Discovery of Highly Active Recombinant PNGase H+ Variants Through the Rational Exploration of Unstudied Acidobacterial Genomes.

Rui-Rui Guo,Josef Voglmeir,Huan-Huan Yang,Ya-Min Du,Li Liu,Gerard Comamala,Kasper Dyrberg Rand,Anne Zeck,Ting Wang,Marius Gramlich

doi:10.3389/fbioe.2020.00741

Abstract

Peptide-N4-(N-acetyl-β-glucosaminyl) asparagine amidases (PNGases, N-glycanases, EC 3.5.1.52) are indispensable tools in releasing N-glycans from glycoproteins. So far, only a limited number of PNGase candidates are available for the structural analysis of glycoproteins and their glycan moieties. Herein, a panel of 13 novel PNGase H+ candidates (the suffix H+ refers to the acidic pH optimum of these acidobacterial PNGases) was tested in their recombinant form for their deglycosylation performance. One candidate (originating from the bacterial species Dyella japonica) showed superior properties both in solution-phase and immobilized on amino-, epoxy- and nitrilotriacetate resins when compared to currently acidic available PNGases. The high expression yield compared to a previously described PNGase H+, broad substrate specificity, and good storage stability of this novel N-glycanase makes it a valuable tool for the analysis of protein glycosylation.

Highlights

The study of protein N-glycosylation is an active area of biotechnological research, given that all antibodies, and many biotherapeutics as well as extracellular and cell surface proteins bear these post-translational modifications (Overington et al, 2006; Saldova et al, 2017)
The enzymatic activity of Peptide-N4-(N-acetyl-β-glucosaminyl) asparagine amidases (PNGases) H+ from Terriglobus roseus could be successfully used for conventional N-glycan analysis protocols (Wang et al, 2017; Du et al, 2018), its use in a wider range of analytical workflows was hampered by difficulties in optimizing expression and purification of the enzyme at larger scales
With the rapid progress and the availability of wholegenome sequences in recent years, >70 acidobacterial PNGase H+ homologs can be currently found using BLAST (Altschul et al, 1990) in March 2020. This pool of candidate genes, allows the evaluation of alternative PNGase candidate genes for their biotechnological potential. In this Brief Research Report, we studied the activity and stability of 12 unstudied acidobacterial PNGase H+ candidates and we compared them to the previously described PNGase from Terriglobus roseus, both in solution and immobilized on various solid supports

Summary

Introduction

The study of protein N-glycosylation is an active area of biotechnological research, given that all antibodies, and many biotherapeutics as well as extracellular and cell surface proteins bear these post-translational modifications (Overington et al, 2006; Saldova et al, 2017). The enzymatic activity of PNGase H+ from Terriglobus roseus could be successfully used for conventional N-glycan analysis protocols (Wang et al, 2017; Du et al, 2018), its use in a wider range of analytical workflows was hampered by difficulties in optimizing expression and purification of the enzyme at larger scales. With the rapid progress and the availability of wholegenome sequences in recent years, >70 acidobacterial PNGase H+ homologs can be currently found using BLAST (Altschul et al, 1990) in March 2020 This pool of candidate genes, allows the evaluation of alternative PNGase candidate genes for their biotechnological potential. In this Brief Research Report, we studied the activity and stability of 12 unstudied acidobacterial PNGase H+ candidates and we compared them to the previously described PNGase from Terriglobus roseus, both in solution and immobilized on various solid supports

Methods

Results

Conclusion