The gastric mucosa of Atlantic salmon (Salmo salar) is abundant in highly active chitinases.

Atlantic salmon (Salmo salar) possesses a genome containing 10 genes encoding chitinases, yet their functional roles remain poorly understood. In other fish species, chitinases have been primarily linked to digestion, but also to other functions, as chitinase-encoding genes are transcribed in a variety of non-digestive organs. In this study, we investigated the properties of two chitinases belonging to the family 18 glycoside hydrolase group, namely Chia.3 and Chia.4, both isolated from the stomach mucosa. Chia.3 and Chia.4, exhibiting 95% sequence identity, proved inseparable using conventional chromatographic methods, necessitating their purification as a chitinase pair. Biochemical analysis revealed sustained chitinolytic activity against ß-chitin for up to 24 h, spanning a pH range of 2 to 6. Moreover, subsequent in vitro investigations established that this chitinase pair efficiently degrades diverse chitin-containing substrates into chitobiose, highlighting the potential of Atlantic salmon to utilize novel chitin-containing feed sources. Analysis of the gastric matrix proteome demonstrates that the chitinases are secreted and rank among the most abundant proteins in the gastric matrix. This finding correlates well with the previously observed high transcription of the corresponding chitinase genes in Atlantic salmon stomach tissue. By shedding light on the secreted chitinases in the Atlantic salmon's stomach mucosa and elucidating their functional characteristics, this study enhances our understanding of chitinase biology in this species. Moreover, the observed capacity to effectively degrade chitin-containing materials implies the potential utilization of alternative feed sources rich in chitin, offering promising prospects for sustainable aquaculture practices.

A thermostable bacterial lytic polysaccharide monooxygenase with high operational stability in a wide temperature range.

BACKGROUND: Lytic polysaccharide monooxygenases (LPMOs) are oxidative, copper-dependent enzymes that function as powerful tools in the turnover of various biomasses, including lignocellulosic plant biomass. While LPMOs are considered to be of great importance for biorefineries, little is known about industrial relevant properties such as the ability to operate at high temperatures. Here, we describe a thermostable, cellulose-active LPMO from a high-temperature compost metagenome (called mgLPMO10). RESULTS: MgLPMO10 was found to have the highest apparent melting temperature (83 °C) reported for an LPMO to date, and is catalytically active up to temperatures of at least 80 °C. Generally, mgLPMO10 showed good activity and operational stability over a wide temperature range. The LPMO boosted cellulose saccharification by recombinantly produced GH48 and GH6 cellobiohydrolases derived from the same metagenome, albeit to a minor extent. Cellulose saccharification studies with a commercial cellulase cocktail (Celluclast®) showed that the performance of this thermostable bacterial LPMO is comparable with that of a frequently utilized fungal LPMO from Thermoascus aurantiacus (TaLPMO9A). CONCLUSIONS: The high activity and operational stability of mgLPMO10 are of both fundamental and applied interest. The ability of mgLPMO10 to perform oxidative cleavage of cellulose at 80 °C and the clear synergy with Celluclast® make this enzyme an interesting candidate in the development of thermostable enzyme cocktails for use in lignocellulosic biorefineries.

A trimodular bacterial enzyme combining hydrolytic activity with oxidative glycosidic bond cleavage efficiently degrades chitin.

Findings from recent studies have indicated that enzymes containing more than one catalytic domain may be particularly powerful in the degradation of recalcitrant polysaccharides such as chitin and cellulose. Some known multicatalytic enzymes contain several glycoside hydrolase domains and one or more carbohydrate-binding modules (CBMs). Here, using bioinformatics and biochemical analyses, we identified an enzyme, Jd1381 from the actinobacterium Jonesia denitrificans, that uniquely combines two different polysaccharide-degrading activities. We found that Jd1381 contains an N-terminal family AA10 lytic polysaccharide monooxygenase (LPMO), a family 5 chitin-binding domain (CBM5), and a family 18 chitinase (Chi18) domain. The full-length enzyme, which seems to be the only chitinase produced by J. denitrificans, degraded both α- and ß-chitin. Both the chitinase and the LPMO activities of Jd1381 were similar to those of other individual chitinases and LPMOs, and the overall efficiency of chitin degradation by full-length Jd1381 depended on its chitinase and LPMO activities. Of note, the chitin-degrading activity of Jd1381 was comparable with or exceeded the activities of combinations of well-known chitinases and an LPMO from Serratia marcescens Importantly, comparison of the chitinolytic efficiency of Jd1381 with the efficiencies of combinations of truncated variants-JdLPMO10 and JdCBM5-Chi18 or JdLPMO10-CBM5 and JdChi18-indicated that optimal Jd1381 activity requires close spatial proximity of the LPMO10 and the Chi18 domains. The demonstration of intramolecular synergy between LPMOs and hydrolytic enzymes reported here opens new avenues toward the development of efficient catalysts for biomass conversion.

Structure and function of a CE4 deacetylase isolated from a marine environment.

Chitin, a polymer of ß(1-4)-linked N-acetylglucosamine found in e.g. arthropods, is a valuable resource that may be used to produce chitosan and chitooligosaccharides, two compounds with considerable industrial and biomedical potential. Deacetylating enzymes may be used to tailor the properties of chitin and its derived products. Here, we describe a novel CE4 enzyme originating from a marine Arthrobacter species (ArCE4A). Crystal structures of this novel deacetylase were determined, with and without bound chitobiose [(GlcNAc)2], and refined to 2.1 Å and 1.6 Å, respectively. In-depth biochemical characterization showed that ArCE4A has broad substrate specificity, with higher activity against longer oligosaccharides. Mass spectrometry-based sequencing of reaction products generated from a fully acetylated pentamer showed that internal sugars are more prone to deacetylation than the ends. These enzyme properties are discussed in the light of the structure of the enzyme-ligand complex, which adds valuable information to our still rather limited knowledge on enzyme-substrate interactions in the CE4 family.

Proteomic investigation of the secretome of Cellvibrio japonicus during growth on chitin.

Studies of the secretomes of microbes grown on insoluble substrates are important for the discovery of novel proteins involved in biomass conversion. However, data in literature and this study indicate that secretome samples tend to be contaminated with cytoplasmic proteins. We have examined the secretome of the Gram-negative soil bacterium Cellvibrio japonicus using a simple plate-based culturing technique that yields samples with high fractions (60-75%) of proteins that are predicted to be secreted. By combining this approach with label-free quantification using the MaxLFQ algorithm, we have mapped and quantified proteins secreted by C. japonicus during growth on α- and ß-chitin. Hierarchical clustering of the detected protein quantities revealed groups of up-regulated proteins that include all five putative C. japonicus chitinases as well as a chitin-specific lytic polysaccharide monooxygenase (CjLPMO10A). A small set of secreted proteins were co-regulated with known chitin-specific enzymes, including several with unknown catalytic functions. These proteins provide interesting targets for further studies aimed at unraveling the enzymatic machineries used by C. japonicus for recalcitrant polysaccharide degradation. Studies of chitin degradation indicated that C. japonicus indeed produces an efficient chitinolytic enzyme cocktail. All MS data have been deposited in the ProteomeXchange with the dataset identifier PXD002843 (http://proteomecentral.proteomexchange.org/dataset/PXD002843).