Strategy for polymorphic control by enzymatic reaction and antisolvent crystallization: effect of aminoacylase on metastable β-glycine formation

Abstract

Although enantioselective resolution by aminoacylase has been established for a long time, the effect of aminoacylase on amino acid polymorphism is still unclear. To explore whether the presence of aminoacylase assists the formation of metastable β-glycine, three approaches were developed: 1) integration of enzymatic reaction with antisolvent crystallization, 2) antisolvent recrystallization with aminoacylase, and 3) antisolvent recrystallization without aminoacylase. The pH value of all the systems was adjusted to 4, 7 or 10 before crystallizing glycine by methanol or ethanol antisolvent addition. β-Glycine could only be formed by enzymatic reaction, while other recrystallization conditions gave mixtures of α- and β-glycine, or α-, β-, γ-glycine, whether aminoacylase was added or not. Aminoacylase which underwent enzymatic reaction was slightly unfolded as evidenced by the red-shift of emission maxima in the fluorescence spectra, suggesting that the change in conformation of aminoacylase could control the polymorphism of glycine in antisolvent crystallization. Moreover, the transformation of the yielded glycine crystals from β-glycine to α- and γ-glycine was inhibited in the water/methanol solution for 24 h at pH ranging from 4 to 10. β-Glycine has been advocated as a promising organic piezoelectric material in recent years. However, its low thermodynamic stability was one of the bottlenecks for production. This study provided not only a reproducible and robust process to produce β-glycine, but also a new application of biocatalysts in crystal growth and design. Aminoacylase was also successfully recycled by centrifugation after reaction, and its catalytic activity was retained.