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Albumin binding is the major cause for the toxicity of protein bound uremic toxins (PBUTs) in uremic patients. Albumin binding property is exploited to address this issue, as some of the extracorporeal dialysis systems use albumin as dialysate. In this line, a detailed study about binding of PBUTs to human serum albumin (HSA) and its domains gives valuable information. The focus of this work emphasizes the mechanism of binding of HSA and its domains with a few selected PBUTs such as hippuric acid (HA), indole acetic acid (IAA) and melatonin. The HSA domains (D2, D3 and D2–3) were expressed in Pichia pastoris and purified by using Albupure matrix. The binding of the expressed domains and HSA, with PBUTs, was measured using surface plasmon resonance and analyzed. All the three domains have significant affinity towards PBUTs, while D3 had greater affinity for all the three selected PBUTs. Docking studies showed that the basic amino acid, lysine, was forming hydrogen bond with PUBTs inorder to stabile these complex. This study would be having therapeutic importance for preparing the extracorporeal dialysis systems, in combination of different domains of HSA to remove the PBUTs.

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.

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.

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.

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 kcat/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.

Plant growth promoting rhizomicroorganisms (PGPR) play an important role in improving plant growth, nutrition and yield of different crops. Chilly is one of the major commercial crops of India with large export potential. The first experiment was conducted with 10 PGPR to investigate their effects on growth and yield of chilly. Although most of the plant parameters studied were statistically not significant, 2 PGPR viz. Paenibacillus polymyxa and Pantoea dispersa showed agronomic improvement in plant growth as compared to control and other treatments. The second experiment was conducted with these 2 PGPR plus 8 more PGPR in order to select the best PGPR for inoculating chilly. Inoculation significantly improved the growth, nutrition and fruit yield as compared to uninoculated control. Considering plant dry biomass and fruit yield, Methylobacterium radiotolerans proved to be the best PGPR. Further screening with M. radiotolerans plus 2 more PGPR viz. Bacillus sonorensis and Paenibacillus elgii on 2 common varieties of chilly resulted in enhanced plant dry biomass, nutrition and fruit yield. The results clearly brought out that B. sonorensis is the most promising PGPR inoculant for chilly. The plant growth promoting traits revealed that B. sonorensis is a P-solubilizer and able to produce indole acetic acid, siderophore, chitinase, hydrogen cyanide and good in biofilm formation.

Harpin

A total of 74 morphologically distinct bacterial colonies were selected during isolation of bacteria from different parts of tomato plant (rhizoplane, phylloplane and rhizosphere) as well as nearby bulk soil. The isolates were screened for plant growth promoting (PGP) traits such as production of indole acetic acid, siderophore, chitinase and hydrogen cyanide as well as phosphate solubilization. Seven isolates viz., NR4, NR6, RP3, PP1, RS4, RP6 and NR1 that exhibited multiple PGP traits were identified, based on morphological, biochemical and 16S rRNA gene sequence analysis, as species that belonged to four genera Aeromonas, Pseudomonas, Bacillus and Enterobacter. All the seven isolates were positive for 1-aminocyclopropane-1-carboxylate deaminase. Isolate NR6 was antagonistic to Fusarium solani and Fusarium moniliforme, and both PP1 and RP6 isolates were antagonistic to F. moniliforme. Except RP6, all isolates adhered significantly to glass surface suggestive of biofilm formation. Seed bacterization of tomato, groundnut, sorghum and chickpea with the seven bacterial isolates resulted in varied growth response in laboratory assay on half strength Murashige and Skoog medium. Most of the tomato isolates positively influenced tomato growth. The growth response was either neutral or negative with groundnut, sorghum and chickpea. Overall, the results suggested that bacteria with PGP traits do not positively influence the growth of all plants, and certain PGP bacteria may exhibit host-specificity. Among the isolates that positively influenced growth of tomato (NR1, RP3, PP1, RS4 and RP6) only RS4 was isolated from tomato rhizosphere. Therefore, the best PGP bacteria can also be isolated from zones other than rhizosphere or rhizoplane of a plant.