- Volume 23 Issue 4
Purification and Characterization of Acetyl Xylan Esterase II from Escherichia coli Cells Harboring Recombinant Plasmid pKMG7
재조합 균주 Escherichia coli가 생산하는 Bacillus stearothermophilus Acetyl Xylan Esterase II의 정제 및 특성
- 김희선 (고려대학교 자연자원대학 유전공학과) ;
- 서정한 (고려대학교 자연자원대학 유전공학과) ;
- 최용진 (고려대학교 자연자원대학 유전공학과)
- Published : 1995.08.01
Acetylxylan esterase II was produced by Escherichia coli HB101 harboring the recombinant plasmid pKMG7 which contained the estII gene of Bacillus stearothermophilus. Optimal medium for the production of the acetylxylan esterase by E. coli HB101/pKMG7 was determined to contain 0.5% galactose, 1% yeast extract and 1% NaCl. The enzyme produced was purified to homogeneity using a combination of 20-50% ammonium sulfate precipitation, DEAE-Sepharose CL-6B chromatography and Sephacryl S-200 gel filtration. The temperature and pH optimum of the esterase were 45$\circ$C and pH 6, respectively. The essential amino acids for the esterase activity were found to be methionine, serine, and cysteine. Molecular weight of the esterase was determined to be 28 kDa by SDS-polyacrylamide gel electrophoresis, and 120 kDa by gel filtration. This suggests that the functional enzyme is a homomeric tetramer. The esterase had an isoelectric point of pH 3.4. The N-terminal amino acid sequence of the enzyme was Ala-Leu-Phe-Glu-Ser-Arg-Phe-Phe-Ser-Glu-Val-Leu-Gly-Leu.
Purification;characterization;acetylxylan esterase II;recombinant plasmid;Bacillus stearothermophilus
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