Chitinase

To investigate the expression patterns of chitinase on SDS-PAGE gel, Paenibacillus ehimensis MA2012 was incubated in gelatin-chitin medium (GCM) at 30 °C for 7 days. Six major bands (Ch3, Ch4, Ch5, Ch6, Ch7, and Ch8) of chitinase isozymes in GC medium appeared on SDS-PAGE gel during the incubation period. Chitinase activity staining of P. ehimensis MA2012 was detected on 2-DE with different pI values (4-11). After DEAE-Sephadex chromatography, eight bands (Ch1 to Ch8) of chitinase isozymes were stained strongly with Calcofluor white M2R at fraction 45. After Sephadex G-75 gel filtration, six bands (Ch3 to Ch8) of chitinase isozymes were stained with Calcofluor white M2R at fractions of 11 to 12. The specific activity of the purified chitinase was 3.8 units mg-1 protein with a purification factor of 0.27. Inhibition rate of the conidial germination of Colletotrichum gloeosporioides was 87% in partial purified chitinase treatment compared with control.

Chitinases are widely distributed in nature and have wide-ranging pharmaceutical and biotechnological applications. This work highlights a real-time and label-free method to assay Chitinase activity via a quartz crystal microbalance with dissipation monitoring (QCM-D) and atomic force microscopy (AFM). The chitin substrate was prepared by spincoating a trimethylsilyl chitin solution onto a silica substrate, followed by regeneration to amorphous chitin (RChi). The QCM-D and AFM results clearly showed that the hydrolysis rate of RChi films increased as Chitinase (from Streptomyces griseus) concentrations increased, and the optimal temperature and pH for Chitinase activity were around 37 °C and 6-8, respectively. The Chitinase showed greater activity on chitin substrates, having a high degree of acetylation, than on chitosan substrates, having a low degree of acetylation.

Pseudomonas protegens strain Pf-5 is a soil bacterium that was first described for its capacity to suppress plant diseases and has since been shown to be lethal to certain insects. Among these is the common fruit fly Drosophila melanogaster, a well-established model organism for studies evaluating the molecular and cellular basis of the immune response to bacterial challenge. Pf-5 produces the insect toxin FitD, but a ΔfitD mutant of Pf-5 retained full toxicity against D. melanogaster in a non-invasive feeding assay, indicating that FitD is not a major determinant of Pf-5's oral toxicity against this insect. Pf-5 also produces a broad spectrum of exoenzymes and natural products with antibiotic activity, whereas a mutant with a deletion in the global regulatory gene gacA produces none of these exoproducts and also lacks toxicity to D. melanogaster. In this study, we made use of a panel of Pf-5 mutants having single or multiple mutations in the biosynthetic gene clusters for seven natural products and two exoenzymes that are produced by the bacterium under the control of gacA.

Multi-auto-transformation vector system has been one of the strategies to produce marker-free transgenic plants without using selective chemicals and plant growth regulators and thus facilitating transgene stacking. In the study reported here, retransformation was carried out in marker-free transgenic potato CV. May Queen containing ChiC gene (isolated from Streptomyces griseus strain HUT 6037) with wasabi defensin (WD) gene (isolated from Wasabia japonica) to pyramid the two disease resistant genes. Molecular analyses of the developed shoots confirmed the existence of both the genes of interest (ChiC and WD) in transgenic plants. Co-expression of the genes was confirmed by RT-PCR, northern blot, and western blot analyses. Disease resistance assay of in vitro plants showed that the transgenic lines co-expressing both the ChiC and WD genes had higher resistance against the fungal pathogens, Fusarium oxysporum (Fusarium wilt) and Alternaria solani (early blight) compared to the non-transformed control and the transgenic lines expressing either of the ChiC or WD genes.