Rational immobilization of lipase by combining the structure analysis and unnatural amino acid insertion

Abstract

Improving the conventional covalent immobilization of enzyme and avoiding random covalent linkage to protect enzyme’s active sites from unwanted covalent linkage at the mean time are the fundamental topics for enzyme immobilization. In this study, unnatural amino acid was introduced into a recombinant lipase and applied for the rational and smart covalent enzyme immobilization. In the first step, Tyr50, 137, 243, 274, and 355 of lipase were replaced with AzPhe unnatural amino acid based on the analysis of enzyme structure. Then, these novel recombinant lipases were coupled to support using strain-promoted azide–alkyne cycloaddition (SPAAC), respectively. Subsequently, both the effect of the immobilization site and the thermo-stability of immobilized lipases were also examined. The relative activities of the immobilized AzPhe-Lip243 and AzPhe-Lip274 were enhanced to 121.33% and 137.06%, respectively, presenting 6.0 and 6.8 fold higher than those of the lipase traditionally immobilized using glutaraldehyde (IM-Lip-GA). In addition, all the immobilized lipases presented better specific activity except for AzPhe-Lip355, whose immobilization site was close to its active site. The rational immobilized lipases also presented better thermo-stability than those by traditionally immobilization method (glutaraldehyde). To sum up, with the aid of protein structure analysis, unnatural amino acid can be rationally inserted into enzyme sequence to inform and direct the covalent enzyme immobilization. This method can be further developed for one-step enzyme purification and immobilization and applied to a broad scope of enzymes.