Improvement of Pichia kudriavzevii Egyptian isolate for keratinase production

Abstract

Background and objective Keratinases are gaining considerable momentum in green technology because of their endowed robustness and multifaceted application potentials, such as valorization of keratinous agro-waste. Therefore, the production of novel keratinases from relative yeasts grown in agro-waste formulated medium is cost-effective and imperative for the sustainability of thriving bioeconomy. Materials and methods A total of 51 yeast isolates were isolated from 10 different poultry farms and assayed for keratinase-specific activity. Molecular identification of the high-efficiency keratinase-producing yeast isolate was done by PCR amplification, employing sequencing of internal transcribed spacer regions of yeast. Mutagenesis induction with ethidium bromide, ultraviolet, and ethyl methane sulfonate (EMS) was done in a multistep mutation-induction process for creating super keratinase-productive mutants. Response surface methodology optimization of culture conditions for high-productive mutant was carried out using different parameters such as incubation time, pH, carbon sources, and nitrogen sources to test keratinase activity. Inter-simple sequence repeat (ISSR-PCR) was applied to study the genetic diversity of isolated Pichia kudriavzevii YK46 compared with their five mutants. Results and conclusion The results indicated that the isolate with the highest keratinase activity was isolate no. 46, which recorded 164.04 U/ml. It was identified as P. kudriavzevii and was submitted to NCBI under accession number ‘OK092586’. It was named as P. kudriavzevii YK46. Results of mutagenesis showed that the best keratinolytic efficiency mutant was designated as EMS-37, which showed an activity of 211.90 U/ml. After response surface methodology optimization of culture conditions for mutant EMS-37, the maximum keratinase activity was noted after an optimized condition at pH 5, 72 h of incubation time, 2.5% glucose, and 2.5% beef extract (as carbon and nitrogen sources), with an activity of 240.172 U/ml (Run3). Inter-simple sequence repeat showed that the highest total and polymorphic with unique bands were revealed in the mutant EMS-37, with 82 and 54 bands, respectively, whereas the mutant EMS-56 showed 72 and 44 bands, respectively, compared with the wild-type strain P. kudriavzevii YK46, with 86 and 58 bands, respectively. The data obtained showed that mutant EMS-37 was the highest producer of keratinase enzyme. It had seven unique bands. These bands might be related to the increase in the productivity of keratinase enzyme.