Triacetyl-chitotriose

Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less established, and one reason for this is a lack of suitable glycans with which to populate arrays. Polysaccharide microarrays are relatively easy to produce because of the ease of immobilizing large polymers noncovalently onto a variety of microarray surfaces, but they lack analytical resolution because polysaccharides often contain multiple distinct carbohydrate substructures. Microarrays of defined oligosaccharides potentially overcome this problem but are harder to produce because oligosaccharides usually require coupling prior to immobilization. We have assembled a library of well characterized plant oligosaccharides produced either by partial hydrolysis from polysaccharides or by de novo chemical synthesis. Once coupled to protein, these neoglycoconjugates are versatile reagents that can be printed as microarrays onto a variety of slide types and membranes. We show that these microarrays are suitable for the high throughput characterization of the recognition capabilities of monoclonal antibodies, carbohydrate-binding modules, and other oligosaccharide-binding proteins of biological significance and also that they have potential for the characterization of carbohydrate-active enzymes.

Endo-type chitinase is the principal enzyme involved in the breakdown of N-acetyl-d-glucosamine-based oligomeric and polymeric materials through hydrolysis. The gene (966-bp) encoding a novel endo-type chitinase (ChiJ), which is comprised of an N-terminal chitin-binding domain type 3 and a C-terminal catalytic glycoside hydrolase family 19 domain, was identified from a fibrolytic intestinal symbiont of the earthworm Eisenia fetida, Cellulosimicrobium funkei HY-13. The highest endochitinase activity of the recombinant enzyme (rChiJ: 30.0 kDa) toward colloidal shrimp shell chitin was found at pH 5.5 and 55 °C and was considerably stable in a wide pH range (3.5–11.0). The enzyme exhibited the highest biocatalytic activity (338.8 U/mg) toward ethylene glycol chitin, preferentially degrading chitin polymers in the following order: ethylene glycol chitin > colloidal shrimp shell chitin > colloidal crab shell chitin. The enzymatic hydrolysis of N-acetyl-β-d-chitooligosaccharides with a degree of polymerization from two to six and colloidal shrimp shell chitin yielded primarily N,N′-diacetyl-β-d-chitobiose together with a small amount of N-acetyl-d-glucosamine. The high chitin-degrading ability of inverting rChiJ with broad pH stability suggests that it can be exploited as a suitable biocatalyst for the preparation of N,N′-diacetyl-β-d-chitobiose, which has been shown to alleviate metabolic dysfunction associated with type 2 diabetes.

The bioconversion of chitin into N-acetyl-d-glucosamine (GlcNAc) using chitinolytic enzymes is one of the important avenues for chitin valorization. However, industrial applications of chitinolytic enzymes have been limited by their poor thermostability. Therefore, it is necessary to discover thermostable chitinolytic enzymes for GlcNAc production from chitin. In this study, two chitinolytic enzyme-encoding genes CaChiT and CaHex from Caldicellulosiruptor acetigenus were identified and heterologously expressed in Escherichia coli. The purified recombinant CaChiT and CaHex showed optimal activities at 70°C and 90°C respectively, and exhibited good thermostability over a range of temperature below 70°C and broad pH stability at pH range of 3.0-8.0. CaChiT and CaHex were active on colloidal chitin, pNP-(GlcNAc)₂, pNP-(GlcNAc)₃, and pNP-GlcNAc, pNP-(GlcNAc)₂, pNP-(GlcNAc)₃, pNP-Glc respectively. Besides, the chitin oligosaccharides and colloidal chitin hydrolysis profiles revealed that CaChiT degraded chitin chains through exo-mode of action. Furthermore, CaChiT and CaHex exhibited a synergistic effect in the degradation of colloidal chitin, reaching 0.60 mg/mL of GlcNAc production after 1 h incubation. These results suggested that a combination of CaChiT and CaHex have great potential for industrial applications in the enzymatic production of GlcNAc from chitin-containing biowastes.

Chitin is an insoluble linear polymer of β(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.

A novel chitosanase gene, designated as PbCsn8, was cloned from Paenibacillus barengoltzii. It shared the highest identity of 73% with the glycoside hydrolase (GH) family 8 chitosanase from Bacillus thuringiensis JAM-GG01. The gene was heterologously expressed in Bacillus subtilis as an extracellular protein, and the highest chitosanase yield of 1, 108 U/mL was obtained by high-cell density fermentation in a 5-L fermentor. The recombinant chitosanase (PbCsn8) was purified to homogeneity and biochemically characterized. PbCsn8 was most active at pH 5.5 and 70°C, respectively. It was stable in a wide pH range of 5.0-11.0 and up to 55°C. PbCsn8 was a bifunctional enzyme, exhibiting both chitosanase and glucanase activities, with the highest specificity towards chitosan (360 U/mg), followed by barley β-glucan (72 U/mg) and lichenan (13 U/mg). It hydrolyzed chitosan to release mainly chitooligosaccharides (COSs) with degree of polymerization (DP) 2-3, while hydrolyzed barley β-glucan to yield mainly glucooligosaccharides with DP > 5. PbCsn8 was further applied in COS production, and the highest COS yield of 79.3% (w/w) was obtained. This is the first report on a GH family 8 chitosanase from P. barengoltzii. The high yield and remarkable hydrolysis properties may make PbCsn8 a good candidate in industrial application.

Listeria monocytogenes possesses two chitinases (LmChiA and LmChiB) belonging to glycoside hydrolase family 18 (GH18). In this study, two chitinase genes (lmchiA and lmchiB) from L. monocytogenes 10403S were cloned and their biochemical characteristics were studied. Using colloidal chitin as substrate, both chitinases exhibited maximum catalytic activity at pH 6-7 with optimum temperature at 50°C. Their activities were stable over broad pH (3-10) and temperature (10-50°C) ranges. Kinetic analysis using [4NP-(GlcNAc)₂] as substrate indicated that LmChiB had an approximately 4-fold lower K_m and 2-fold higher k_cat than LmChiA, suggesting that the catalytic specificity and efficiency of LmChiB were greater than those of LmChiA. LmChiA and LmChiB showed the same reactivity toward oligomeric substrates and exhibited both non-processive endo-acting and processive exo-acting (chitobiosidase) activity on colloidal chitin, chitin oligosaccharides and 4-nitrophenyl substrates. Structure-based sequence alignments and homology modeling of the catalytic domains revealed that both chitinases consisted of an (α/β)₈ TIM barrel fold with a conserved DXDXE motif. The key residues involved in the substrate hydrolysis were conserved with other bacterial chitinases. The site-directed mutagenesis of conserved Asp and Glu residues in DXDXE motif of both chitinases significantly reduced the chitinolytic activity toward colloidal chitin substrate and revealed their critical role in the catalytic mechanism. LmChiA and LmChiB might have potential in chitin waste utilization and biotechnological applications.

Chitinases play important role in chitin bioconversion, while few of them have been put into use due to their poor properties. We aimed to identify and characterize chitinases suitable for N-acetyl chitooligosaccharides (COSs) production from chitin materials. A chitinase gene (SsChi28) from Streptomyces sampsonii XY2-7 was cloned and heterologously expressed in E. coli BL21 (DE3) as an active protein. The deduced protein shared high sequence identities and structure similarities with some glycoside hydrolase family 19 chitinases. The recombinant enzyme (SsChi28) was purified and biochemically characterized. SsChi28 was a monomeric protein with a molecular mass of 30 kDa estimated by SDS-PAGE. It was most active at pH 6.0 and 55°C, respectively, and stable in a wide pH range of 3.5-11.5 and up to 60°C. The enzyme exhibited strict substrate specificities towards ethylene glycol chitin (222.3 U/mg) and colloidal chitin (20.1 U/mg). Besides, it displayed lysozyme activity against Micrococcus lysodeikticus. SsChi28 hydrolyzed colloidal chitin to yield mainly N-acetyl chitobiose, accounting high up to 73% (w/w) in total products. The excellent enzymatic properties of SsChi28 may make it potential in chitin bioconversion (especially for N-acetyl COS production), as well as in biological control of fungal diseases.

In this study, a chitinase gene, Chit46 from a mycoparasitic fungus Trichoderma harzianum was successfully expressed in Pichia pastoris with a high heterologous chitinase production of 31.4 U/mL, much higher than the previous reports. The active center and substrate binding pocket of the recombinant Chit46 (rChit46) were analyzed and the effects of pH, temperature, metal ions and glycosylation on its activity were tested. rChit46 effectively hydrolyzed colloidal chitin with a high conversion rate of 80.5% in 3 h and the chitin hydrolysates were mainly composed of (GlcNAc)₂ (94.8%), which make it a good candidate for the green recycling of chitinous waste. rChit46 could also significantly inhibit growth of the phytopathogenic fungus Botrytis cinerea, which endowed it with the potential as a biocontrol agent.

Background: Lytic polysaccharide monooxygenases (LPMOs) opened a new horizon for biomass deconstruction. They use a redox mechanism not yet fully understood and the range of substrates initially envisaged to be the crystalline polysaccharides is steadily expanding to non-crystalline ones. Results: The enzyme KpLPMO10A from the actinomycete Kitasatospora papulosa was cloned and overexpressed in Escherichia coli cells in the functional form with native N-terminal. The enzyme can release oxidized species from chitin (C1-type oxidation) and cellulose (C1/C4-type oxidation) similarly to other AA10 members from clade II (subclade A). Interestingly, KpLPMO10A also cleaves isolated xylan (not complexed with cellulose, C4-type oxidation), a rare activity among LPMOs not described yet for the AA10 family. The synergistic effect of KpLPMO10A with Celluclast ® and an endo-β-1,4-xylanase also supports this finding. The crystallographic elucidation of KpLPMO10A at 1.6 Å resolution along with extensive structural analyses did not indicate any evident diference with other characterized AA10 LPMOs at the catalytic interface, tempting us to suggest that these enzymes might also be active on xylan or that the ability to attack both crystalline and non-crystalline substrates involves yet obscure mechanisms of substrate recognition and binding. Conclusions: This work expands the spectrum of substrates recognized by AA10 family, opening a new perspective for the understanding of the synergistic efect of these enzymes with canonical glycoside hydrolases to deconstruct ligno(hemi)cellulosic biomass.

Microbial chitinases (EC 3.2.1.14) are known to hydrolyse the chitinous gut epithelium of insects and cell walls of many fungi. In this study, seven chitinases from different bacteria and fungi were produced, characterized and their biocontrol abilities against graminaceous stem borers Eldana saccharina, Chilo partellus and Sesamia calamistis were assessed. All chitinases were stable over broad ranges of pH and temperature, however, recombinant fungal chitinases were more acid-stable than the bacterial counterparts. Chitinases from the thermophilic filamentous fungi Thermomyces lanuginosus SSBP (Chit1) and from Bacillus licheniformis (Chit lic) caused 70% and 80% mortality, respectively, in second instar larvae of E. saccharina. Six of the seven partially-purified microbial chitinases inhibited Aspergillus niger, A. flavus, A. alliaceus, A. ochraceus, Fusarium verticillioides and Mucor sp. Overall, microbial chitinases show promise as biocontrol agents of fungi and stalk–boring lepidopterans.

CIA17 is a PP2-like, homodimeric lectin made up of 17 kDa subunits present in the phloem exudate of ivy gourd (Coccinia indica). Isothermal titration calorimetric (ITC) studies on the interaction of chitooligosaccharides [(GlcNAc)_2–6] showed that the dimeric protein has two sugar binding sites which recognize chitotriose with ~70-fold higher affinity than chitobiose, indicating that the binding site is extended in nature. ITC, atomic force microscopic and non-denaturing gel electrophoresis studies revealed that the high-affinity interaction of CIA17 with chitohexaose (K_a = 1.8 × 10⁷ M⁻¹) promotes the formation of protein oligomers. Computational studies involving homology modeling, molecular docking and molecular dynamics simulations on the binding of chitooligosaccharides to CIA17 showed that the protein binding pocket accommodates up to three GlcNAc residues. Interestingly, docking studies with chitohexaose indicated that its two triose units could interact with binding sites on two protein molecules to yield dimeric complexes of the type CIA17-(GlcNAc)₆-CIA17, which can extend in length by the binding of additional chitohexaose and CIA17 molecules. These results suggest that PP2 proteins play a role in plant defense against insect/pathogen attack by directly binding with the higher chain length chitooligosaccharides and forming extended, filamentous structures, which facilitate wound sealing.

A thermostable chitinase Chi1 from Myceliophthora thermophila C1 was homologously produced and characterized. Chitinase Chi1 shows high thermostability at 40°C (>140 h 90% activity), 50°C (>168 h 90% activity), and 55°C (half-life 48 h). Chitinase Chi1 has broad substrate specificity, and converts chitin, chitosan, modified chitosan and chitin oligosaccharides. The activity of chitinase Chi1 is strongly affected by the degree of deacetylation (DDA), molecular weight (Mw) and side chain modification of chitosan. Chitinase Chi1 releases mainly (GlcNAc)₂ from insoluble chitin and chito-oligosaccharides with a polymerization degree (DP) ranging from 2 to 12 from chitosan, in a processive way. Chitinase Chi1 shows higher activity towards chitin oligosaccharides (GlcNAc)_4-6 than towards (GlcNAc)₃ and is inactive for (GlcNAc)₂. During hydrolysis, oligosaccharides bind at subsites -2 to +2 in the enzyme’s active site. Chitinase Chi1 can be used for chitin valorisation and for production of chitin- and chito-oligosaccharides at industrial scale.

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 family 18 Glycoside Hydrolase (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 GH18 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.

Thermobia domestica belongs to an ancient group of insects and has a remarkable ability to digest crystalline cellulose without microbial assistance. By investigating the digestive proteome of Thermobia, we have identified over twenty members of an uncharacterized family of lytic polysaccharide monooxygenases (LPMOs). We show that this LPMO family spans across several clades of the Tree of Life, is of ancient origin and was recruited by early arthropods with possible roles in remodelling endogenous chitin scaffolds during development and metamorphosis. Based on our in-depth characterization of Thermobia’s LPMOs, we propose that diversification of these enzymes towards cellulose digestion might have endowed ancestral insects with an effective biochemical apparatus for biomass degradation, allowing the early colonization of land during the Paleozoic Era. The vital role of LPMOs in modern agricultural pests and disease vectors offers new opportunities to help tackle global challenges in food security and the control of infectious diseases.

Thermostable enzymes are a promising alternative for chemical catalysts currently used for the production of N-acetylglucosamine (GlcNAc) from chitin. In this study, a novel thermostable β-N-acetylglucosaminidase MthNAG was cloned and purified from the thermophilic fungus Myceliophthora thermophila C1. MthNAG is a protein with a molecular weight of 71 kDa as determined with MALDI-TOF-MS. MthNAG has the highest activity at 50°C and pH 4.5. The enzyme shows high thermostability above the optimum temperature: at 55°C (144 h, 75% activity), 60°C (48 h, 85% activity; half-life 82 h), and 70°C (24 h, 33% activity; half-life 18 h). MthNAG releases GlcNAc from chitin oligosaccharides (GlcNAc)_2-5, p-nitrophenol derivatives of chitin oligosaccharides (GlcNAc)_1-3-pNP, and the polymeric substrates swollen chitin and soluble chitosan. The highest activity was detected towards (GlcNAc)₂. MthNAG released GlcNAc from the non-reducing end of the substrate. We found that MtHNAG and Chitinase Chi1 from M. thermophila C1 synergistically degraded swollen chitin and released GlcNAc in concentration of approximately 130 times higher than when only MthNAG was used. Therefore, chitinase Chi1 and MthNAG have great potential in the industrial production of GlcNAc.

Lytic polysaccharide monooxygenases (LPMOs) contribute to enzymatic conversion of recalcitrant polysaccharides such as chitin and cellulose and may also play a role in bacterial infections. Some LPMOs are multimodular, the implications of which remain only partly understood. We have studied the properties of a tetra‐modular LPMO from the food poisoning bacterium Bacillus cereus (named BcLPMO10A). We show that BcLPMO10A, comprising an LPMO domain, two fibronectin‐type III (FnIII)‐like domains, and a carbohydrate‐binding module (CBM5), is a powerful chitin‐active LPMO. While the role of the FnIII domains remains unclear, we show that enzyme functionality strongly depends on the CBM5, which, by promoting substrate binding, protects the enzyme from inactivation. BcLPMO10A enhances the activity of chitinases during the degradation of α-chitin.

Chitin, one of Nature’s most abundant biopolymers, can be obtained by either traditional chemical pulping or by extraction using the ionic liquid (IL) 1-ethyl-3-methylimidazolium acetate. The IL extraction and coagulation process provides access to a unique chitin, with an open hydrated gel-like structure. Here, enzymatic hydrolysis of this chitin hydrogel, dried shrimp shell, chitin extracted from shrimp shells using IL and then dried, and commercial chitin was carried out using chitinase from Streptomyces griseus. The enzymatic hydrolysis of shrimp shells resulted only in the monomer N-acetylglucosamine, while much higher amounts of the dimer (N, N′-diacetylchitobiose) compared to the monomer were detected when using all forms of ‘pure’ chitin. Interestingly, small amounts of the trimer (N, N′,N′′-triacetylchitotriose) were also detected when the IL-chitin hydrogel was used as substrate. Altogether, our findings indicate that the product distribution and yield are highly dependent on the substrate selected for the reaction and its hydrated state.

We isolated a laminarin-degrading cold-adapted bacterium strain LA from coastal seawater in Sagami Bay, Japan and identified it as a Pseudoalteromonas species. We named the extracellular laminarinase LA-Lam, and purified and characterized it. LA-Lam showed high degradation activity for Laminaria digitata laminarin in the ranges of 15-50°C and pH 5.0-9.0. The major terminal products degraded from L. digitata laminarin with LA-Lam were glucose, laminaribiose, and laminaritriose. The degradation profile of laminarioligosaccharides with LA-Lam suggested that the enzyme has a high substrate binding ability toward tetrameric or larger saccharides. Our results of the gene sequence and the SDS-PAGE analyses revealed that the major part of mature LA-Lam is a catalytic domain that belongs to the GH16 family, although its precursor is composed of a signal peptide, the catalytic domain, and three-repeated unknown regions.

Curdlan is a water-insoluble microbial exo-polysaccharide that is hardly degraded. The gene CcGluE encoding an endo-β-1 →3-glucanase consisting of 412 amino acids (44 kDa) from Cellulosimicrobium cellulans E4-5 was cloned and expressed in Escherichia coli. The recombinant CcGluE hydrolysed curdlan powder effectively. CcGluE shows high endo-β-1 →3 glucanase activity and low β-1,4 and β-1,6 glucanase activities with broad substrate specificity for glucan, including curdlan, laminarin and β-1 →3/1 →6-glucan, and the highest catalytic activity for curdlan. Moreover, the hydrolytic products of curdlan were glucan oligosaccharides with degrees of polymerisation of 2-13, and the main products were glucobiose and glucotriose. Degradation mode analysis indicated that CcGluE is more likely to hydrolyse glucopentaose and revealed that CcGluE was an endo-glucanase. Furthermore, upon combination with a homogenising pre-treatment method with curdlan, the degradation efficiency of CcGluE for curdlan powder was greatly improved 7.1-fold, which laid a good foundation for the utilisation of curdlan.

In plants, chitooligosaccharide-binding phloem exudate lectins play an important role in the defense mechanism against parasites. Here, we investigated the thermal and chaotrope-induced unfolding of cucumber (Cucumis sativus) phloem exudate lectin (CPL). Circular dichroism (CD) spectroscopic studies indicate that the secondary and tertiary structures of CPL are essentially unaltered up to 90°C. Consistent with this, differential scanning calorimetric studies revealed that CPL is highly thermostable and undergoes a cooperative thermal unfolding transition centered at 97.6°C. The unfolding process was calorimetrically irreversible, and could be described by a non-two-state model, suggesting that upon undergoing a reversible unfolding transition the protein attains a final state in an irreversible step. The ratio of calorimetric and van’t Hoff enthalpies (ΔH_c/ΔH_v/) was >1.0, suggesting that the two monomers in the dimeric protein unfold at the same temperature. CD spectra recorded at different pH indicated that the secondary and tertiary structures of the protein are nearly unaltered in the pH range 3.0-10.0. Guanidine hydrochloride-induced unfolding studies indicate that chemical denaturation of CPL can also be described by a two-state process, without involving any intermediate. The stability of CPL to high temperatures and large variations of pH appear to be particularly suited for its role in plant defense.