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

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.

Engineering better catalytic activity and acidic adaptation into Kluyveromyces marxianus exoinulinase using site‐directed mutagenesis

“…The recombinant inulinase (INU1) from K. marxianus NCYC2887 overexpressed by Saccharomyces cerevisiae showed an apparent MW of 98 kDa, bigger than its theoretical 59 kDa 35 . The increase of apparent MW may probably be attributed to the post‐translational N‐glycosylation, as demonstrated in the exo‐inulinase from K. cicerisporus expressed in Pichia pastoris X‐33 with theoretical MW of 60.54 kDa and apparent MW of 79.5 kDa 36 …”

Removal of N-terminal tail changes the thermostability of the low-temperature-active exo-inulinase InuAGN25

“…To enhance thermostability, K. marxianus exo-inulinase was fused to the inulin-binding module from the N-terminal region of Bacillus macerans cycloinulinooligosaccharide fructanotransferase [ 8 ]. Ma et al reported that the thermostability of Kluyveromyces cicerisporus exo-inulinase was changed when five N-glycosylation sites (Asn362, Asn370, Asn399, Asn467, and Asn526) at the C-terminus were substituted with glutamines [ 21 ]. This study firstly showed that thermostability of exo-inulinases were affected by N-terminal tail.…”

The Important Roles Played in Substrate Binding of Aromatic Amino Acids in Exo-Inulinase From Kluyveromyces cicerisporus CBS 4857

“…The three-dimensional model of KcINU1 was obtained as described previously ( Ma et al, 2019 ). The model was automatically built through the Swiss-Model server 3 using the crystal structure of exo-inulinase INU1 (PDB code 6J0T) as the template which sharing 98.2% sequence identity with the KcINU1.…”

“…Site-directed mutations were performed using RF cloning method the same as previously described ( Ma et al, 2019 ). The pPICZαA-rKcINU1 recombinant plasmid was used as the template, and primers ( Supplementary Table 1 ) used in RF-cloning were designed by online service tool 4 .…”

“…Previously, we have identified a novel exo-inulinase, named KcINU1, from Kluyveromyces cicerisporus and demonstrated the residues D53 and E238 (numbered D30 and E115, respectively, in previous paper which did not include the signal peptide sequence) involved in the catalytic activity of the enzyme ( Ma et al, 2016 ). Further experiments showed that N-glycosylation significantly affected the enzymatic activity and thermostability of the enzyme ( Ma et al, 2019 ). However, the enzyme-substrate interaction and catalytic mechanism is still unclear.…”