Revisiting the AA14 family of lytic polysaccharide monooxygenases and their catalytic activity.

Lytic polysaccharide monooxygenases (LPMOs) belonging to the AA14 family are believed to contribute to the enzymatic degradation of lignocellulosic biomass by specifically acting on xylan in recalcitrant cellulose-xylan complexes. Functional characterization of an AA14 LPMO from Trichoderma reesei, TrAA14A, and a re-evaluation of the properties of the previously described AA14 from Pycnoporus coccineus, PcoAA14A, showed that these proteins have oxidase and peroxidase activities that are common for LPMOs. However, we were not able to detect activity on cellulose-associated xylan or any other tested polysaccharide substrate, meaning that the substrate of these enzymes remains unknown. Next to raising questions regarding the true nature of AA14 LPMOs, the present data illustrate possible pitfalls in the functional characterization of these intriguing enzymes.

Genomic and Proteomic Study of Andreprevotia ripae Isolated from an Anthill Reveals an Extensive Repertoire of Chitinolytic Enzymes.

Chitin is an abundant natural polysaccharide that is hard to degrade because of its crystalline nature and because it is embedded in robust co-polymeric materials containing other polysaccharides, proteins, and minerals. Thus, it is of interest to study the enzymatic machineries of specialized microbes found in chitin-rich environments. We describe a genomic and proteomic analysis of Andreprevotia ripae, a chitinolytic Gram-negative bacterium isolated from an anthill. The genome of A. ripae encodes four secreted family GH19 chitinases of which two were detected and upregulated during growth on chitin. In addition, the genome encodes as many as 25 secreted GH18 chitinases, of which 17 were detected and 12 were upregulated during growth on chitin. Finally, the single lytic polysaccharide monooxygenase (LPMO) was strongly upregulated during growth on chitin. Whereas 66% of the 29 secreted chitinases contained two carbohydrate-binding modules (CBMs), this fraction was 93% (13 out of 14) for the upregulated chitinases, suggesting an important role for these CBMs. Next to an unprecedented multiplicity of upregulated chitinases, this study reveals several chitin-induced proteins that contain chitin-binding CBMs but lack a known catalytic function. These proteins are interesting targets for discovery of enzymes used by nature to convert chitin-rich biomass. The MS proteomic data have been deposited in the PRIDE database with accession number PXD025087.

Chromatographic Assays for the Enzymatic Degradation of Chitin.

Chitin is an insoluble linear polymer of ß(1→4)-linked N-acetylglucosamine. Enzymatic cleavage of chitin chains can be achieved using hydrolytic enzymes, called chitinases, and/or oxidative enzymes, called lytic polysaccharide monooxygenases (LPMOs). These two groups of enzymes have different modes of action and yield different product types that require different analytical methods for detection and quantitation. While soluble chromogenic substrates are readily available for chitinases, proper insight into the activity of these enzymes can only be obtained by measuring activity toward their polymeric, insoluble substrate, chitin. For LPMOs, only assays using insoluble chitin are possible and relevant. Working with insoluble substrates complicates enzyme assays from substrate preparation to product analysis. Here, we describe typical set-ups for chitin degradation reactions and the chromatographic methods used for product analysis. Graphical abstract: Overview of chromatographic methods for assessing the enzymatic degradation of chitin.

Can we make Chitosan by Enzymatic Deacetylation of Chitin?

Chitin, an insoluble linear polymer of ß-1,4-N-acetyl-d-glucosamine (GlcNAc; A), can be converted to chitosan, a soluble heteropolymer of GlcNAc and d-glucosamine (GlcN; D) residues, by partial deacetylation. In nature, deacetylation of chitin is catalyzed by enzymes called chitin deacetylases (CDA) and it has been proposed that CDAs could be used to produce chitosan. In this work, we show that CDAs can remove up to approximately 10% of N-acetyl groups from two different (α and ß) chitin nanofibers, but cannot produce chitosan.

Proteomic Detection of Carbohydrate-Active Enzymes (CAZymes) in Microbial Secretomes.

Secretomes from microorganisms growing on biomass contain carbohydrate-active enzymes (CAZymes) of potential biotechnological interest. By analyzing such secretomes, we may discover key enzymes involved in degradation processes and potentially infer the mode-of-action of biomass conversion. Some of these enzymes may have predicted functions in carbohydrate degradation, while others may not, while yet exhibiting a similar expression pattern; these latter enzymes constitute potential novel enzymes involved in the degradation process and provide a basis for further biochemical exploration. Hence, secretomes represent an important source for the study of both predicted and novel CAZymes. Here we describe a plate-based culturing technique that allows for collection of protein fractions that are highly enriched for secreted proteins, bound or unbound to the substrate, and which minimizes contamination by intracellular proteins trough unwanted cell lysis.

Bioconversion of chitosan into chito-oligosaccharides (CHOS) using family 46 chitosanase from Bacillus subtilis (BsCsn46A).

BsCsn46A, a GH family 46 chitosanase from Bacillus subtilis had been previously shown to have potential for bioconversion of chitosan to chito-oligosaccharides (CHOS). However, so far, in-depth analysis of both the mode of action of this enzyme and the composition of its products were lacking. In this study, we have employed size exclusion chromatography, 1H NMR, and mass spectrometry to reveal that BsCsn46A can rapidly cleave chitosans with a wide-variety of acetylation degrees, using a non-processive endo-mode of action. The composition of the product mixtures can be tailored by varying the degree of acetylation of the chitosan and the reaction time. Detailed analysis of product profiles revealed differences compared to other chitosanases. Importantly, BsCsn46A seems to be one of the fastest chitosanases described so far. The detailed analysis of preferred endo-binding modes using H218O showed that a hexameric substrate has three productive binding modes occurring with similar frequencies.

Systems analysis of the glycoside hydrolase family 18 enzymes from Cellvibrio japonicus characterizes essential chitin degradation functions.

Understanding the strategies used by bacteria to degrade polysaccharides constitutes an invaluable tool for biotechnological applications. Bacteria are major mediators of polysaccharide degradation in nature; however, the complex mechanisms used to detect, degrade, and consume these substrates are not well-understood, especially for recalcitrant polysaccharides such as chitin. It has been previously shown that the model bacterial saprophyte Cellvibrio japonicus is able to catabolize chitin, but little is known about the enzymatic machinery underlying this capability. Previous analyses of the C. japonicus genome and proteome indicated the presence of four glycoside hydrolase family 18 (GH18) enzymes, and studies of the proteome indicated that all are involved in chitin utilization. Using a combination of in vitro and in vivo approaches, we have studied the roles of these four chitinases in chitin bioconversion. Genetic analyses showed that only the chi18D gene product is essential for the degradation of chitin substrates. Biochemical characterization of the four enzymes showed functional differences and synergistic effects during chitin degradation, indicating non-redundant roles in the cell. Transcriptomic studies revealed complex regulation of the chitin degradation machinery of C. japonicus and confirmed the importance of CjChi18D and CjLPMO10A, a previously characterized chitin-active enzyme. With this systems biology approach, we deciphered the physiological relevance of the glycoside hydrolase family 18 enzymes for chitin degradation in C. japonicus, and the combination of in vitro and in vivo approaches provided a comprehensive understanding of the initial stages of chitin degradation by this bacterium.

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

Enzymatic conversion of chitin, a ß-1,4 linked polymer of N-acetylglucosamine, is of major interest in areas varying from the biorefining of chitin-rich waste streams to understanding how medically relevant fungi remodel their chitin-containing cell walls. Although numerous chitinolytic enzymes have been studied in detail, relatively little is known about enzymes capable of deacetylating chitin. We describe the structural and functional characterization of a 237 residue deacetylase (AnCDA) from Aspergillus nidulans FGSC A4. AnCDA acts on chito-oligomers, crystalline chitin, chitosan, and acetylxylan, but not on peptidoglycan. The K m and k cat of AnCDA for the first deacetylation of penta-N-acetyl-chitopentaose are 72 µM and 1.4 s-1, respectively. Combining mass spectrometry and analyses of acetate release, it was shown that AnCDA catalyses mono-deacetylation of (GlcNAc)2 and full deacetylation of (GlcNAc)3-6 in a non-processive manner. Deacetylation of the reducing end sugar was much slower than deacetylation of the other sugars in chito-oligomers. These enzymatic characteristics are discussed in the light of the crystal structure of AnCDA, providing insight into how the chitin deacetylase may interact with its substrates. Interestingly, AnCDA activity on crystalline chitin was enhanced by a lytic polysaccharide monooxygenase that increases substrate accessibility by oxidative cleavage of the chitin chains.

Proteomic data on enzyme secretion and activity in the bacterium Chitinophaga pinensis.

The secretion of carbohydrate-degrading enzymes by a bacterium sourced from a softwood forest environment has been investigated by mass spectrometry. The findings are discussed in full in the research article "Proteomic insights into mannan degradation and protein secretion by the forest floor bacterium Chitinophaga pinensis" in Journal of Proteomics by Larsbrink et al. ([1], doi: 10.1016/j.jprot.2017.01.003). The bacterium was grown on three carbon sources (glucose, glucomannan, and galactomannan) which are likely to be nutrient sources or carbohydrate degradation products found in its natural habitat. The bacterium was grown on solid agarose plates to mimic the natural behaviour of growth on a solid surface. Secreted proteins were collected from the agarose following trypsin-mediated hydrolysis to peptides. The different carbon sources led to the secretion of different numbers and types of proteins. Most carbohydrate-degrading enzymes were found in the glucomannan-induced cultures. Several of these enzymes may have biotechnological potential in plant cell wall deconstruction for biofuel or biomaterial production, and several may have novel activities. A subset of carbohydrate-active enzymes (CAZymes) with predicted activities not obviously related to the growth substrates were also found in samples grown on each of the three carbohydrates. The full dataset is accessible at the PRIDE partner repository (ProteomeXchange Consortium) with the identifier PXD004305, and the full list of proteins detected is given in the supplementary material attached to this report.

Genomic, proteomic and biochemical analysis of the chitinolytic machinery of Serratia marcescens BJL200.

The chitinolytic machinery of Serratia marcescens BJL200 has been studied in detail over the last couple of decades, however, the proteome secreted by this Gram-negative bacterium during growth on chitin has not been studied in depth. In addition, the genome of this most studied chitinolytic Serratia strain has until now, not been sequenced. We report a draft genome sequence for S. marcescens BJL200. Using label-free quantification (LFQ) proteomics and a recently developed plate-method for assessing secretomes during growth on solid substrates, we find that, as expected, the chitin-active enzymes (ChiA, B, C, and CBP21) are produced in high amounts when the bacterium grows on chitin. Other proteins produced in high amounts after bacterial growth on chitin provide interesting targets for further exploration of the proteins involved in degradation of chitin-rich biomasses. The genome encodes a fourth chitinase (ChiD), which is produced in low amounts during growth on chitin. Studies of chitin degradation with mixtures of recombinantly produced chitin-degrading enzymes showed that ChiD does not contribute to the overall efficiency of the process. ChiD is capable of converting N,N'-diacetyl chitobiose to N-acetyl glucosamine, but is less efficient than another enzyme produced for this purpose, the Chitobiase. Thus, the role of ChiD in chitin degradation, if any, remains unclear.

Proteomic insights into mannan degradation and protein secretion by the forest floor bacterium Chitinophaga pinensis.

Together with fungi, saprophytic bacteria are central to the decomposition and recycling of biomass in forest environments. The Bacteroidetes phylum is abundant in diverse habitats, and several species have been shown to be able to deconstruct a wide variety of complex carbohydrates. The genus Chitinophaga is often enriched in hotspots of plant and microbial biomass degradation. We present a proteomic assessment of the ability of Chitinophaga pinensis to grow on and degrade mannan polysaccharides, using an agarose plate-based method of protein collection to minimise contamination with exopolysaccharides and proteins from lysed cells, and to reflect the realistic setting of growth on a solid surface. We show that select Polysaccharide Utilisation Loci (PULs) are expressed in different growth conditions, and identify enzymes that may be involved in mannan degradation. By comparing proteomic and enzymatic profiles, we show evidence for the induced expression of enzymes and PULs in cells grown on mannan polysaccharides compared with cells grown on glucose. In addition, we show that the secretion of putative biomass-degrading enzymes during growth on glucose comprises a system for nutrient scavenging, which employs constitutively produced enzymes. SIGNIFICANCE OF THIS STUDY: Chitinophaga pinensis belongs to a bacterial genus which is prominent in microbial communities in agricultural and forest environments, where plant and fungal biomass is intensively degraded. Such degradation is hugely significant in the recycling of carbon in the natural environment, and the enzymes responsible are of biotechnological relevance in emerging technologies involving the deconstruction of plant cell wall material. The bacterium has a comparatively large genome, which includes many uncharacterised carbohydrate-active enzymes. We present the first proteomic assessment of the biomass-degrading machinery of this species, focusing on mannan, an abundant plant cell wall hemicellulose. Our findings include the identification of several novel enzymes, which are promising targets for future biochemical characterisation. In addition, the data indicate the expression of specific Polysaccharide Utilisation Loci, induced in the presence of different growth substrates. We also highlight how a constitutive secretion of enzymes which deconstruct microbial biomass likely forms part of a nutrient scavenging process.

Mode of action of a family 75 chitosanase from Streptomyces avermitilis.

Chitooligosaccharides (CHOS) are oligomers composed of glucosamine and N-acetylglucosamine with several interesting bioactivities that can be produced from enzymatic cleavage of chitosans. By controlling the degree of acetylation of the substrate chitosan, the enzyme, and the extent of enzyme degradation, CHOS preparations with limited variation in length and sequence can be produced. We here report on the degradation of chitosans with a novel family 75 chitosanase, SaCsn75A from Streptomyces avermitilis . By characterizing the CHOS preparations, we have obtained insight into the mode of action and subsite specificities of the enzyme. The degradation of a fully deacetylated and a 31% acetylated chitosan revealed that the enzyme degrade these substrates according to a nonprocessive, endo mode of action. With the 31% acetylated chitosan as substrate, the kinetics of the degradation showed an initial rapid phase, followed by a second slower phase. In the initial faster phase, an acetylated unit (A) is productively bound in subsite -1, whereas deacetylated units (D) are bound in the -2 subsite and the +1 subsite. In the slower second phase, D-units bind productively in the -1 subsite, probably with both acetylated and deacetylated units in the -2 subsite, but still with an absolute preference for deacetylated units in the +1 subsite. CHOS produced in the initial phase are composed of deacetylated units with an acetylated reducing end. In the slower second phase, higher amounts of low DP fully deacetylated oligomers (dimer and trimer) are produced, while the higher DP oligomers are dominated by compounds with acetylated reducing ends containing increasing amounts of internal acetylated units. The degradation of chitosans with varying degrees of acetylation to maximum extents of degradation showed that increasingly longer oligomers are produced with increasing degree of acetylation, and that the longer oligomers contain sequences of consecutive acetylated units interspaced by single deacetylated units. The catalytic properties of SaCsn75A differ from the properties of a previously characterized family 46 chitosanase from S. coelicolor (ScCsn46A).