Biocatalytic behavior of a new Aspergillus niger whole-cell biocatalyst with high operational stability during the synthesis of green biosolvent isopropyl esters

Abstract

Using microbial cell-surface displayed enzymes to produce chemicals is a promising green chemistry procedure, but few studies evaluated filamentous fungi cell-surface display systems. Here, a novel Aspergillus niger whole-cell biocatalyst with excellent operational stability was constructed using a heterologous anchor protein, and the details about its practical catalytic characteristics during the synthesis of green biosolvent isopropyl esters (IPEs) were investigated. Candida antarctica lipase B (CALB) was chosen as a model enzyme, fused to a Saccharomyces cerevisiae glycosylphosphatidylinositol protein, SED1, and displayed on the A. niger surface, the hydrolytic activity of the A. niger mycelium-surface displayed CALB reached 440.30U/g dry cell. In solvent-free esterifications of medium-long chain fatty acids and isopropanol, the A. niger/CALB-Flag-SED1 whole-cell biocatalyst showed great organic substrate tolerance, high thermostability, no significant lag time in a dry initial condition and no synthetic activity decrease after five batch reactions at 65°C. The highest yields of isopropyl laurate, isopropyl myristate, and isopropyl palmitate, 79.21, 81.62, and 81.41%, respectively, were obtained after a 6-h reaction. Acetone washing helped to remove the excessive water accumulating during esterification and benefitted the operational stability. The relative acid conversions of the three IPEs in batch 5 were 99.79, 99.10, and 96.83%, respectively, of those in batch 1. The A. niger/CALB-Flag-SED1 whole-cell biocatalyst is a promising alternative to commercial immobilized products, and SED1 can serve as an efficient anchor module to display industrial enzymes in an A. niger expression system.