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.

HarpinPss encapsulation in chitosan nanoparticles for improved bioavailability and disease resistance in tomato.

HarpinPss, an elicitor from Pseudomonas syringae pv. syringae, induces systemic acquired resistance in non-host plants, providing resistance to phytopathogens. Poor assimilation of harpinPss is a major constraint in foliar application as biopesticide. We, therefore, prepared harpinPss-loaded chitosan nanoparticles (H-CSNPs) to improve permeability and bio-availability of harpinPss in tomato. H-CSNPs showed high encapsulation efficiency (90%), improved stability (p < 0.01) and bioavailability of harpinPss (p < 0.01). Treatment with H-CSNPs resulted in sustained induction of peroxidase, phenylalanine ammonia lyase and decreased Rhizoctonia solani infection (p < 0.05). Transcripts of several genes involved in defense response were differentially expressed in harpinPss, CSNPs and H-CSNPs treatments. While, genes involved in jasmonic acid (JA) metabolism were up-regulated during harpinPss and H-CSNP spray treatments, indicating the role of JA pathway in triggering harpin-mediated defense responses. Furthermore, the entry of CSNPs into the cell and localization of harpinPss into chloroplast was tracked using rhodamine-labelled CSNPs encapsulated with GFP tagged harpinPss. The results of this study indicate use of H-CSNPs is effective for sustained-release of harpinPss and provides resistance for prolonged duration.

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.