Cloning, purification and characterization of a thermostable carboxylesterase from Anoxybacillus sp PDF1


Ay F., KARAOĞLU H. , Irian K., ÇANAKÇI S., BELDÜZ A. O.

PROTEIN EXPRESSION AND PURIFICATION, cilt.80, sa.1, ss.74-79, 2011 (SCI İndekslerine Giren Dergi) identifier identifier identifier

  • Yayın Türü: Makale / Tam Makale
  • Cilt numarası: 80 Konu: 1
  • Basım Tarihi: 2011
  • Doi Numarası: 10.1016/j.pep.2011.06.019
  • Dergi Adı: PROTEIN EXPRESSION AND PURIFICATION
  • Sayfa Sayıları: ss.74-79

Özet

The gene encoding a carboxylesterase from Anoxybacillus sp., PDF1, was cloned and sequenced. The recombinant protein was expressed in Escherichia coli BL21, under the control of isopropyl-beta-D-thiogalactopyranoside-inducible T7 promoter. The enzyme, designated as PDF1Est, was purified by heat shock and ion-exchange column chromatography. The molecular mass of the native protein, as determined by SDS-PAGE, was about 26 kDa. PDF1Est was active under a broad pH range (pH 5.0-10.0) and a broad temperature range (25-90 degrees C), and it had an optimum pH of 8.0 and an optimum temperature of 60 degrees C. The enzyme was thermostable carboxylesterase, and did not lose any activity after 30 min of incubation at 60 degrees C. The enzyme exhibited a high level of activity with p-nitrophenyl butyrate with apparent K-m, V-max, and K-cat values of 0.348 +/- 0.030 mM, 3725.8 U/mg, and 1500 +/- 54.50/s, respectively. The effect of some chemicals on the esterase activity indicated that Anoxybacillus sp. PDF1 produce an carboxylesterase having serine residue in active site and -SH groups in specific sites, which are required for its activity. (C) 2011 Elsevier Inc. All rights reserved.