Proteomics-based screening and functional characterizationof novel enzymes involved in the degradation of woodcomponents
Motoyuki SHIMIZU
Faculty of Agriculture, Meijo University
Research aims
Filamentous fungi, including Aspergillus nidulans and A. oryzae, are popular model
eukaryotes used in numerous genetic and biological studies. Interestingly, these fungi
produce various cellulolytic and hemicellulolytic enzymes, which have recently become
important natural resources for industrial biorefinery processes and the production of
second-generation biofuels from plant biomass. Since hemicelluloses have complex
structures, a variety of hemicellulolytic enzymes with different substrate specificities isnecessary to achieve complete degradation. Based on sequence similarity, thecarbohydrate-active enzymes (CAZy) database (http://www.cazy.org/) classifies these
enzymes into four families: the glycoside hydrolase (GH), polysaccharide lyase (PL),carbohydrate esterase (CE), and auxiliary activity (AA) families.
Here, we analyzed the secretome of A. nidulans in response to various
polysaccharides. Furthermore, we investigated the enzymatic function of a secretedhypothetical protein 1 (HP1). HP1 shared no significant homology with proteins ofknown function but displayed a rhamnogalacturonan (RG) lyase activity. Therefore, wewould like to propose that HP1 is a novel RG lyase, which defines a new family of PL.
Methods
The A. nidulans wild-type strain A26 was grown for 48 h at 37°C in minimum media
containing as the sole carbon source 1% microcrystalline cellulose (MCC),
carboxymethyl cellulose (CMC), chitin, xylan, glucomannan (GlcM), or
β-galactomannan (GalM). The secreted proteins from culture filtrates were thendigested with trypsin, and the resulting peptides were analyzed using liquid
chromatography-tandem mass spectrometry (LC-MS/MS). MS/MS spectra wereanalyzed using the MASCOT search engine. The recombinant HP1 was isolated fromthe Pichia pastoris yeast strain KM71H, purified, and characterized.

Results
Using LC-MS/MS, we analyzed the secretome of A. nidulans grown on various single
carbon sources (Fig. 1). We were able to identify 446 unique proteins (Fig. 1), including
members of the GH, PL, CE, and AA families of carbohydrate-active enzymes,proteases, lipases, and proteins sharing homology with diverse proteins with known
functions (Other). We also identified 82 hypothetical proteins (HPs), which have noputative conserved domains and share no sequence similarity with other known proteins(Fig. 1). Amongst these, we used E. coli or P. pastoris expression systems to express
and purify HP1 to HP30.

Fig. 1. Analysis of the secretome of A. nidulans in response tovarious polysaccharides.
In this study, we present our analyses of the function of HP1. On SDS-PAGE, thepurified HP1 migrated as a single band with a molecular mass of 38 kDa (Fig. 2A). Invitro, HP1 showed significant catalytic activity towards rhamnogalacturonan I (RGI)
degradation (Fig. 2B, and 2C), but had no detectable activity on MCC, CMC, chitin,xylan, GlcM, or GalM (data not shown). We next tested the catalytic activity of HP1 on
α-1,5-arabinan, arabinogalactan, β-1,4-galactan, polygalacturonic acid, RGI, and linear
RG (At-RG). Our thin layer chromatography (TLC) analysis of the reaction products
showed that HP1 was active on β-1,4-galactan (which contained 13% RG), RGI, andAt-RG (Fig. 2C). Considering that polysaccharides can be degraded by hydrolases,lyases and lytic polysaccharide monooxygenases, MS analysis of the reaction products
can be used to determine the type of reaction catalyzed by HP1. Therefore, we analyzed
the soluble products resulting from the degradation of At-RG by HP1 using
MALDI-TOF MS (data not shown). The molecular weights of the three products
detected corresponded to those of the Na+ adducts of unsaturated galacturonosylrhamnose (∆GR), ∆GRGR, and ∆GRGRGR (data not shown), which suggests that HP1
has lyase activity. Moreover, we were able to confirm the lyase activity of HP1 on

At-RG by measuring the change in absorbance at 235 nm resulting from the formationof unsaturated oligosaccharides (data not shown). Together, these results show that HP1
is an RG lyase and belongs to a new family of PL in the CAZy classification(http://www.cazy.org/).

Fig. 2. Activity of purified HP1 towards various glycans. A) SDS-PAGEof HP1; B) Diagram showing the structure of RGI; C) TLC
analysis of the soluble products resulting from the catalytic
activity of HP1 on various glycans composing RGI.

Conclusion
We recently discovered a novel β-1,4-mannanase called Man134A, which is secreted
by A. nidulans and shared no sequence homology with proteins of known function(including β-mannanases belonging to the GH5, GH26, and GH113 families)[1-3]. In thisstudy, we report the identification and characterization of a novel RG lyase, which
belongs to a new family of PL based on the CAZy classification. Together, thesefindings show that filamentous fungi remain an essential resource for the discovery andcharacterization of enzymes involved in the degradation of polysaccharides.

References
1) Shimizu, M. et al. (2015) Novel β-1,4-mannanase belonging to a new glycosidehydrolase family in Aspergillus nidulans. J. Biol. Chem. 290: 27914-27927.
2) Sakai, K. et al. (2017) Biochemical characterization of thermostable
β-1,4-mannanase belonging to the glycoside hydrolase family 134 from Aspergillus
oryzae. Appl. Microbiol. Biotechnol. 101: 3237-3245.
3) Sakai, K. et al. (2018) Characterization of pH-tolerant and thermostable GH 134
β-1,4-mannanase SsGH134 possessing carbohydrate binding module 10. J. Biosci.
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