Production of Thermostable Organic Solvent Tolerant Keratinolytic Protease from Thermoactinomyces sp. RM4: IAA Production and Plant Growth Promotion

Abstract

There are several reports about the optimization of protease production, but only few have optimized the production of organic solvent tolerant keratinolytic proteases that show remarkable exploitation in the development of the non-polluting processes in biotechnological industries. The present study was carried with aim to optimize the production of a thermostable organic solvent tolerant keratinolytic protease Thermoactinomyces sp. RM4 utilizing chicken feathers. Thermoactinomyces sp. RM4 isolated from the soil sample collected from a rice mill wasteyard site near Kashipur, Uttrakhand was identified on the basis of 16S rDNA analysis. The production of organic solvent tolerant keratinolytic protease enzyme by Thermoactinomyces sp. RM4 was optimized by varying physical culture conditions such as pH (10.0), temperature (60°C), inoculum percentage (2%), feather concentration (2%) and agitation rate (2 g) for feather degradation. The result showed that Thermoactinomyces sp. RM4 potentially produces extra-cellular thermostable organic solvent tolerant keratinolytic protease in the culture medium. Further, the feather hydrolysate from keratinase production media showed plant growth promoting activity by producing indole-3-acetic acid itself. The present findings suggest that keratinolytic protease from Thermoactinomyces sp. RM4 offers enormous industrial applications due to its organic solvent tolerant property in peptide synthesis, practical role in feather degradation and potential function in plant growth promoting activity, which might be a superior candidate to keep ecosystem healthy and functional.

Keywords: Thermoactinomyces; ecosystem health; feather degradation; indole-3-acetic acid production; keratinase; organic solvent stability.

FIGURE 1

Scanning electron microscopy of Thermoactinomyces sp. RM4.

FIGURE 2

Factors affecting keratinase enzyme production and feather degradation in keratinase production media. (A) Effect of pH; (B) Effect of temperature; (C) Effect of inoculum percentage; (D) Effect of feather concentration.

FIGURE 3

Feather degradation, keratinase activity, and soluble protein released under different rates of agitation, and SEM of degraded feather. (A) Effect of agitation rate; (B–E) SEM of feather degradation in keratinase production media.

FIGURE 4

Purification of keratinase from Thermoactinomyces sp. RM4. (A) Elution profile of keratinase of Thermoactinomyces sp. strain RM4 in Sephadex G-75 column; (B) SDS–PAGE assay and the activity of purified Keratinase from Thermoactinomyces sp. RM4. Lane1: standard protein markers; Lane 2: purified proteases; Lane 3: zymography of purified enzyme.

FIGURE 5

Characterization of keratinase from Thermoactinomyces sp. RM4. (A,B) Effect of pH on the activity and the stability of Keratinase enzyme; (C,D) Effect of temperature on keratinase activity and stability.

FIGURE 6

Characterization of keratinase from Thermoactinomyces sp. RM4. (A) Effect of different metal ions on keratinase enzyme activity at different concentrations of 0.1, 1.0, and 5.0 mM; (B) Effect of different chemicals on enzyme activity at different concentrations of 0.1, 1.0, and 5.0 mM; (C) Substrate specificity of keratinase; (D) Effect of organic solvents on enzyme activity.

FIGURE 7

Plant growth promoting activity of feather hydrolysate from keratinase production media. (A) Indole-3-acetic acid (IAA) production in culture media supplemented with different concentrations of L- tryptophan during incubation of 6 days; (B) Effect of feather hydrolysate supplementation in soil on seed germination; (C) Effect of feather hydrolysate supplementation in soil on plant growth.