Unique -glycosylation of a Recombinant Exo-inulinase from and Its Effect on Enzymatic Activity and Thermostability

Overview

Journal J Biol Eng

Publisher Biomed Central

Specialty Biomedical Engineering

Date 2019 Nov 19

PMID 31737090

Citations 6

Authors

Junyan Ma

Qian Li

Haidong Tan

Hao Jiang

Kuikui Li

Lihua Zhang

Quan Shi

Heng Yin

Affiliations

Soon will be listed here.

Abstract

Background: Inulinase can hydrolyze polyfructan into high-fructose syrups and fructoligosaccharides, which are widely used in food, the medical industry and the biorefinery of . In the present study, a recombinant exo-inulinase (rKcINU1), derived from CBS4857, was proven as an -linked glycoprotein, and the removal of -linked glycan chains led to reduced activity.

Results: Five -glycosylation sites with variable high mannose-type oligosaccharides (ManGlcNAc) 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 -sandwich domain, which has been proven to be more conducive to the occurrence of glycosylation modification than the N-terminus domain. Single-site -glycosylation mutants with Asn substituted by Gln were obtained, and the Mut with all five -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 ) occurred, and other single mutations resulted in the decrease in the specific activity to various extents, among which N467Q demonstrated the lowest enzyme activity.

Conclusion: The increased enzyme activity in N362Q, combined with thermostability testing, 3D modeling, kinetics data and secondary structure analysis, implied that the -linked glycan chains at the Asn-362 position functioned negatively, mainly as a type of steric hindrance toward its adjacent glycans to bring rigidity. Meanwhile, the -glycosylation at the other four sites positively regulated enzyme activity caused by altered substrate affinity by means of fine-tuning the -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 -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.

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