Abstract

The thermostable esterase from the bacterium Ureibacillus thermosphaericus was expressed with Trx tag from plasmid pET32b-estUT1 under T7 promoter in E. coli BL21(DE3). The specific activity and relative thermal stability of the tagged enzyme increased from 45.2 to 65.8% (1 h at 70°С). The additional TrxA tag does not affect the pH optimum of enzyme activity and substrate specificity. At the same time, the absence of the TrxA tag resulted in a significant increase in the stability of estUT1 in during incubation with various chemicals, including ethanol and methanol. The maximum catalytic efficiency (k_cat/K_M) for esterase was observed in the absence of the TrxA tag and was 280.0 s⁻¹ mM⁻¹. Thereby fusion with TrxA tag promotes the enzyme secretion in the dissolved form, but reduces its thermal stability.

中文翻译：

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嗜热链球菌的热稳定酯酶estUT1：TrxA标签对酶特性的影响

摘要

在大肠杆菌BL21（DE3）中，在T7启动子下，利用来自质粒pET32b-estUT1的Trx标签，表达来自嗜热球菌的热稳定酯酶。标记酶的比活性和相对热稳定性从45.2增加到65.8％（在70°C下1 h）。额外的TrxA标签不会影响酶活性和底物特异性的最适pH。同时，TrxA标签的缺失导致在与各种化学物质（包括乙醇和甲醇）孵育期间，estUT1的稳定性显着提高。在没有TrxA标签的情况下观察到酯酶的最大催化效率（k _cat / K _M）为280.0 s ^-1mM ^-1。从而与TrxA标签融合促进了溶解形式的酶分泌，但是降低了其热稳定性。