Zhang Qiang, Miao Renyun, Liu Tianhai, Huang Zhongqian, Peng Weihong, Gan Bingcheng, Zhang Xiaoping, Tan Hao
1College of Resources, Sichuan Agricultural University, Chengdu, China.
2National-local Joint Engineering Laboratory of Breeding and Cultivation of Edible and Medicinal Fungi, Soil and Fertilizer Institute, Sichuan Academy of Agricultural Sciences, Chengdu, China.
3 Biotech. 2019 May;9(5):171. doi: 10.1007/s13205-019-1688-6. Epub 2019 Apr 9.
The aim of this study is to determine the key laccase-encoding gene in the life cycle of SCYDJ1-A1, and to characterize the biochemical properties of the laccase. Two laccase-like multicopper oxidase (LMCO) genes were identified in the genome of SCYDJ1-A1 as putative laccase-encoding genes. The two genes, belonging to Auxiliary Activity family 1 subfamily 3, were named as MiLacA and MiLacB. Phylogenetic analysis of deduced amino acid sequences showed that MiLacA is closest to a LMCO of 22J1, while MiLacB had low similarity with known LMCOs. Real-time quantitative PCR results showed that MiLacA was expressed at much higher levels than MiLacB throughout the entire course of artificial cultivation. MiLacA was overexpressed in as a recombinant protein. Biochemical characterization of the purified enzyme showed that MiLacA simultaneously possessed laccase and polyphenol-oxidase activities. MiLacA could be strongly inhibited by Fe, which is unusual. The optimum pH was four and optimum temperature was 60 °C. The enzyme retained over 74% of the laccase activity after 16-h incubation at 60 °C, which means that its thermostability is at the forefront among the currently known laccases. Our findings may help to elucidate how the laccase of is involved in decaying lignin in plant litter, and could also provide a candidate thermostable laccase for potential industrial application.
本研究的目的是确定SCYDJ1 - A1生命周期中的关键漆酶编码基因，并对漆酶的生化特性进行表征。在SCYDJ1 - A1的基因组中鉴定出两个漆酶样多铜氧化酶（LMCO）基因作为推定的漆酶编码基因。这两个基因属于辅助活性家族1亚家族3，被命名为MiLacA和MiLacB。推导氨基酸序列的系统发育分析表明，MiLacA与22J1的一种LMCO最接近，而MiLacB与已知的LMCO相似度较低。实时定量PCR结果表明，在整个人工培养过程中，MiLacA的表达水平远高于MiLacB。MiLacA作为重组蛋白在大肠杆菌中过表达。纯化酶的生化特性表明，MiLacA同时具有漆酶和多酚氧化酶活性。MiLacA可被铁强烈抑制，这是不寻常的。最适pH为4，最适温度为60℃。该酶在60℃孵育16小时后仍保留超过74%的漆酶活性，这意味着其热稳定性在目前已知的漆酶中处于领先地位。我们的研究结果可能有助于阐明SCYDJ1 - A1的漆酶如何参与植物凋落物中木质素的降解，也可为潜在的工业应用提供一种候选的热稳定漆酶。
