Cloning and expression of keratinase gene from a marine-derived Pseudomonas aeruginosa Gxun-7 and enzymatic properties of the recombinant enzyme

Abstract:

[Background] Keratinase is a kind of hydrolase that specifically degrades keratin, and has important application potential in animal feed, biological fertilizer, medicine, washing, tanning, and environmental treatment. [Objective] The keratinase gene of Pseudomonas aeruginosa Gxun-7 from marine environment was cloned and expressed, and the enzymatic properties of recombinant enzyme were investigated, which laid a foundation for the application of keratinase in industrial production. [Methods] Based on the putative keratinase gene of P. aeruginosa Gxun-7 genome, primers were designed to obtain keratinase gene kp2. The recombinant expression plasmid pET22b- kp2 was constructed, and transformed into E. coli RosettagamiB (DE3) for induced expression. Meanwhile, the expression conditions of the recombinant expression strain were optimized. The recombinant keratinase was isolated and purified by nickel column and its enzymatic properties were studied. [Results] The molecular weight of recombinant keratinase was about 33 kDa, and the optimal temperature was 40 ℃ at pH 8.0. The recombinant keratinase had good stability at temperature of 30-60 ℃ and pH 6.5-8.0. Metal ions including Co²⁺ and Cu²⁺, and chemical reagents including sodium dodecyl sulfonate (SDS), ethylenediaminetetraacetic acid (EDTA), and phenylmethylsulfonyl fluoride (PMSF) inhibited the enzyme activity, whereas Mg²⁺, K⁺, mercaptoethanol, and dithiothreitol (DTT) promoted the enzyme activity. The recombinant keratinase showed good salt tolerance, and the relative enzyme activity was 87.55% under the action of 12.5% NaCl. When casein was used as substrate, the K _m and V _max of the enzyme were 60.92 mg/mL and 9.70 U/mL. [Conclusion] The recombinant keratinase from the marine-derived P. aeruginosa Gxun-7 has good stability in temperature, alkali and salt, which can be applied to industrial production in the future.