Diacetyl-chitobiose

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.

A chitinase gene (PxChi52) from Paenibacillus xylanexedens Z2-4 was cloned and heterologously expressed in Escherichia coli BL21 (DE3). PxChi52 shared the highest identity of 91% with a glycoside hydrolase family 18 chitinase (ChiD) from Bacillus circulans. The recombinant enzyme (PxChi52) was purified and biochemically characterized. PxChi52 had a molecular mass of 52.8 kDa. It was most active at pH 4.5 and 65°C, respectively, and stable in a wide pH range of 4.0-13.0 and up to 50°C. The enzyme exhibited the highest specific activity of 16.0 U/mg towards colloidal chitin, followed by ethylene glycol chitin (5.4 U/mg) and ball milled chitin (0.4 U/mg). The K_m and V_max values of PxChi52 towards colloidal chitin were determined to be 3.06 mg/mL and 71.38 U/mg, respectively, PxChi52 hydrolyzed colloidal chitin and chitooligosaccharides with degree of polymerization 2-5 to release mainly N-acetyl chitobiose. In addition, PxChi52 displayed inhibition effects on the growth of some phytopathogenic fungi, including Alternaria alstroemeriae, Botrytis cinerea, Rhizoctonia solani, Sclerotinia sclerotiorum and Valsa mali. The unique properties of PxChi52 may enable it potential application in agriculture field as a biocontrol agent.

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.

Cytophaga hutchinsonii is an aerobic cellulolytic soil bacterium that rapidly digests crystalline cellulose. The predicted mechanism by which C. hutchinsonii digests cellulose differs from that of other known cellulolytic bacteria and fungi. The genome of C. hutchinsonii contains 22 glycoside hydrolase (GH) genes, which may be involved in cellulose degradation. One predicted GH with uncertain specificity, CHU_0961, is a modular enzyme with several modules. In this study, phylogenetic tree of the catalytic modules of the GH9 enzymes showed that CHU_0961 and its homologues formed a new group (group C) of GH9 enzymes. The catalytic module of CHU_0961 (CHU_0961B) was identified as a 1,4-β-D-glucan glucohydrolase (EC 3.2.1.74) that has unique properties compared with known GH9 cellulases. CHU_0961B showed highest activity against barley glucan, but low activity against other polysaccharides. Interestingly, CHU_0961B showed similar activity against ρ-nitrophenyl β-D-cellobioside (ρ-NPC) and ρ-nitrophenyl β-D-glucopyranoside. CHU_0961B released glucose from the nonreducing end of cello-oligosaccharides, ρ-NPC, and barley glucan in a nonprocessive exo-type mode. CHU_0961B also showed same hydrolysis mode against deacetyl-chitooligosaccharides as against cello-oligosaccharides. The k_cat/K_m values for CHU_0961B against cello-oligosaccharides increased as the degree of polymerization increased, and its k_cat/K_m for cellohexose was 750 times higher than that for cellobiose. Site-directed mutagenesis showed that threonine 321 in CHU_0961 played a role in hydrolyzing cellobiose to glucose. CHU_0961 may act synergistically with other cellulases to convert cellulose to glucose on the bacterial cell surface. The end product, glucose, may initiate cellulose degradation to provide nutrients for bacterial proliferation in the early stage of C. hutchinsonii growth.

The importance of the gut microbiota in human health has led to an increased interest to study probiotic bacteria. Fermented food is a source of already established probiotics, but it also offers an opportunity to discover new taxa. Four strains of Weissella sp. isolated from Indian fermented food have been genome sequenced and classified into the species W. cibaria based on whole-genome phylogeny. The genome of W. cibaria strain 92, known to utilise xylooligosaccharides and produce lactate and acetate, was analysed to identify genes for oligosaccharide utilisation. Clusters including genes involved in transportation, hydrolysis and metabolism of xylooligosaccharides, arabinooligosaccharides and β-glucosides were identified. Growth on arabinobiose and laminaribiose was detected. A 6-phospho-β-glucosidase clustered with a phosphotransferase system was found upregulated during growth on laminaribiose, indicating a mechanism for laminaribiose utilisation. The genome of W. cibaria strain 92 harbours genes for utilising the phosphoketolase pathway for the production of both acetate and lactate from pentose and hexose sugars but lacks two genes necessary for utilising the pentose phosphate pathway. The ability of W. cibaria strain 92 to utilise several types of oligosaccharides derived from dietary fibres, and produce lactate and acetate makes it interesting as a probiotic candidate for further evaluation.

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.

Lytic polysaccharide monooxygenases (LPMOs) catalyze oxidative cleavage of recalcitrant polysaccharides such as cellulose and chitin and play an important role in the enzymatic degradation of biomass. Although it is clear that these monocopper enzymes have extended substrate-binding surfaces for interacting with their fibrous substrates, the structural determinants of LPMO substrate specificity remain largely unknown. To gain additional insight into substrate specificity in LPMOs, here we generated a mutant library of a cellulose-active family AA10 LPMO from Streptomyces coelicolor A3(2) (ScLPMO10C, also known as CelS2) having multiple substitutions at five positions on the substrate-binding surface that we identified by sequence comparisons. Screening of this library using a newly-developed MS-based high-throughput assay helped identify multiple enzyme variants that contained four substitutions and exhibited significant chitinolytic activity and a concomitant decrease in cellulolytic activity. The chitin-active variants became more rapidly inactivated during catalysis than a natural chitin-active AA10 LPMO, an observation likely indicative of suboptimal substrate binding leading to autocatalytic oxidative damage of these variants. These results reveal several structural determinants of LPMO substrate specificity and underpin the notion that productive substrate binding by these enzymes is complex, depending on a multitude of amino acids located on the substrate-binding surface.

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.

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.

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.

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.

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.

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.

Serratia marcescens PRNK-1, which has strong chitinolytic activity, was isolated from cockroaches (Periplaneta Americana L.). The chitinase from S. marcescens PRNK-1 was characterized after incubation in a 0.5% colloidalchitin medium at 30°C for 3 days. The molecular weights of three bands after staining for chitinase activity were approximately 34, 41, and 48 kDa on an SDS-PAGE gel. S. marcescens PRNK-1 strain strongly inhibited hyphal growth of Rhizoctonia solani and Fusarium oxysporum. Thin-layer chromatography(TLC) and high performance liquid chromatograph (HPLC) analyses were conducted to investigate the degradation patterns of N-acetyl-chitooligosaccharides by PRNK-1 chitinase. The N-acetyl-chitooligosaccharides: N-acetyl-chitin dimer (GlcNAc)₂, N-acetyl-chitin trimer (GlcNAc)₃, and N-acetyl-chitin tetramer (GlcNAc)₄ were degraded to (GlcNAc)_1-3 on a TLC plate. In an additional experiment, (GlcNAc)₆ was degraded to (GlcNAc)_1-4 on a TLC plate. The optimal temperature for chitinase activity of the PRNK-1 was 50°C, producing 32.8 units/mL. As seen via TLC, the highest degradation of (GlcNAc)₄ by PRNK-1 chitinase occurred with 50°C incubation. The optimal pH for chitinase activity of PRNK-1 was pH 5.5, producing 24.6 units/mL. As seen via TLC, the highest degradation of (GlcNAc)₄ by PRNK-1 chitinase occurred at pH 5.0-6.0. These results indicate that chitinase produced from S. marcescens PRNK-1 strain showed strong antifungal activity and potential of production of N-acetyl-chitooligosaccharides.

Partially acetylated chitosan oligosaccharides (paCOS) have various potential applications in agriculture, biomedicine and pharmaceutics due to their suitable bioactivities. One method to produce paCOS is partial chemical hydrolysis of chitosan polymers, but that leads to poorly defined mixtures of oligosaccharides. However, the effective production of defined paCOS is crucial for fundamental research and for developing applications. A more promising approach is enzymatic depolymerization of chitosan using chitinases or chitosanases, as the substrate specificity of the enzyme determines the composition of the oligomeric products. Protein-engineering of these enzymes to alter their substrate specificity can overcome the limitations associated with naturally occurring enzymes and expand the spectrum of specific paCOS that can be produced. Here, engineering the substrate specificity of Bacillus sp. MN chitosanase is described for the first time. Two muteins with active site substitutions can accept N-acetyl-D-glucosamine units at their subsite (-2), which is impossible for the wildtype enzyme.