Purification and Characterization of Acetyl Xylan Esterase from Escherichia coli Cells Harboring the Recombinant Plasmid pKMG6

제조합 균주 Escherochia coli가 생산하는 Bacillus stearothermophilus Acetyl Xylan Esterase의 정제 및 특성

  • 김인숙 (고려대학교 자연자원대학 유전공학과) ;
  • 이철우 (고려대학교 자연자원대학 유전공학과) ;
  • 최용진 (고려대학교 자연자원대학 유전공학과)
  • Published : 1994.10.01


Acetyl xylan esterase was produced by E. coli HB101 harboring a recombinant plasmid pKMG6 which contained the estI gene of Bacillus stearothermophilus. The maximum production was observed when the E. coli strain was grown at 37$\circC for 12 hours in the medium containing 0.5% acetyl xylan, 1.0% tryptons, 1.0% sodium chloride, and 0.5% yeast extract. The esterase produced was purified to homogeneity using a combination of ammonium sulfate fractionation, DEAE Sepharose CL-6B ion exchange chromatography and Sephacryl S-200 gel filtration. The native enzyme had an apparent molecular mass of 60 kd and was composed of two identical subunits of 29 kd. The N-terminal amino acid sequence of the polypeptide was Ala-X-Leu-Gln- Ile-Gln-Phe-X-X-Gln. The acetyl esterase displayed a pH optimum of 6.5 and a temperature opti- mum of 45$\circC. The heavy metal ions such as Ag$^{++}$, Hg$^{++}$ and Cu$^{++}$ inhibited nearly completely the activity of the esterase, and no specific metal ion was found to be required for the enzyme activity. The enzyme readily cleaved MAS, $\beta$-D-glucose pentaacetate, $\alpha$-naphthyl acetate, $\rho$-nitrophenyl acetate as well as acetyl xylan, but had no activity on $\rho$-nitrophenyl propionate, $\beta$-nitrophenyl butyrate or $\beta$-nitrophenyl valerate. The Km and Vmax values for MAS were 2.87 mM and 11.55 $\mu$mole/min, respectively. Synergistic behavior was demonstrated with a combination of xylanase and esterase from B. stearothermophilus in hydrolyzing acetyl xylan.


Purification;characterization;acetyl xylan esterase;plasmid pKMG6;Bacillus strarothermophilus


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