Simultaneous analysis of C1 and C4 oxidized oligosaccharides, the products of lytic polysaccharide monooxygenases acting on cellulose

Abstract

Lytic polysaccharide monooxygenases play a pivotal role in enzymatic deconstruction of plant cell wall material due to their ability to catalyze oxidative cleavage of glycosidic bonds. LPMOs may release different products, often in small amounts, with various oxidation patterns (C1 or C4) and with varying stabilities, making accurate analysis of product profiles a major challenge. So far, HPAEC has been the method of choice but it has limitations with respect to analysis of C4-oxidized products. Here, we compare various HPLC methods and present procedures that allow efficient separation of intact C1- and C4-oxidized products. We demonstrate that both PGC and HILIC (in WAX-mode) can separate C1- and C4-oxidized products and that PGC gives superior chromatographic performance. In contrast to HPAEC, these methods are directly compatible with mass spectroscopy and charged aerosol detection (CAD), which enables online peak validation and quantification with LOD levels in the low ng range. While the novel methods show lower resolution than HPAEC, this is compensated by easy peak identification, allowing, for example, discrimination between chromatographically highly similar native and C4-oxidized cello-oligomers. HPAEC-MS studies revealed chemical oxidation of C4-geminal diol products, which implies that peaks commonly believed to be C4-oxidized cello-oligomers, in fact are on-column generated derivatives. Non-destructive separation of C4-oxidized cello-oligosaccharides on the PGC column allowed us, for the first time, to isolate C4-oxidized standards. HPAEC fractionation of a purified C4-oxidized tetramer revealed that on-column decomposition leads to formation of the native trimer, which may explain why product mixtures generated by C4-oxidizing LPMOs seem to be rich in native oligosaccharides when analyzed by HPAEC. The findings and methods described here will aid in future studies in the emerging LPMO field.

A Lytic Polysaccharide Monooxygenase with Broad Xyloglucan Specificity from the Brown-Rot Fungus Gloeophyllum trabeum and Its Action on Cellulose-Xyloglucan Complexes

“…5B. It is noteworthy that the native species observed with HPAEC may also originate from C-4-oxidized products, as it has recently been shown that C-4-oxidized products are unstable and tend to lose the oxidized monosugar under the chromatographic conditions used here (34). …”

“…Peaks were assigned based on previous assignments by Isaksen et al (50); native cello-oligosaccharides are labeled as Glc n , where n is the degree of polymerization (DP). Note that it has recently been shown that C-4-oxidized products are unstable under these chromatographic conditions and that the peaks labeled C-4 are, in fact, diagnostic degradation products (34). The fractions of native products are high because C-4-oxidized products tend to lose the oxidized monosugar under these chromatographic conditions.…”

“…Such products can be detected using various types of chromatography (33, 34) or mass spectrometry (5). The activity of strictly C-4-oxidizing LPMOs may also be quantified directly using a standard method for detection of the newly generated reducing ends (13).…”

Recombinant expression of thermostable processive MtEG5 endoglucanase and its synergism with MtLPMO from Myceliophthora thermophila during the hydrolysis of lignocellulosic substrates

“…Higher amounts of G2 were detected in the 1st h of the reaction, while after 24-h incubation, the main non-oxidized product was glucose. Regarding the release of oxidative products, peaks were assigned based on previous assignments [57, 58] and comparison with the activity patterns of Nc LPMO9C from Neurospora crassa [59] and Pc LPMO9D from Phanerochaete chrysosporium [60] (Fig. 7).…”

Recent liquid chromatographic approaches and developments for the separation and purification of carbohydrates

“…A more comprehensive discussion on PGC supports is available 30 , as is a more exhaustive discussion on comparisons between PGC supports versus reversed-phase and HILIC ones for glycan analysis 31 . Since PGC columns remain attractive for polar analytes, these packings have seen use in the separation of glycans isolated from mammalian sources 32,33 and have applications in the separation of other carbohydrates such as underivatized oligosaccharide anomers in vegetable matrices 34 , cello-oligosaccharides 35,36 and fructan mixtures 37 . While these PGC packings remain somewhat unexplored for carbohydrate purification, a few drawbacks associated with these supports have come to light, including the loss of retention/variability in retention times 31,38 and poor stability of the packing material under acidic mobile phase conditions 39 .…”