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

Structure of chitinase D from Serratia proteamaculans reveals the structural basis of its dual action of hydrolysis and transglycosylation

Chitinases are known to hydrolyze chitin polymers into smaller chitooligosaccharides. Chitinase from bacterium Serratia proteamaculans (SpChiD) is found to exhibit both hydrolysis and transglycosylation activities. SpChiD belongs to family 18 of glycosyl hydrolases (GH-18). The recombinant SpChiD was crystallized and its three-dimensional structure was determined at 1.49 Å resolution. The structure was refined to an R-factor of 16.2%. SpChiD consists of 406 amino acid residues. The polypeptide chain of SpChiD adopts a (β/α)8 triosephosphate isomerase (TIM) barrel structure. SpChiD contains three acidic residues, Asp149, Asp151 and Glu153 as part of its catalytic scheme. While both Asp149 and Glu153 adopt single conformations, Asp151 is observed in two conformations. The substrate binding cleft is partially obstructed by a protruding loop, Asn30 - Asp42 causing a considerable reduction in the number of available subsites in the substrate binding site. The positioning of loop, Asn30 - Asp42 appears to be responsible for the transglycosylation activity. The structure determination indicated the presence of sulfone Met89 (SMet89). The sulfone methionine residue is located on the surface of the protein at a site where extra domain is attached in other chitinases. This is the first structure of a single domain chitinase with hydrolytic and transglycosylation activities

Improving efficiency and sustainability of chitin bioconversion through a combination of Streptomyces secretomes and mechanical-milling

Chitin, particularly α-chitin, is the most abundant and highly recalcitrant form, fortified by an intricate network of hydrogen bonds. Efficient valorization of α-chitin requires a mild pre-treatment and enzymatic hydrolysis. Streptomyces spp. secrete chitin-active CAZymes that can efficiently tackle the recalcitrant problem of chitin biomass. To better understand the potential of Streptomyces spp., a comparative analysis was performed between the novel isolate, Streptomyces sp. UH6 and the well-known chitin degraders, S. coelicolor and S. griseus. Growth studies and FE-SEM analysis revealed that all three Streptomyces spp. could utilize and degrade both α- and β-chitin. Zymogram analysis showed expression of 5-7 chitinases in the secretomes of Streptomyces strains. The chitin-active-secretomes produced by Streptomyces sp. UH6 and S. griseus were optimally active at acidic pH (pH 4.0 and 5.0) and 50°C. Time-course degradation of α- and β-chitin with the secretomes generated N-acetyl-D-glucosamine (GlcNAc) and N,N-diacetylchitobiose [(GlcNAc)2] as the predominant products. Further, the highly crystalline α-chitin was subjected to pre-treatment by ball-milling, which reduced the crystallinity from 88% to 56.6% and increased the BET surface area by 3-folds. Of note, the activity of all three Streptomyces secretomes was improved by a mild pre-treatment, while Streptomyces sp. UH6 secretome displayed improved GlcNAc and (GlcNAc)2 yields by 14.4 and 9.6-folds, respectively. Overall, our results suggest that the Streptomyces chitin-active-secretomes, particularly Streptomyces sp. UH6, can be deployed for efficient valorization of chitin biomass and to establish an economically feasible and eco-friendly process for valorizing highly recalcitrant α-chitin

Thermodynamic insights into the role of aromatic residues in chitooligosaccharide binding to the transglycosylating chitinase-D from Serratia proteamaculans

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The first evidence of cryo-milling improving enzymatic production of chitooligosaccharides from chitin biomass

Cryo-milling offers an energy-efficient approach to process α-chitin, the most prevalent and well-structured chitin form. In contrast to ball-milling, cryo-milling operates at low temperatures (<123 K), preventing heat-related charring and chitooligosaccharide loss. This study explored various cryo-milling conditions\u27 impact on α-chitin, resulting in reduced crystalline plane intensity, increased surface area and smaller particle size without substrate breakdown or functional group loss. Additionally, the study investigated cryo-milled α-chitin\u27s enzymatic hydrolysis for the first time. Using Paenibacillus sp. LS1\u27s chitin-active-secretome, cryo-milled α-chitin exhibited a twofold increase in GlcNAc yield compared to unmilled α-chitin. Hydrolysis with recombinant Chi5 from Paenibacillus sp. LS1 resulted in twofold and fourfold increments in (GlcNAc)2 and GlcNAc yield, respectively. Overall, cryo-milling emerges as a gentle and effective alternative to ball-milling. It reduces α-chitin crystallinity and particle size, and increases the available surface area for enhanced enzymatic saccharification, thereby facilitating chitooligosaccharide production

Thermodynamic insights into the role of aromatic residues in chitooligosaccharide binding to the transglycosylating chitinase-D from Serratia proteamaculans

publishedVersio

Transglycosylation by chitinase D from Serratia proteamaculans improved through altered substrate interactions

We describe improvement of the transglycosylation (TG) by chitinase D from Serratia proteamaculans (SpChiD). The SpChiD produced less proportion of TG products up to 90 min, with 2 mM chitotetraose as the substrate and hydrolysed the products subsequently. Of the five residues targeted at the catalytic center, E159D resulted in substantial loss of both hydrolytic and TG activities. Y160A resulted in a product profile similar to SpChiD and a rapid turnover of substrate with slightly increased TG activity. Rest of the three mutants, M226A, Y228A and R284A displayed improved TG and decreased hydrolytic ability. Four of the five amino acid substitutions, F64W, F125A, G119S and S116G, at the catalytic groove increased TG activity, while W120A completely lost the TG activity with a concomitant increase in hydrolysis. Mutation of W247 at the solvent accessible region significantly reduced the hydrolytic activity with increased TG activity. The mutants M226A, Y228A, F125A, S116G, F64W, G119S, R284, and W247A accumulated approximately double the concentration of TG products like chitopentaose and chitohexaose, compared to SpChiD, respectively. The double mutant E159D/F64W regained the activity with accumulation of 6.0% of chitopentaose at 6 h, similar to SpChiD at 30 min. Loss of chitobiase activity was unique to Y228A. Substitution of amino acids at catalytic center and/or groove substantially improved the TG activity of SpChiD, both in terms of quantity of TG products produced and the extended duration of TG activity

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 50 ns 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

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