Rational immobilization of l-threonine aldolase from Bacillus nealsonii for efficiently synthesis of l-syn-3-[4-(methylsulfonyl)] phenylserine

Abstract

l-syn-3-[4-(methylsulfonyl)] phenylserine is a key industrial intermediate for the synthesis of florfenicol, thiamphenicol, and other products. l-Threonine aldolase can synthesize l-syn-3-[4-(methylsulfonyl)] phenylserine from glycine and 4-methylsulphonyl benzaldehyde directly. However, the selectivity of free l-threonine aldolase was difficult to meet the requirement of industrialization. Here, we selected the suitable site to insert His tag as immobilized site to improve the selectivity of l-threonine aldolase based on the molecular dynamics simulation and prior protein engineering. The phenanthroline was coupled to the surface of magnetic nanoparticles coated with silicon and thus enabled the selective and oriented immobilization of His-tagged l-threonine aldolase. Due to magnetic nanoparticles coated with silicon as supports, it was easy to separate the residual reactant and impurities from the immobilized enzyme by magnetic attraction. Moreover, the immobilization was combined with the purification of free enzymes through directional immobilization, which avoided heterozyme linking supports and improved the utilization efficiency of supports. Our study demonstrates an effective approach: how to analyze and select enzyme-targeted ligation sites in conjunction with previous modifications of engineered bacteria, and the immobilized enzyme obtained also provided a good candidate for the industrial production of l-syn-3-[4-(methylsulfonyl)] phenylserine.