Abstract

Microbial esterases are a highly desirable tool for numerous biosynthetic and biotechnological applications requiring ester bond cleavage. Once identified, microbial esterases are often produced recombinantly in Escherichia coli to enhance yield and ease of purification. In this study a polyhistidine-tagged SGNH esterase gene (AaSGNH1), originating from the cyanobacterium Aphanizomenon flos-aquae, was cloned into an over-expression plasmid and expressed in BL21(DE3) cells. The recombinant esterase enzyme was produced as inactive inclusion bodies which were insoluble in 8 M urea but readily solubilized by the detergent Empigen BB^®. Crucially, the procurement of active enzyme required controlled removal of detergent during column chromatography and dialysis steps. The refolded esterase was characterized with respect to its ability to catalyze the cleavage of p-nitrophenol esters of different chain lengths (C2, C8, C16). In addition, the temperature and pH optima were determined and it was found that the enzyme was most active at low temperatures (5–15 °C) and under alkaline conditions (pH 8–10). It was found that the kinetic properties of AaSGNH1 were remarkably similar to other SGNH esterases described thereby validating that the protein was effectively refolded. Overall, this study provides a simple strategy for isolating cold-active recombinant esterase enzyme when expressed as inclusion bodies.

摘要

微生物酯酶是许多需要酯键断裂的生物合成和生物技术应用的理想工具。一旦鉴定，微生物酯酶通常在大肠杆菌中重组生产，以提高产量和易于纯化。在这项研究中，将来自蓝藻Aphanizomenon flos-aquae的多组氨酸标记的 SGNH 酯酶基因 ( Aa SGNH1)克隆到过表达质粒中并在 BL21(DE3) 细胞中表达。重组酯酶以非活性包涵体形式产生，不溶于 8 M 尿素，但易被洗涤剂 Empigen BB^溶解. 至关重要的是，活性酶的采购需要在柱层析和透析步骤中控制去污剂的去除。重折叠的酯酶的特征在于其催化切割不同链长（C2、C8、C16）的对硝基苯酚酯的能力。此外，确定了温度和最适 pH 值，发现该酶在低温（5-15 °C）和碱性条件（pH 8-10）下最活跃。发现Aa SGNH1 的动力学特性与描述的其他 SGNH 酯酶非常相似，从而验证了该蛋白质被有效地重折叠。总的来说，这项研究提供了一种简单的策略，用于分离表达为包涵体的冷活性重组酯酶。