共培养是提高漆酶发酵水平的一种有效手段,实验室前期研究发现,较之单培养,在阿魏蘑与胶红酵母的共培养过程中,阿魏蘑漆酶基因lacc2和lacc6的转录水平发生了明显变化。通过克隆得到阿魏蘑漆酶基因lacc2和lacc6,异源表达后对2个重组漆酶LACC2和LACC6进行分离纯化和酶学性质分析。以2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐[2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonate), ABTS]为底物时,LACC2和LACC6的最适反应温度和pH相同,分别为50 ℃和3.0;低浓度的K+、Cu2+、Co2+和Mn2+对2个重组酶有不同程度的促进,但对LACC2的促进作用更为明显;与LACC6相比,LACC2对有机试剂和表面活性剂的耐受力更强,但更容易受到抑制剂的影响;动力学研究发现,LACC2和LACC6的最适底物均为ABTS,Km值分别为0.13和0.24 mmol/L。研究结果为共培养过程中胶红酵母促进阿魏蘑产漆酶的作用机理分析提供了同工酶酶学性质方面的重要基础数据,有利于促进机理的全面解析。
Co-culture is an effective way to improve laccases fermentation level. The previous study showed that the transcription levels of lacc2 and lacc6 changed significantly during co-culture. In this study, lacc2 and lacc6 were successfully cloned and expressed in Pichia pastoris GS115. The crude enzyme solution of LACC2 and LACC6 were purified to investigate the enzymatic properties. When ABTS was used as the substrate, the optimal temperatures and pH of LACC2 and LACC6 were 50°C and 3.0, respectively. Moreover, low concentrations of K+, Cu2+, Co2+ and Mn2+ enhanced the activities of LACC2 more than LACC6. In addition, LACC2 showed better tolerance to organic reagents and surfactants than LACC6, while LACC2 was more vulnerable to the inhibitors than LACC6. The enzyme kinetics results showed that ABTS was the optimal substrate of LACC2 and LACC6, and the Km of LACC2 and LACC6 were 0.13 mmol/L and 0.24 mmol/L, respectively. This study provided important basic data on the properties of isoenzymes for the mechanism analysis of R. mucilaginosa enhanced P. eryngii var. ferulae laccases production during co-culture, which was beneficial to the comprehensive analysis of the promotion mechanism.
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