ABSTRACT
In this study, five keratinolytic bacteria were isolated from poultry farm waste of Eastern Province, Saudi Arabia. The highest keratinase activity was obtained at 40-45 °C, pH 8-9, feather concentration 0.5-1%, and using white chicken feather as keratin substrate for 72 h. Enhancement of keratinase activity through physical mutagen UV radiation and/or chemical mutagen ethyl methanesulfonate (EMS) resulted in five mutants with 1.51-3.73-fold increased activity over the wild type. When compared with the wild type, scanning electron microscopy validated the mutants' effectiveness in feather degradation. Bacterial isolates are classified as members of the S8 family peptidase Bacillus cereus group based on sequence analysis of the 16S rRNA and keratinase genes. Interestingly, keratinase KerS gene shared 95.5-100% identity to keratinase, thermitase alkaline serine protease, and thermophilic serine protease of the B. cereus group. D137N substitution was observed in the keratinase KerS gene of the mutant strain S13 (KerS13uv+ems), and also seven substitution variations in KerS26 and KerS26uv of strain S26 and its mutant S26uv. Functional analysis revealed that the subtilisin-like serine protease domain containing the Asp/His/Ser catalytic triad of KerS gene was not affected by the predicted substitutions. Prediction of physicochemical properties of KerS gene showed instability index between 17.5-19.3 and aliphatic index between 74.7-75.7, which imply keratinase stability and significant thermostability. The docking studies revealed the impact of substitutions on the superimposed structure and an increase in binding of mutant D137N of KerS13uv+ems (affinity: -7.17; S score: -6.54 kcal/mol) and seven mutants of KerS26uv (affinity: -7.43; S score: -7.17 kcal/mol) compared to the wild predicted structure (affinity: -6.57; S score: -6.68 kcal/mol). Together, the keratinolytic activity, similarity to thermostable keratinases, and binding affinity suggest that keratinases KerS13uv+ems and KerS26uv could be used for feather processing in the industry.
ABSTRACT
Pot experiments were conducted to evaluate the possible roles of nitrogen fixation and/or enhanced mineral uptake by Azospirillum lipoferum and Bacillus polymexa inoculation in improving salt tolerance of maize plants. Plants were inoculated and grown under salt stress (osmotic potential: -0.3, -0.6, -0.9 and -1.2 Mpa). Both microorganisms were able to fix nitrogen up to -0.9 Mpa salinity level accompanied with increased total N-yield compared with the control plants. In order to investigate the role of bacterial inoculation on enhanced mineral uptake, the growth and some physiological parameters of inoculated plants were compared with plants fertilized by K and P foliar application. Plant inoculation with the N2-fixers or plant spraying with KH2PO4 resulted in an increase in fresh and dry matter as well as water content of plants. Treated plants exhibited changed plant mineral content which was associated with increased Mg/K and decreased P/K, Ca/K and Na/K ratios. This was accompanied by accumulation of soluble sugars, amino acids in shoots and roots of plants resulting in a concomitant increase in the osmotic potential of the cell sap as a possible mechanism of adaptation to salinity.