Abstract
The main keratinase (kerA) gene from the Bacillus licheniformis S90 was optimized by two codon optimization strategies and expressed in Pichia pastoris in order to improve the enzyme production compared to the preparations with the native kerA gene. The results showed that the corresponding mutations (synonymous codons) according to the codon bias in Pichia pastoris were successfully introduced into keratinase gene. The highest keratinase activity produced by P. pastoris pPICZαA-kerAwt, pPICZαA-kerAopti1 and pPICZαA-kerAopti2 was 195 U/ml, 324 U/ml and 293 U/ml respectively. In addition, there was no significant difference in biomass concentration, target gene copy numbers and relative mRNA expression levels of every positive strain. The molecular weight of keratinase secreted by recombinant P. pastori was approx. 39 kDa. It was optimally active at pH 7.5 and 50°C. The recombinant keratinase could efficiently degrade both α-keratin (keratin azure) and β-keratin (chicken feather meal). These properties make the P. pastoris pPICZαA-kerAopti1 a suitable candidate for industrial production of keratinases.
Highlights
Keratins are major component of cuticulum i.e. feathers, wool, scales, hair, hoofs, nails and so on
Due to intensive cross-linking by hydrogen, hydrophobic and cystine disulfide bonds, keratins are recalcitrant and poorly degraded by most proteinases, e.g. papain, collagenase, pepsin and trypsin [1,2]
The keratinases obtained from various sources have been widely used in medicine, detergent, cosmetics, leather and biodegradable plastic manufacture [3,4]
Summary
Keratins are major component of cuticulum i.e. feathers, wool, scales, hair, hoofs, nails and so on. In spite of the considerably stability, keratin can be efficiently degraded by many microorganisms because of the secretion of keratinases [2,3]. Keratinases are metallo or serine proteinases which can degrade the insoluble structure forming keratin substrates. Keratinases were used to degrade the prion for treatment of bovine spongiform encephalopathy, scrapie and human Creutzfeldt-Jakob disease [5]. Keratinases have been developed by poultry and feed industries to improve the properties of poultry diets [6,7]. The requirement for keratinases has been recently increased. A myriad of bacteria, fungi and actinomycetes were found to naturally secrete keratinases [2,3,4]
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