Abstract

This report examines the soluble expression of a novel feruloyl esterase, BpFae, from Burkholderia pyrrocinia in Escherichia coli overexpression systems using three expression vectors, pET28a, pCold-TF and pGEX-4T-1. BpFae was overexpressed as insoluble inclusion bodies when pET28a was used as the expression vector. BpFae was overexpressed in the soluble form when using pCold-TF; however, the overexpressed protein showed negligible activity. Recombinant BpFae was produced in the soluble form and active when E. coli cells were transformed with the pGEX-4T-1-BpFae vector. Optimal conditions for soluble overexpression of recombinant BpFae were determined, and the highest activity of recombinant BpFae was 2.54 U mL⁻¹. Multiple sequence alignment, phylogenetic analysis and construction of a three-dimensional model indicated that BpFae is a unique FAE from B. pyrrocinia with underlying research value.

摘要

本报告使用三种表达载体pET28a，pCold-TF和pGEX-4T-1在大肠杆菌过表达系统中研究了伯克霍尔德菌的新阿魏酸酯酶BpFae的可溶性表达。当将pET28a用作表达载体时，BpFae过表达为不溶性包涵体。使用pCold-TF时，BpFae以可溶形式过表达；然而，过表达的蛋白质显示微不足道的活性。当用pGEX-4T-1-BpFae载体转化大肠杆菌细胞时，重组BpFae以可溶形式产生并具有活性。确定了重组BpFae可溶性过量表达的最佳条件，重组BpFae的最高活性为2.54 U mL ^-1。多序列比对，系统发育分析和三维模型的结构表明，BpFae是从一个独特的FAE B. pyrrocinia与研究价值的下面。