cutinase. In this study, eight cutinase genes were cloned from Sclerotinia sclerotiorum. The most active gene SsCut-52 was obtained by PCR combined with RT-PCR, expressed in Escherichia coli BL21 and purified by Ni-NTA affinity chromatography to study its characteristics and pathogenicity. Sscut-52 had a total length of 768 bp and 17 signal peptides at the N terminals. Phylogenetic analysis showed that its amino acid sequence had the highest homology with Botrytis keratinase cutinase and was closely related to Rutstroemia cutinase. Sscut-52 was highly expressed during the process of infecting plants by Sclerotinia sclerotiorum. Moreover, the expression level of Sscut-52 was higher than those of other cutinase genes in the process of sclerotia formation from mycelium. The heterologously expressed cutinase existed in the form of inclusion body. The renatured SsCut-52 was active at pH 4.0–10.0, and mostly active at pH 6.0, with a specific activity of 3.45 U/mg achieved. The optimum temperature of SsCut-52 was 20–30 ℃, and less than 60% of the activity could be retained at temperatures higher than 50 ℃. Plant leaf infection showed that SsCut-52 may promote the infection of Banlangen leaves by Sclerotinia sclerotiorum.