Expression of Bacillus pumilus keratinase rK27 in Bacillus subtilis: enzyme application for developing renewable flocculants from bone meal

In this study thermostable keratinase rK(27) of Bacillus pumilus KS12 was expressed and secreted in Bacillus subtilis WB980 expression system under the control of xylose promoter (PxylA). The concentration of the recombinant keratinase rK(27) produced by B. subtilis reached 4,432 U/mL after 24 h of culture at 37 A degrees C and 200 rpm with 0.5 % xylose at an initial concentration of 0.3 OD600nm. Using the one-factor-at-a-time approach, we achieved an improvement in enzyme yield of up to 3.4-fold (15,390 U/mL) in the presence of 3 % yeast extract and 0.5 % tryptone. The enzyme was purified to homogenity using nickel affinity chromatography with a 3.63-fold purity and 80 % recovery. The purified enzyme rK(27) hydrolyzed 1 g bone meal after 12 h at 40 A degrees C, pH 9, with a maximum protein release of 37.3 mg/g bone meal; in comparison subtilisin Carlsberg hydrolyzed 19.3 mg/g bone meal and proteinase K hydrolyzed 6.2 mg/g bone meal. The hydrolysate obtained after hydrolysis of bone by rK(27) was found to be effective as a flocculant at 0.1 mg in a 10 % (w/v) kaolin solution when compared with hydrolysates obtained from substilisin Carlsberg and proteinase K, which were effective at 0.5 mg and > 2 mg, respectively.