The Ustilago maydis AA10 LPMO is active on fungal cell wall chitin.

Lytic polysaccharide monooxygenases (LPMOs) can perform oxidative cleavage of glycosidic bonds in carbohydrate polymers (e.g., cellulose, chitin), making them more accessible to hydrolytic enzymes. While most studies have so far mainly explored the role of LPMOs in a (plant) biomass conversion context, alternative roles and paradigms begin to emerge. The AA10 LPMOs are active on chitin and/or cellulose and mostly found in bacteria and in some viruses and archaea. Interestingly, AA10-encoding genes are also encountered in some pathogenic fungi of the Ustilaginomycetes class, such as Ustilago maydis, responsible for corn smut disease. Transcriptomic studies have shown the overexpression of the AA10 gene during the infectious cycle of U. maydis. In fact, U. maydis has a unique AA10 gene that codes for a catalytic domain appended with a C-terminal disordered region. To date, there is no public report on fungal AA10 LPMOs. In this study, we successfully produced the catalytic domain of this LPMO (UmAA10_cd) in Pichia pastoris and carried out its biochemical characterization. Our results show that UmAA10_cd oxidatively cleaves α- and ß-chitin with C1 regioselectivity and boosts chitin hydrolysis by a GH18 chitinase from U. maydis (UmGH18A). Using a biologically relevant substrate, we show that UmAA10_cd exhibits enzymatic activity on U. maydis fungal cell wall chitin and promotes its hydrolysis by UmGH18A. These results represent an important step toward the understanding of the role of LPMOs in the fungal cell wall remodeling process during the fungal life cycle.IMPORTANCELytic polysaccharide monooxygenases (LPMOs) have been mainly studied in a biotechnological context for the efficient degradation of recalcitrant polysaccharides. Only recently, alternative roles and paradigms begin to emerge. In this study, we provide evidence that the AA10 LPMO from the phytopathogen Ustilago maydis is active against fungal cell wall chitin. Given that chitin-active LPMOs are commonly found in microbes, it is important to consider fungal cell wall as a potential target for this enigmatic class of enzymes.

Comprehensive Genome Analysis of Cellulose and Xylan-Active CAZymes from the Genus Paenibacillus: Special Emphasis on the Novel Xylanolytic Paenibacillus sp. LS1.

Xylan is the most abundant hemicellulose in hardwood and graminaceous plants. It is a heteropolysaccharide comprising different moieties appended to the xylose units. Complete degradation of xylan requires an arsenal of xylanolytic enzymes that can remove the substitutions and mediate internal hydrolysis of the xylan backbone. Here, we describe the xylan degradation potential and underlying enzyme machinery of the strain, Paenibacillus sp. LS1. The strain LS1 was able to utilize both beechwood and corncob xylan as the sole source of carbon, with the former being the preferred substrate. Genome analysis revealed an extensive xylan-active CAZyme repertoire capable of mediating efficient degradation of the complex polymer. In addition to this, a putative xylooligosaccharide ABC transporter and homologues of the enzymes involved in the xylose isomerase pathway were identified. Further, we have validated the expression of selected xylan-active CAZymes, transporters, and metabolic enzymes during growth of the LS1 on xylan substrates using qRT-PCR. The genome comparison and genomic index (average nucleotide identity [ANI] and digital DNA-DNA hybridization) values revealed that strain LS1 is a novel species of the genus Paenibacillus. Lastly, comparative genome analysis of 238 genomes revealed the prevalence of xylan-active CAZymes over cellulose across the Paenibacillus genus. Taken together, our results indicate that Paenibacillus sp. LS1 is an efficient degrader of xylan polymers, with potential implications in the production of biofuels and other beneficial by-products from lignocellulosic biomass. IMPORTANCE Xylan is the most abundant hemicellulose in the lignocellulosic (plant) biomass that requires cooperative deconstruction by an arsenal of different xylanolytic enzymes to produce xylose and xylooligosaccharides. Microbial (particularly, bacterial) candidates that encode such enzymes are an asset to the biorefineries to mediate efficient and eco-friendly deconstruction of xylan to generate products of value. Although xylan degradation by a few Paenibacillus spp. is reported, a complete genus-wide understanding of the said trait is unavailable till date. Through comparative genome analysis, we showed the prevalence of xylan-active CAZymes across Paenibacillus spp., therefore making them an attractive option towards efficient xylan degradation. Additionally, we deciphered the xylan degradation potential of the strain Paenibacillus sp. LS1 through genome analysis, expression profiling, and biochemical studies. The ability of Paenibacillus sp. LS1 to degrade different xylan types obtained from different plant species, emphasizes its potential implication in lignocellulosic biorefineries.

Genome analysis of Streptomyces sp. UH6 revealed the presence of potential chitinolytic machinery crucial for chitosan production.

Chitosan and its derivatives have numerous applications in wastewater treatment as bio-coagulants, flocculants and bio-adsorbents against both particulate and dissolved pollutants. Chitinolytic bacteria secrete an array of enzymes, which play crucial role in chitin to chitosan conversion. Consequently, there is a growing demand for identification and characterization of novel bacterial isolates with potential implications in chitosan production. We describe genomic features of the new isolate Streptomyces sp. UH6. Analysis of the 6.51 Mb genome revealed the GC content as 71.95% and presence of 6990 coding sequences of which 63% were functionally annotated. Further, we identified two possible chitin-utilization pathways, which employ secreted enzymes like lytic polysaccharide monooxygenases and family-18 glycoside hydrolases (GHs). More importantly, the genome has six family-4 polysaccharide deacetylases with probable role in chitin to chitosan conversion, as well as two chitosanases belonging to GH46 and GH75 families. In addition, the gene clusters, dasABC and ngcEFG coding for transporters, which mediate the uptake of N,N'-diacetylchitobiose and N-acetyl-d-glucosamine were identified. Several genes responsible for hydrolysis of other polysaccharides and fermentation of sugars were also identified. Taken together, the phylogenetic and genomic analyses suggest that the isolate Streptomyces sp. UH6 secretes potential chitin-active enzymes responsible for chitin to chitosan conversion.

On the expansion of biological functions of lytic polysaccharide monooxygenases.

Lytic polysaccharide monooxygenases (LPMOs) constitute an enigmatic class of enzymes, the discovery of which has opened up a new arena of riveting research. LPMOs can oxidatively cleave the glycosidic bonds found in carbohydrate polymers enabling the depolymerisation of recalcitrant biomasses, such as cellulose or chitin. While most studies have so far mainly explored the role of LPMOs in a (plant) biomass conversion context, alternative roles and paradigms begin to emerge. In the present review, we propose a historical perspective of LPMO research providing a succinct overview of the major achievements of LPMO research over the past decade. This journey through LPMOs landscape leads us to dive into the emerging biological functions of LPMOs and LPMO-like proteins. We notably highlight roles in fungal and oomycete plant pathogenesis (e.g. potato late blight), but also in mutualistic/commensalism symbiosis (e.g. ectomycorrhizae). We further present the potential importance of LPMOs in other microbial pathogenesis including diseases caused by bacteria (e.g. pneumonia), fungi (e.g. human meningitis), oomycetes and viruses (e.g. entomopox), as well as in (micro)organism development (including several plant pests). Our assessment of the literature leads to the formulation of outstanding questions, promising for the coming years exciting research and discoveries on these moonlighting proteins.

Chemoenzymatic production of chitooligosaccharides employing ionic liquids and Thermomyces lanuginosus chitinase.

The aim of this work was to study a two-step chemoenzymatic method for production of short chain chitooligosaccharides. Chitin was chemically pretreated using sulphuric acid, sodium hydroxide and two different ionic liquids, 1-Ethyl-3-methylimidazolium bromide and Trihexyltetradecylphosphonium bis(2,4,4-trimethylpentyl)phosphinate under mild processing conditions. Pretreated chitin was further hydrolyzed employing purified chitinase from Thermomyces lanuginosus ITCC 8895. Trihexyltetradecylphosphonium bis(2,4,4-trimethylpentyl)phosphinate treated chitin appeared amorphous and resulted in generation of 1.10 ± 0.89 mg ml-1 of (GlcNAc)2 and 1.07 ± 0.92 mg ml-1 of (GlcNAc)3. Further derivation of optimum conditions through two-factor-9 run experiments resulted in to 1.5 and 1.3 fold increments in (GlcNAc)2 and (GlcNAc)3 production, respectively. 0.1 g of both (GlcNAc)2 and (GlcNAc)3 has been purified from the Trihexyltetradecylphosphonium bis(2,4,4-trimethylpentyl)phosphinate pretreated chitin (1 g) employing cation exchange chromatography. The present study will lay the foundation for development of a green sustainable solution for cost effective upcycling of coastal residual resources to chito-bioactives.

Deciphering the antagonistic effect of Streptomyces spp. and host-plant resistance induction against charcoal rot of sorghum.

MAIN CONCLUSION: The findings of this study suggest that the selected five strains of Streptomyces spp. could be used for biological control of charcoal rot disease in sorghum. Two strains each of Streptomyces albus (CAI-17 and KAI-27) and Streptomyces griseus (KAI-26 and MMA-32) and one strain of Streptomyces cavourensis (SAI-13) previously reported to have plant growth-promotion activity in chickpea, rice and sorghum were evaluated for their antagonistic potential against Macrophomina phaseolina, which causes charcoal rot in sorghum. The antagonistic potential of these strains against M. phaseolina was assessed through dual culture assay, metabolite production assay, blotter paper assay in greenhouse and field disease screens. In both dual culture and metabolite production assays, the selected strains significantly inhibited the growth of M. phaseolina (63-74%). In the blotter paper assay, all the five strains of Streptomyces spp. inhibited the pathogen (80-90%). When these five strains were tested for their antagonistic potential under the greenhouse (two times) and field (two seasons) conditions by toothpick method of inoculation, significant differences were observed for charcoal rot severity. Principal component analysis capturing 91.3% phenotypic variations, revealed that the shoot samples treated with both Streptomyces and the pathogen exhibited significantly enhanced antioxidant parameters including superoxide dismutase, catalase, ascorbate peroxidase, guaiacol peroxidase, glutathione reductase, phenylalanine ammonia-lyase, polyphenol oxidase, and total phenolic contents when compared to shoot samples treated with only M. phaseolina. Scanning electron microscope analysis revealed that the phloem and xylem tissues of the Streptomyces treated stem samples were intact compared to that of pathogen inoculated plants. This study indicated that the selected strains of Streptomyces spp. have the potential for biological control of charcoal rot disease in sorghum.

Chemoenzymatic Synthesis of Chito-oligosaccharides with Alternating N-d-Acetylglucosamine and d-Glucosamine.

Chito-oligosaccharides (CHOS) are homo- or hetero-oligomers of N-acetylglucosamine (GlcNAc, A) and d-glucosamine (GlcN, D). Production of well-defined CHOS-mixtures, or even pure CHOS, with specific lengths and sugar compositions, is of great interest since these oligosaccharides have interesting bioactivities. While direct chemical synthesis of CHOS is not straightforward, chemo-enzymatic approaches have shown some promise. We have used engineered glycoside hydrolases to catalyze oligomerization of activated DA building blocks through transglycosylation reactions. The building blocks were generated from readily available (GlcNAc)2-para-nitrophenol through deacetylation of the nonreducing end sugar with a recombinantly expressed deacetylase from Aspergillus niger (AnCDA9). This approach, using a previously described hyper-transglycosylating variant of ChiA from Serratia marcescens (SmChiA) and a newly generated transglycosylating variant of Chitinase D from Serratia proteamaculans (SpChiD), led to production of CHOS containing up to ten alternating D and A units [(DA)2, (DA)3, (DA)4, and (DA)5]. The most abundant compounds were purified and characterized. Finally, we demonstrate that (DA)3 generated in this study may serve as a specific inhibitor of the human chitotriosidase. Inhibition of this enzyme has been suggested as a therapeutic strategy against systemic sclerosis.

Efficient conversion of crystalline chitin to N-acetylglucosamine and N,N'-diacetylchitobiose by the enzyme cocktail produced by Paenibacillus sp. LS1.

Direct conversion of crystalline chitin to N-acetylglucosamine and the related chitooligomers through enzymatic approaches is gaining importance owing to their various biological applications. Here we report the crystalline chitin degradation ability of chitinolytic cocktail produced by the isolate Paenibacillus sp. LS1. Growth studies of the isolate in presence of different chitin substrates revealed preference for ß-chitin and colloidal chitin. FE-SEM micrographs showed formation of visible perforations on the crystalline chitin particles by the isolate. Further, zymogram analysis revealed the presence of six potential chitinase isozymes. The enzyme-cocktail produced by the isolate was optimally active at 50 °C in 50 mM sodium acetate, pH-4.0. Time-course hydrolysis of crystalline α- and ß-chitin with the Paenibacillus sp. LS1 enzyme-cocktail produced N-acetylglucosamine and N,N'-diacetylchitobiose as the predominant products. Taken together, our results confirm that the Paenibacillus sp. LS1 enzyme-cocktail would have potential application in eco-friendly enzymatic approaches for efficient saccharification of crystalline chitin.

Chitinase-E from Chitiniphilus shinanonensis generates chitobiose from chitin flakes.

Enzymatic deconstruction of chitin to chitobiose is of significant interest in view of its various biological applications. Here we report detailed insights into the chitin degradation by chitinase-E from a chitinolytic bacterium Chitiniphilus shinanonensis (CsChiE). CsChiE was optimally active at 50 °C in 50 mM sodium phosphate pH-7.0. It showed a kcat and overall catalytic efficiency (Kcat/Km) as 3.9 × 103 s-1 and 0.6 × 103 s-1 mg-1 mL, respectively, on colloidal chitin (CC). CsChiE efficiently hydrolyzed crystalline polymers like α-, ß- and CC and released chitobiose as the predominant product (11.3 mM on CC). Further, CsChiE displayed substantial activity towards the unmilled crab shell chitin waste (chitin-flakes) and generated chitobiose. Activity studies on chitooligosaccharides revealed that CsChiE produced chitobiose as the major product. Our results indicate that the multi-modular CsChiE is a non-processive exo-chitinase which is more suitable to generate chitobiose from a variety of chitinous substrates including unprocessed chitin-flakes.

Structural and Thermodynamic Signatures of Ligand Binding to the Enigmatic Chitinase D of Serratia proteamaculans.

The Gram-negative bacteria Serratia marcescens and Serratia proteamaculans have efficient chitinolytic machineries that degrade chitin into N-acetylglucosamine (GlcNAc), which is used as a carbon and energy source. The enzymatic degradation of chitin in these bacteria occurs through the synergistic action of glycoside hydrolases (GHs) that have complementary activities; an endo-acting GH (ChiC) making random scissions on the polysaccharide chains and two exo-acting GHs mainly targeting single reducing (ChiA) and nonreducing (ChiB) chain ends. Both bacteria produce low amounts of a fourth GH18 (ChiD) with an unclear role in chitin degradation. Here, we have determined the thermodynamic signatures for binding of (GlcNAc)6 and the inhibitor allosamidin to SpChiD as well as the crystal structure of SpChiD in complex with allosamidin. The binding free energies for the two ligands are similar (Δ Gr° = -8.9 ± 0.1 and -8.4 ± 0.1 kcal/mol, respectively) with clear enthalpic penalties (Δ Hr° = 3.2 ± 0.1 and 1.8 ± 0.1 kcal/mol, respectively). Binding of (GlcNAc)6 is dominated by solvation entropy change (- TΔ Ssolv° = -17.4 ± 0.4 kcal/mol) and the conformational entropy change dominates for allosamidin binding (- TΔ Sconf° = -9.0 ± 0.2 kcal/mol). These signatures as well as the interactions with allosamidin are very similar to those of SmChiB suggesting that both enzymes are nonreducing end-specific.

A carbohydrate binding module-5 is essential for oxidative cleavage of chitin by a multi-modular lytic polysaccharide monooxygenase from Bacillus thuringiensis serovar kurstaki.

Conversion of crystalline chitin to soluble sugar molecules, using lytic polysaccharide mono-oxygenases (LPMOs) has emerged as a new avenue for the production of biofuels. The present study describes the role of accessory domains in a multi-modular LPMO from Bacillus thuringiensis serovar kurstaki (BtLPMO10A). The full length BtLPMO10A (BtLPMO10A-FL) possesses an N-terminal LPMO of AA10 family (BtAA10) and a C-terminal CBM5 (BtCBM5) connected via two fibronectin (Fn) III domains (aligned as AA10-FnIII-FnIII-CBM5 from N- to C-terminus). To determine the role of individual domains, we generated truncation mutants of BtLPMO10A-FL. Substrate binding and kinetic studies revealed that BtCBM5 was involved in increasing binding efficiency of BtAA10 which otherwise has feeble binding towards ß-chitin and could not bind to α-chitin. Furthermore, binding assays also indicated that the presence of CBM5 increases the binding efficiency of BtLPMO10A-FL under extreme pH conditions. FnIII domains neither bind nor assist BtLPMO10A-FL in chitin binding and serve as linkers in BtLPMO10A-FL. BtLPMO10A-FL and BtAA10 generated oxidized chito-oligosaccharides from the insoluble ß-chitin substrate. It is concluded that BtCBM5 is responsible for increasing binding efficiency of BtLPMO10A-FL, whereas; BtAA10 domain is accountable for oxidative cleavage of recalcitrant chitin.

Key Residues Affecting Transglycosylation Activity in Family 18 Chitinases: Insights into Donor and Acceptor Subsites.

Understanding features that determine transglycosylation (TG) activity in glycoside hydrolases is important because it would allow the construction of enzymes that can catalyze controlled synthesis of oligosaccharides. To increase TG activity in two family 18 chitinases, chitinase D from Serratia proteamaculans ( SpChiD) and chitinase A from Serratia marcescens ( SmChiA), we have mutated residues important for stabilizing the reaction intermediate and substrate binding in both donor and acceptor sites. To help mutant design, the crystal structure of the inactive SpChiD-E153Q mutant in complex with chitobiose was determined. We identified three mutations with a beneficial effect on TG activity: Y28A (affecting the -1 subsite and the intermediate), Y222A (affecting the intermediate), and Y226W (affecting the +2 subsite). Furthermore, exchange of D151, the middle residue in the catalytically important DXDXE motif, to asparagine reduced hydrolytic activity ≤99% with a concomitant increase in apparent TG activity. The combination of mutations yielded even higher degrees of TG activity. Reactions with the best mutant, SpChiD-D151N/Y226W/Y222A, led to rapid accumulation of high levels of TG products that remained stable over time. Importantly, the introduction of analogous mutations at the same positions in SmChiA (Y163A equal to Y28A and Y390F similar to Y222A) had similar effects on TG efficiency. Thus, the combination of the decreasing hydrolytic power, subsite affinity, and stability of intermediate states provides a powerful, general strategy for creating hypertransglycosylating mutants of retaining glycoside hydrolases.

Active-site mutations improved the transglycosylation activity of Stenotrophomonas maltophilia chitinase A.

Transglycosylation (TG) by family 18 chitinases is of special interest due to the many biological applications of long-chain chitooligosaccharides (CHOS). In the current study, the TG activity of chitinase A from Stenotrophomonas maltophilia (StmChiA) was improved through structure-guided mutations within and around the active site. Three independent mutants were created, targeting Trp residues from the -3 and -1 subsites and the central catalytic Asp from the DxDxE motif of StmChiA. The former was replaced with Ala and the latter with Asn. Changes in the hydrolytic and TG activities of the enzymes were assessed by monitoring the product profile of each mutant by high-performance liquid chromatography. All three mutants showed increased TG activity. Increased in the higher TG activity of mutant W306A was accompanied by increased hydrolysis. However, this mutant also accumulated substantial amounts of TG products during the first 15-30min of the reaction. In contrast, mutants D464N and W679A showed reduced hydrolysis, which was accompanied by the gradual accumulation of TG products up to 12h. Molecular docking studies with chitohexaose showed that the side chains of Trp residues mediate stacking interactions with sugar residues from the -3 and -1 subsites, indicating the importance of these residues in the enzymatic activity of StmChiA. Overall, mutants of the glycon-binding site (W306A and W679A) appear to produce long-chain CHOS more efficiently than the catalytic mutant D464N.

A new chitinase-D from a plant growth promoting Serratia marcescens GPS5 for enzymatic conversion of chitin.

The current study describes heterologous expression and biochemical characterization of single-modular chitinase-D from Serratia marcescens (SmChiD) with unprecedented catalytic properties which include chitobiase and transglycosylation (TG) activities besides hydrolytic activity. Without accessory domains, SmChiD, hydrolyzed insoluble polymeric chitin substrates like colloidal, α- and ß-chitin. Activity studies on CHOS with degree of polymerization (DP) 2-6 as substrate revealed that SmChiD hydrolyzed DP2 with a chitobiase activity and showed TG activity on CHOS with DP3-6, producing longer chain CHOS. But, the TG products were further hydrolyzed to shorter chain CHOS with DP1-2 products. SmChiD with its unique catalytic properties, could be a potential enzyme for the production of long chain CHOS and also for the preparation of efficient enzyme cocktails for chitin degradation.

Production of bioactive chitosan oligosaccharides using the hypertransglycosylating chitinase-D from Serratia proteamaculans.

The biological activities of chitosan and its oligosaccharides are greatly influenced by properties such as the degree of polymerization (DP), degree of acetylation (DA) and pattern of acetylation (PA). Here, structurally diverse chitosan oligosaccharides from chitosan polymers (DA=35% or 61%) were generated using Serratia proteamaculans wild-type chitinase D (SpChiD) and the W114A mutant which lacks transglycosylase activity. The crude oligosaccharide mixtures and purified fractions with specific DP and DA ranges were tested for their ability to induce an oxidative burst in rice cell suspension cultures. The crude mixtures were more active when produced by the W114A mutant whereas the purified fractions were more active when produced by wild-type SpChiD. Neither hydrolysis nor transglycosylation by SpChiD was inhibited in the presence of fully-deacetylated oligosaccharides, suggesting that SpChiD could be exploited to generate oligosaccharides with defined DA and PA values.

Inverse relationship between chitobiase and transglycosylation activities of chitinase-D from Serratia proteamaculans revealed by mutational and biophysical analyses.

Serratia proteamaculans chitinase-D (SpChiD) has a unique combination of hydrolytic and transglycosylation (TG) activities. The TG activity of SpChiD can be used for large-scale production of chito-oligosaccharides (CHOS). The multiple activities (hydrolytic and/or chitobiase activities and TG) of SpChiD appear to be strongly influenced by the substrate-binding cleft. Here, we report the unique property of SpChiD substrate-binding cleft, wherein, the residues Tyr28, Val35 and Thr36 control chitobiase activity and the residues Trp160 and Trp290 are crucial for TG activity. Mutants with reduced (V35G and T36G/F) or no (SpChiDΔ30-42 and Y28A) chitobiase activity produced higher amounts of the quantifiable even-chain TG product with degree of polymerization (DP)-6, indicating that the chitobiase and TG activities are inversely related. In addition to its unprecedented catalytic properties, unlike other chitinases, the single modular SpChiD showed dual unfolding transitions. Ligand-induced thermal stability studies with the catalytically inactive mutant of SpChiD (E153A) showed that the transition temperature increased upon binding of CHOS with DP2-6. Isothermal titration calorimetry experiments revealed the exceptionally high binding affinities for E153A to CHOS with DP2-6. These observations strongly support that the architecture of SpChiD substrate-binding cleft adopted to control chitobiase and TG activities, in addition to usual chitinase-mediated hydrolysis.

Catalytic efficiency of chitinase-D on insoluble chitinous substrates was improved by fusing auxiliary domains.

Chitin is an abundant renewable polysaccharide, next only to cellulose. Chitinases are important for effective utilization of this biopolymer. Chitinase D from Serratia proteamaculans (SpChiD) is a single domain chitinase with both hydrolytic and transglycosylation (TG) activities. SpChiD had less of hydrolytic activity on insoluble polymeric chitin substrates due to the absence of auxiliary binding domains. We improved catalytic efficiency of SpChiD in degradation of insoluble chitin substrates by fusing with auxiliary domains like polycystic kidney disease (PKD) domain and chitin binding protein 21 (CBP21). Of the six different SpChiD fusion chimeras, two C-terminal fusions viz. ChiD+PKD and ChiD+CBP resulted in improved hydrolytic activity on α- and ß-chitin, respectively. Time-course degradation of colloidal chitin also confirmed that these two C-terminal SpChiD fusion chimeras were more active than other chimeras. More TG products were produced for a longer duration by the fusion chimeras ChiD+PKD and PKD+ChiD+CBP.

Biotechnological approaches for field applications of chitooligosaccharides (COS) to induce innate immunity in plants.

Plants have evolved mechanisms to recognize a wide range of pathogen-derived molecules and to express induced resistance against pathogen attack. Exploitation of induced resistance, by application of novel bioactive elicitors, is an attractive alternative for crop protection. Chitooligosaccharide (COS) elicitors, released during plant fungal interactions, induce plant defenses upon recognition. Detailed analyses of structure/function relationships of bioactive chitosans as well as recent progress towards understanding the mechanism of COS sensing in plants through the identification and characterization of their cognate receptors have generated fresh impetus for approaches that would induce innate immunity in plants. These progresses combined with the application of chitin/chitosan/COS in disease management are reviewed here. In considering the field application of COS, however, efficient and large-scale production of desired COS is a challenging task. The available methods, including chemical or enzymatic hydrolysis and chemical or biotechnological synthesis to produce COS, are also reviewed.

Mutagenesis and molecular dynamics simulations revealed the chitooligosaccharide entry and exit points for chitinase D from Serratia proteamaculans.

BACKGROUND: Transglycosylation (TG) activity is a property of glycosyl hydrolases (GHs) with which new glycosidic bonds are introduced between donor and acceptor sugar molecules. This special property of the GHs has potential to generate longer chain chitooligosaccharides (CHOS) that show elicitor activity in plants. We hypothesize that TG activity could be improved by retaining the substrate for a longer duration in the catalytic site. METHODS: Four variants of chitinase D from Serratia proteamaculans (SpChiD) i.e. G119S, G119W, W120A and G201W were analyzed in detail for improved TG activity using high performance liquid chromatography (HPLC) and high resolution mass spectrometry (HRMS). The results were strongly supported by 50ns molecular dynamics (MD) simulations and estimated solvated interaction energies (SIE). RESULTS: The mutant G119W lost much of both hydrolytic and TG activities, while the mutant G201W displayed increased TG. The trajectory of MD simulations of the mutant G119W showed that the indole rings of two adjacent Trp residues create a major hindrance for the DP4 movement towards the catalytic center. Increased van der Waals (vdW) and coulombic interactions between DP4 substrate and the Trp-201 resulted in enhanced TG activity with the mutant G201W. The average number of hydrogen bonds observed for the DP4 substrate was increased for the mutants G119W and G201W compared to SpChiD. CONCLUSION: The increase in TG activity could be due to partial blocking of product exit of SpChiD. GENERAL SIGNIFICANCE: This new approach can be used for generating mutants of GHs with improved TG activity to produce longer chain oligosaccharides.