Glycosylation influences activity, stability and immobilization of the feruloyl esterase 1a from Myceliophthora thermophila

Cyrielle Bonzom,Ekaterina Mirgorodskaya,Lisbeth Olsson,Stefan Uthoff,Sun-Li Chong,Silvia Hüttner,Alexander Steinbüchel,Raymond M D Verhaert

doi:10.1186/s13568-019-0852-z

Abstract

Heterologous protein production is widely used in industrial biotechnology. However, using non-native production hosts can lead to enzymes with altered post-translational modifications, such as glycosylation. We have investigated how production in a non-native host affects the physicochemical properties and enzymatic activity of a feruloyl esterase from Myceliophthora thermophila, MtFae1a. The enzyme was produced in two microorganisms that introduce glycosylation (M. thermophila and Pichia pastoris) and in Escherichia coli (non-glycosylated). Mass spectrometric analysis confirmed the presence of glycosylation and revealed differences in the lengths of glycan chains between the enzymes produced in M. thermophila and P. pastoris. The melting temperature and the optimal temperature for activity of the non-glycosylated enzyme were considerably lower than those of the glycosylated enzymes. The three MtFae1a versions also exhibited differences in specific activity and specificity. The catalytic efficiency of the glycosylated enzymes were more than 10 times higher than that of the non-glycosylated one. In biotechnology, immobilization is often used to allow reusing enzyme and was investigated on mesoporous silica particles. We found the binding kinetics and immobilization yield differed between the enzyme versions. The largest differences were observed when comparing enzymes with and without glycosylation, but significant variations were also observed between the two differently glycosylated enzymes. We conclude that the biotechnological value of an enzyme can be optimized for a specific application by carefully selecting the production host.

Highlights

Increasing interest is being shown by industry in utilizing enzymes for various applications, as they catalyze reactions with high specificity, and are considered environmentally friendly
Different production hosts yield enzyme versions with different molecular weights The gene coding for the feruloyl esterase 1a from Myceliophthora thermophila, MtFae1a, was introduced into three different microorganisms: (i) its native host, M. thermophila, (ii) the methylotrophic yeast Pichia pastoris, and (iii) the bacterium Escherichia coli
The enzyme was successfully produced in all three host organisms, yielding the three enzyme versions: M-Fae, P-Fae and E-Fae

Summary

Introduction

Increasing interest is being shown by industry in utilizing enzymes for various applications, as they catalyze reactions with high specificity, and are considered environmentally friendly. To prepare sufficient quantities of enzymes for biotechnological applications, heterologous protein production is often used. Glycosylation may affect protein folding (Rudd et al 1994; Hoffmann and Flörke 1998; Mitra et al 2006; Benoit et al 2006; Hanson et al 2009), stability (Chu et al 1978), aggregation (Schülke and Schmid 1988; Bosques and Imperiali 2003), substrate binding (Goettig 2016), the structural dynamics (Lee et al 2015), and the catalytic activity (Skropeta 2009)

Methods

Results

Conclusion