Deciphering the thermotolerance of chitinase O from Chitiniphilus shinanonensis by in vitro and in silico studies.

Biochemical and biophysical studies revealed that chitinase O from Chitiniphilus shinanonensis (CsChiO) exhibits considerable thermotolerance, possibly due to the formation of a stable structural conformation. CsChiO is an exochitinase with a temperature optimum of 70 °C. The secondary structures of CsChiO and its catalytic domain (Cat-CsChiO) are only marginally affected upon heating up to 90 °C, as revealed by circular dichroism (CD) spectroscopy. Differential scanning calorimetric (DSC) studies revealed that CsChiO exhibits two endothermic transitions at ca. 51 °C (Tm1) and 59 °C (Tm2), whereas Cat-CsChiO shows a single endothermic transition at 52 °C. Together, the CD and DSC analyses suggested that the catalytic domain of CsChiO undergoes a thermotropic transition at ~52 °C from native state to another stable structural conformation. Results from molecular dynamic simulations corroborated that Cat-CsChiO adopts a stable structural conformation above 50 °C by partial unfolding. Thermotolerant CsChiO would be useful for the conversion of chitin, which is highly abundant, to biologically active COS. This study unveiled the adaptability of enzymes/proteins in nature to perform biological functions at elevated temperatures.

Elicitation of defense response by transglycosylated chitooligosaccharides in rice seedlings.

Long-chain chitooligosaccharides (COS) with degree of polymerization (DP) more than 4 are known to have potential biological activities. A hyper-transglycosylating mutant of an endo-chitinase from Serratia proteamaculans (SpChiD-Y28A) was used to synthesize COS with DP6 and DP7 using COS DP5 as substrate. Purified COS with DP5-7 were tested to elicit the defense response in rice seedlings. Among the COS used, DP7 strongly induced oxidative burst response as well as peroxidase, and phenylalanine ammonia lyase activites. A few selected marker genes in salicylic acid (SA)- and jasmonic acid-dependent pathways were evaluated by real-time PCR. The expression levels of pathogenesis-related (PR) genes PR1a and PR10 and defense response genes (chitinase1, peroxidase and ß -1,3-glucanase) were up regulated upon COS treatment in rice seedlings. The DP7 induced Phenylalanine ammonia lyase and Isochorismate synthase 1 genes, with concomitant increase of Mitogen-activated protein kinase 6 and WRKY45 transcription factor genes indicated the possible role of phosphorylation in the transmission of a signal to induce SA-mediated defense response in rice.

Midgut aminopeptidase N expression profile in castor semilooper (Achaea janata) during sublethal Cry toxin exposure.

The midgut of lepidopteran larvae is a multifunctional tissue that performs roles in digestion, absorption, immunity, transmission of pathogens and interaction with ingested various molecules. The proteins localized at the inner apical brush border membrane are primarily digestive proteases, but some of them, like aminopeptidase N, alkaline phosphatase, cadherins, ABC transporter C2, etc., interact with Crystal (Cry) toxins produced by Bacillus thuringiensis (Bt). In the present study, aminopeptidase N (APN) was characterized as Cry-toxin-interacting protein in the larval midgut of castor semilooper, Achaea janata. Transcriptomic and proteomic analyses revealed the presence of multiple isoforms of APNs (APN1, 2, 4, 6 and 9) which have less than 40% sequence similarity but show the presence of characteristic 'GAMENEG' and zinc-binding motifs. Feeding a sublethal dose of Cry toxin caused differential expression of various APN isoform. Further, 6thgeneration Cry-toxin-exposed larvae showed reduced expression of APN2. This report suggests that A. janata larvae exploit altered expression of APNs to overcome the deleterious effects of Cry toxicity, which might facilitate toxin tolerance in the long run.

New Class of Chitosanase from Bacillus amyloliquefaciens for the Generation of Chitooligosaccharides.

Chitooligosaccharides (COS) generated from either chitin (chitin oligosaccharides) or chitosan (chitosan oligosaccharides) have a wide range of applications in agriculture, medicine, and other fields. Here, we report the characterization of a chitosanase from Bacillus amyloliquefaciens (BamCsn) and the importance of a tryptophan (Trp), W204, for BamCsn activity. BamCsn hydrolyzed the chitosan polymer by an endo mode. It also hydrolyzed chitin oligosaccharides and interestingly exhibited transglycosylation activity on chitotetraose and chitopentaose. Mutation of W204, a nonconserved amino acid in chitosanases, to W204A abolished the hydrolytic activity of BamCsn, with a change in the structure that resulted in a decreased affinity for the substrate and impaired the catalytic ability. Phylogenetic analysis revealed that BamCsn could belong to a new class of chitosanases that showed unique properties like transglycosylation, cleavage of chitin oligosaccharides, and the presence of W204 residues, which is important for activity. Chitosanases belonging to the BamCsn class showed a high potential to generate COS from chitinous substrates.

Selection and mutational analyses of the substrate interacting residues of a chitinase from Enterobacter cloacae subsp. cloacae (EcChi2) to improve transglycosylation.

Transglycosylation (TG) by Enterobacter cloacae subsp. cloacae chitinase 2 (EcChi2) has been deciphered by site-directed mutagenesis. EcChi2 originally displayed feeble TG with chitin oligomer with a degree of polymerization (DP4), for a short duration. Based on the 3D modelling and molecular docking analyses, we altered the substrate interactions at the substrate-binding cleft, catalytic center, and catalytic groove of EcChi2 by mutational approach to improve TG. The mutation of W166A and T277A increased TG by EcChi2 and also affected its catalytic efficiency on the polymeric substrates. Whereas, R171A had a drastically decreased hydrolytic activity but, retained TG activity. In the increased hydrolytic activity of the T277A, altered interactions with the substrates played an indirect role in the catalysis. Mutation of the central Asp, in the conserved DxDxE motif, to Ala (D314A) and Asn (D314N) conversion yielded DP5-DP8 TG products. The quantifiable TG products (DP5 and DP6) increased to 8% (D314A) and 7% (D314N), resulting in a hyper-transglycosylating mutant. Mutation of W276A and W398A resulted in the loss of TG activity, indicating that the aromatic residues (W276 and W398) at +1 and +2 subsites are essential for the TG activity of EcChi2.

Efficient conversion of α-chitin by multi-modular chitinase from Chitiniphilus shinanonensis with KOH and KOH-urea pretreatment.

Enzymatic conversion of α-chitin to high-value chitooligosaccharides (COS) was up to 7.2 % by a slow-acting endo-chitinase (uni-modular) after KOH or KOH-urea pretreatment. Here, we report a better source for efficient conversion of α-chitin, with KOH/KOH-urea (20K2 or 20KU2) pretreatment, by a multi-modular chitinase (CsChiG) from Chitiniphilus shinanonensis. The CsChiG and its catalytic domain (Cat-CsChiG) converted 20KU2 substrate to soluble COS with an efficiency of 43.1 % and 11.8 %, respectively. Deletion of the chitin binding domain has reduced the conversion of untreated and colloidal chitin substrates by 4-5 folds, and for 20K2 and 20KU2 substrates it was only two folds decrease. A combination of KOH or KOH-urea pretreatment, followed by enzymatic hydrolysis with multi-modular chitinases, thus appears a promising approach to convert the abundantly available chitin to highly useful COS.

Pretreatment with KOH and KOH-urea enhanced hydrolysis of α-chitin by an endo-chitinase from Enterobacter cloacae subsp. cloacae.

Chitin is the second most abundant and renewable polysaccharide, next to cellulose. Hydrolysis of abundant and highly crystalline α-chitin, pretreated with KOH and KOH-urea aqueous solutions, by a single modular endo-chitinase from Enterobacter cloacae subsp. cloacae (EcChi1) was investigated. The hydrolysis of untreated α-chitin and colloidal chitin by EcChi1 produced N-acetylglucosamine and N, N'-diacetylchitobiose, whereas, hydrolysis of treated substrates generated N, N', N''-triacetylchitotriose, in addition to N-acetylglucosamine and N, N'-diacetylchitobiose. The total amount of chitooligosaccharides (COS) generated by EcChi1 from pretreated substrates was 10 to 25-fold higher compared to untreated α-chitin at 24 h (depending on the solvent type and state of substrate). EcChi1 released higher amount of DP1 and DP2 products on treated α-chitin, with a fold change of 45 and 18, respectively. Treatment of α-chitin with KOH/KOH-urea is, therefore, a promising approach for an efficient conversion of rich source of chitin to soluble COS by chitinases like EcChi1.

A transglycosylating chitinase from Chitiniphilus shinanonensis (CsChiL) hydrolyzes chitin in a processive manner.

Chitin, mostly extracted from shrimp waste, is the second most abundant biopolysaccharide, next only to cellulose. Enzymatic conversion of chitin into useful bioactive molecules such as chitooligosaccharides (COS) has potential biotechnological applications. The current study describes the characterization of a single modular GH18 chitinase from Chitiniphilus shinanonensis (CsChiL). CsChiL was optimally active at 50 °C in sodium citrate buffer, pH 6.0 and active over a broad pH range (6-10). In addition to hydrolysis, CsChiL displayed chitobiase and transglycosylation activities on COS with degree of polymerization (DP) 2 and 4-6, respectively. CsChiL hydrolyzed chitin polymers (α, ß, and colloidal chitin) in a processive manner. Molecular dynamics simulations and residue-wise binding energy contributions provided structural insights and molecular basis of inherent transglycosylation activity by CsChiL. Overall, CsChiL could be useful in generation of COS from the chitin obtained from shrimp waste with potential applications in agriculture and food industries.

Applicability of endochitinase of Flavobacterium johnsoniae with transglycosylation activity in generating long-chain chitooligosaccharides.

Chitin and its derivatives are used for a variety of applications. Flavobacterium johnsoniae UW101 is an aerobic Gram-negative bacterium. Genome analysis of F. johnsoniae UW101 revealed the presence of 10 glycoside hydrolases (GHs) that may degrade or modify chitin. The gene encoding chitinase B (FjchiB), which encodes a single catalytic GH18 domain has been cloned and heterologously expressed in Escherichia coli. FjChiB was optimally active in 50 mM sodium citrate buffer (pH 6.0) at 40 °C. FjChiB was salt-tolerant and catalytically versatile, with substrate specificity towards 75% DDA (degree of de-acetylation) chitosan, followed by colloidal chitin. Chitotetraose (DP4) was the shortest of the oligomeric substrates used by FjChiB. The Km and Vmax values of FjChiB for colloidal chitin were 49.38 mg/ml and 11.2 nanokat mg-1, respectively. The overall catalytic efficiency (kcat/Km) of FjChiB was 1.40 × 103 mg-1 ml s-1. FjChiB exhibited transglycosylation (TG) with chitopentaose (DP5) and chitohexaose (DP6) substrates. The TG by FjChiB was fine-tuned by introducing a tryptophan (G106W) and asparagine (D148N) in the highly conserved catalytic groove and catalytic center, respectively. Hydrolytic products profile and homology modelling indicated that FjChiB is an endochitinase that holds promise for the conversion of chitin into useful products through both TG and/or hydrolysis.

Carboxy-terminal glycosyl hydrolase 18 domain of a carbohydrate active protein of Chitinophaga pinensis is a non-processive exochitinase.

The recombinant C-terminal domain of chitinase C of Chitinophaga pinensis (CpChiC-GH18C) exhibits the highest activity at pH 6.0 and 35 °C, with a Km of 76.13 (mg-1 ml), a kcat of 10.16 (s-1), and a kcat/Km of 0.133 (mg-1 ml s-1) on colloidal chitin. Analysis of degradation of (GlcNAc)3-6 oligomers shows that CpChiC-GH18C releases (GlcNAc)2 as the main product, indicating an exo-type cleavage pattern. CpChiC-GH18C hydrolyzes the chitin polymers yielding GlcNAc, (GlcNAc)2, and (GlcNAc)3 as end products with no sign of processivity. Circular dichroism spectra indicate that the secondary and tertiary structures of CpChiC-GH18C are unaltered up to 45 °C and the protein denatures without an intermediate state. The urea-induced unfolding is a two-state process and the unfolding of native CpChiC-GH18C occurs in a single step. Among the metal ions tested, Hg2+ completely inhibits the enzyme activity. The chemical modulators, p-hydroxymercuribenzoic acid and N-bromosuccinimide considerably decrease the enzyme activity. Sequence analysis and homology modeling suggest that CpChiC-GH18C lacks a tryptophan residue at the aglycon site. Further, the CpChiC-GH18C has a shallow and open groove, suggesting that CpChiC-GH18C is non-processive exo-type chitinase with properties suitable for the bioconversion of chitin waste.

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.

Transglycosylation by a chitinase from Enterobacter cloacae subsp. cloacae generates longer chitin oligosaccharides.

Humans have exploited natural resources for a variety of applications. Chitin and its derivative chitin oligosaccharides (CHOS) have potential biomedical and agricultural applications. Availability of CHOS with the desired length has been a major limitation in the optimum use of such natural resources. Here, we report a single domain hyper-transglycosylating chitinase, which generates longer CHOS, from Enterobacter cloacae subsp. cloacae 13047 (EcChi1). EcChi1 was optimally active at pH 5.0 and 40 °C with a Km of 15.2 mg ml-1, and k cat/Km of 0.011× 102 mg-1 ml min-1 on colloidal chitin. The profile of the hydrolytic products, major product being chitobiose, released from CHOS indicated that EcChi1 was an endo-acting enzyme. Transglycosylation (TG) by EcChi1 on trimeric to hexameric CHOS resulted in the formation of longer CHOS for a prolonged duration. EcChi1 showed both chitobiase and TG activities, in addition to hydrolytic activity. The TG by EcChi1 was dependent, to some extent, on the length of the CHOS substrate and concentration of the enzyme. Homology modeling and docking with CHOS suggested that EcChi1 has a deep substrate-binding groove lined with aromatic amino acids, which is a characteristic feature of a processive enzyme.

Amino Groups of Chitosan Are Crucial for Binding to a Family 32 Carbohydrate Binding Module of a Chitosanase from Paenibacillus elgii.

We report here the role and mechanism of specificity of a family 32 carbohydrate binding module (CBM32) of a glycoside hydrolase family 8 chitosanase from Paenibacillus elgii (PeCsn). Both the activity and mode of action of PeCsn toward soluble chitosan polymers were not different with/without the CBM32 domain of P. elgii (PeCBM32). The decreased activity of PeCsn without PeCBM32 on chitosan powder suggested that PeCBM32 increases the relative concentration of enzyme on the substrate and thereby enhanced enzymatic activity. PeCBM32 specifically bound to polymeric and oligomeric chitosan and showed very weak binding to chitin and cellulose. In isothermal titration calorimetry, the binding stoichiometry of 2 and 1 for glucosamine monosaccharide (GlcN) and disaccharide (GlcN)2, respectively, was indicative of two binding sites in PeCBM32. A three-dimensional model-guided site-directed mutagenesis and the use of defined disaccharides varying in the pattern of acetylation suggested that the amino groups of chitosan and the polar residues Glu-16 and Glu-38 of PeCBM32 play a crucial role for the observed binding. The specificity of CBM32 has been further elucidated by a generated fusion protein PeCBM32-eGFP that binds to the chitosan exposing endophytic infection structures of Puccinia graminis f. sp. tritici Phylogenetic analysis showed that CBM32s appended to chitosanases are highly conserved across different chitosanase families suggesting their role in chitosan recognition and degradation. We have identified and characterized a chitosan-specific CBM32 useful for in situ staining of chitosans in the fungal cell wall during plant-fungus interaction.