梯棱羊肚菌AA1_2家族多铜氧化酶基因的异源表达与生化鉴定

典型的漆酶通常属于辅助活性酶第一家族第一亚族（auxiliary activity family 1 subfamily 1,简称AA1_1家族）,而AA1_2家族的多铜氧化酶通常拥有将二价铁氧化成三价铁的活性,部分AA1_2家族酶蛋白兼具漆酶活性。梯棱羊肚菌全基因组只有一个AA1_2家族酶基因,该基因编码的酶蛋白是否拥有漆酶功能尚未清楚。本研究主要从酶生化特性的角度,结合酶基因的表达规律,对该基因的功能进行初探。对该AA1_2家族基因在梯棱羊肚菌生长发育不同阶段的表达水平进行实时定量PCR检测;将该基因编码序列克隆到表达载体中在大肠杆菌中异源表达,层析获得纯化的酶蛋白,对酶蛋白的生化特性进行了鉴定。发现该AA1_2多铜氧化酶基因在外源营养袋和土壤中的营养菌丝里低表达,在菇原基和子实体中表达较活跃。异源表达获得纯化的酶蛋白分子量约64kDa,表现出亚铁氧化酶（EC 1.16.3.1）与漆酶（EC 1.10.3.2）双重活性。其亚铁氧化酶活性在pH 4最高,漆酶活性在pH 6最高。亚铁氧化酶活性与漆酶活性的最适温度均为30℃左右,在30℃温育16h后仍保留70%以上活性。亚铁氧化酶和漆酶活性受Mn ²⁺、Hg ²⁺和Pb ²⁺抑制。对蛋白质变性剂SDS、尿素的耐受性较强。本研究通过酶学证据证实了梯棱羊肚菌AA1_2家族多铜氧化酶基因编码的酶蛋白具有亚铁氧化酶-漆酶双重活性,系在子囊菌大型真菌中首次发现,为进一步研究铁元素代谢与漆酶活性在羊肚菌子实体形成与发育过程中的作用提供了启示。

Heterogenous overexpression and biochemical characterization of the AA1_2 family multicopper oxidase of Morchella importuna

Typical laccases usually belong to the subfamily 1 of auxiliary activity family 1 (the AA1_1 family), while the multicopper oxidases in AA1_2 family usually possess an activity to oxidize Fe ²⁺ to Fe ³⁺, and some of the known AA1_2 enzymes simultaneously possess a laccase activity. The whole genome of Morchella importuna only possess a single gene of AA1_2 family. Whether the enzymatic protein encoded by this AA1_2 gene possesses a laccase function remains undetermined. This study is aiming at understanding the functions of the gene from its expression profiling in combination with biochemical characterization of the encoding enzyme. Gene expression levels at different stages during the growth and development of M. importuna were determined by quantitative real-time PCR. The protein-coding sequence of the gene was cloned into a pET vector and overexpressed in Escherichia coli cells. Purified enzymatic protein was obtained by chromatography and then determined for its biochemical characteristics. The results showed that the AA1_2 family multicopper oxidase gene had low expression levels in the vegetative mycelia presented in exogenous nutrient bags and the soils, and high expression levels in primordia and fruiting bodies. The purified enzymatic protein had a molecular weight near 64kDa, showing bifunctional activities of ferroxidase (EC 1.16.3.1) and laccase (EC 1.10.3.2). The ferroxidase activity showed the maximum level at pH 4, while the laccase activity showed the maximum level at pH 6. Both enzymatic activities were optimum at 30°C, and retained over 70% of the activities after incubation for 16h at 30°C. Both the ferroxidase and laccase activities could be inhibited by Mn ²⁺, Pb ²⁺ and Hg ²⁺. The enzymatic protein showed a high tolerance to the protein denaturants SDS and urea. The findings confirmed that the enzymatic protein encoded by the AA1_2 family multicopper oxidase gene possessed bifunctional ferroxidase-laccase activities with enzymological evidences, which is the first report in macrofungi of Ascomycota, and might provide insights into further studies on the involvement of iron metabolism as well as laccase activity in the formation and development of morel fruiting body.