Unique N-glycosylation of a recombinant exo-inulinase from Kluyveromyces cicerisporus and its effect on enzymatic activity and thermostability

Junyan Ma,Quan Shi,Qian Li,Hao Jiang,Lihua Zhang,Haidong Tan,Heng Yin,Kuikui Li

doi:10.1186/s13036-019-0215-y

Abstract

BackgroundInulinase can hydrolyze polyfructan into high-fructose syrups and fructoligosaccharides, which are widely used in food, the medical industry and the biorefinery of Jerusalem artichoke. In the present study, a recombinant exo-inulinase (rKcINU1), derived from Kluyveromyces cicerisporus CBS4857, was proven as an N-linked glycoprotein, and the removal of N-linked glycan chains led to reduced activity.ResultsFive N-glycosylation sites with variable high mannose-type oligosaccharides (Man3–9GlcNAc2) were confirmed in the rKcINU1. The structural modeling showed that all five glycosylation sites (Asn-362, Asn-370, Asn-399, Asn-467 and Asn-526) were located at the C-terminus β-sandwich domain, which has been proven to be more conducive to the occurrence of glycosylation modification than the N-terminus domain. Single-site N-glycosylation mutants with Asn substituted by Gln were obtained, and the Mut with all five N-glycosylation sites removed was constructed, which resulted in the loss of all enzyme activity. Interestingly, the N362Q led to an 18% increase in the specific activity against inulin, while a significant decrease in thermostability (2.91 °C decrease in Tm) occurred, and other single mutations resulted in the decrease in the specific activity to various extents, among which N467Q demonstrated the lowest enzyme activity.ConclusionThe increased enzyme activity in N362Q, combined with thermostability testing, 3D modeling, kinetics data and secondary structure analysis, implied that the N-linked glycan chains at the Asn-362 position functioned negatively, mainly as a type of steric hindrance toward its adjacent N-glycans to bring rigidity. Meanwhile, the N-glycosylation at the other four sites positively regulated enzyme activity caused by altered substrate affinity by means of fine-tuning the β-sandwich domain configuration. This may have facilitated the capture and transfer of substrates to the enzyme active cavity, in a manner quite similar to that of carbohydrate binding modules (CBMs), i.e. the chains endowed the β-sandwich domain with the functions of CBM. This study discovered a unique C-terminal sequence which is more favorable to glycosylation, thereby casting a novel view for glycoengineering of enzymes from fungi via redesigning the amino acid sequence at the C-terminal domain, so as to optimize the enzymatic properties.

Highlights

Inulinase can hydrolyze polyfructan into high-fructose syrups and fructoligosaccharides, which are widely used in food, the medical industry and the biorefinery of Jerusalem artichoke
Exo-inulinases belong to the family of glycoside hydrolase 32 (GH32), a family including other inulinases, invertases, β-fructofuranosidases and fructosyltransferases, which share a common structural feature: a N-terminal five-blade β-propeller catalytic domain with four antiparallel β-strands exhibiting ‘W’ topology for each blade, surrounding the catalytic active center, followed by a Cterminal β-sandwich domain constituting with two antiparallel six-stranded β-sheets [4]
The β-sandwich domain of a dimeric β-fructofuranosidase from Schwanniomyces occidentalis was found to be involved in substrate binding through the interaction between β-sandwich domain of the adjacent subunit within the dimer with the substrate [6]; and the C-terminal domain (BsCBM66) of the exo-acting levanase SacC from Bacillus subtilis proved to belong to CBM66 with the function of identifying and binding substrate, as well as facilitating the orienting of the catalytic domain to the substrate [8, 9], thereby enhancing enzymatic activity through increasing the concentration of the appended enzymes in the vicinity of the substrate

Summary

Introduction

Inulinase can hydrolyze polyfructan into high-fructose syrups and fructoligosaccharides, which are widely used in food, the medical industry and the biorefinery of Jerusalem artichoke. The β-sandwich domain of a dimeric β-fructofuranosidase from Schwanniomyces occidentalis was found to be involved in substrate binding through the interaction between β-sandwich domain of the adjacent subunit within the dimer with the substrate [6]; and the C-terminal domain (BsCBM66) of the exo-acting levanase SacC from Bacillus subtilis proved to belong to CBM66 with the function of identifying and binding substrate, as well as facilitating the orienting of the catalytic domain to the substrate [8, 9], thereby enhancing enzymatic activity through increasing the concentration of the appended enzymes in the vicinity of the substrate It was just the extensive interactions of BsCBM66 with the terminal fructose moiety (Fru-3) of levantriose that conferred SacC the substrate specificity [7]

Methods

Results

Conclusion