Annals of Microbiology

, Volume 64, Issue 3, pp 1257–1266 | Cite as

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

  • Rinky Rajput
  • Rani GuptaEmail author
Original Article


In this study thermostable keratinase rK27 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 rK27 produced by B. subtilis reached 4,432 U/mL after 24 h of culture at 37 °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 rK27 hydrolyzed 1 g bone meal after 12 h at 40 °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 rK27 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.


Keratinase Bacillus pumilus Bacillus subtilis WB980 Bone meal Flocculants 



Financial assistance from University of Delhi, Misc. R & D Grant and Department of Biotechnology, New Delhi through project no. BT/PR012505/PID/06/01/2009 is duly acknowledged.

Supplementary material

13213_2013_770_MOESM1_ESM.doc (64 kb)
ESM 1 (DOC 64 kb)
13213_2013_770_MOESM2_ESM.doc (30 kb)
ESM 2 (DOC 30 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg and the University of Milan 2013

Authors and Affiliations

  1. 1.Department of MicrobiologyUniversity of DelhiNew DelhiIndia

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