Conversion of α-Chitin Substrates with Varying Particle Size and Crystallinity Reveals Substrate Preferences of the Chitinases and Lytic Polysaccharide Monooxygenase of Serratia marcescens

Abstract

1Industrial depolymerization of chitinous biomass generally requires numerous steps and the 2 use of deleterious substances. Enzymatic methods provide an alternative, but fundamental 3 knowledge that could direct potential development of industrial enzyme cocktails is scarce. 4We have studied the contribution of mono-component chitinases (ChiA, -B, and -C) and the 5 lytic polysaccharide monooxygenase (LPMO) from Serratia marcescens on depolymerization 6 of -chitin substrates with varying particle size and crystallinity that were generated using a 7 converge mill. For all chitinases activity was positively correlated to a decline in particle size 8 and crystallinity. Especially ChiC, the only non-processive endo-chitinase from the S. 9 marcescens chitinolytic machinery, benefited from mechanical pretreatment. Combining the 10 chitinases revealed clear synergies for all substrates tested. CBP21, the chitin-active LPMO 11 from S. marcescens, increased solubilization of substrates with high degrees of crystallinity 12 when combined with each of the three chitinases, but this synergy was reduced upon decline 13 in crystallinity. 14 15 16 KEYWORDS 17 -chitin, mechanical pretreatment, chitinase, GH18, lytic polysaccharide monooxygenases, 18

Listeria monocytogenes has a functional chitinolytic system and an active lytic polysaccharide monooxygenase

“…B–F). The relative rate of Lm LPMO10 was similar to that of the well‐characterized LPMO, CBP21 from S. marcescens (Fig. B–F).…”

“…These product profiles are essentially identical to those observed for CBP21 on the same substrates , and also show a lack of high degree of polymerization (DP) products for α‐chitin compared to β‐chitin. This has been suggested to be caused by the tighter packing of chitin chains in the α‐allomorph of these substrates, which prevents release of long oligosaccharides as a result of adhesion to the insoluble substrate .…”

“…Lytic polysaccharide monooxygenases (LPMOs) represent a new enzyme family that is important for the efficient degradation of recalcitrant polysaccharides like chitin by a variety of bacterial species . The enzymes are copper‐dependent and cleave polysaccharide chains embedded in the crystalline regions of the substrate that are generally inaccessible to glycoside hydrolases .…”

Structure and function of a broad-specificity chitin deacetylase from Aspergillus nidulans FGSC A4

“…As an alternative source of β-chitin we used squid pen chitin purchased from Yaegaki (Hayashida, Japan) exposed to cutter milling for 60 sec for particle size reduction (See ref. 41 for methods). Soluble chitosans with a degree of N -acetylation of 61% and 64% (F A 0.61 and 0.64), with a random distribution of N -acetylated and de- N -acetylated units 31 , were a gift from Professor Kjell M. Vårum from the Department of Biotechnology, Norwegian University of Science and Technology.…”

Activation of enzymatic chitin degradation by a lytic polysaccharide monooxygenase

“…Under our conditions, there may be enough points for attack by the endo-acting ChiC, and the help from CBP21 is unnecessary initially. Interestingly, Nakagawa and co-workers observed that CBP21 hardly influenced the activity of ChiA on α-chitin 18. This finding was rationalized by possible different face preferences on the chitinous substrate for the two different enzymes.Previously, it has been shown that none of the chitinases of S. marcescens are capable of completely degrading the substrates used in our study by themselves 12,19.…”