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KMID : 0545119960060060414
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Journal of Microbiology and Biotechnology
1996 Volume.6 No. 6 p.414 ~ p.419
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Purification and Characterization of Clostridium thermocellum Xylanase from Recombinant Escherichia coli
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KOO, BON-JOON
OH, HWA-GYUN/CHO, KI-HAENG/YANG, CHANG-KUN/JUNG, KYUNG-HWA/RYU, DAI-YOUNG
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Abstract
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The xylnX gene encoding a xylanase from Clostridium thermocellum ATSS27405 was cloned in the plasmid pJH27, an E. coli-Bacillus shuttle vector and the resultant recombinant plasmid, pJX18 was transformed into E. coli HB101. The overexpressed xylanase was found to be secreted into the periplasmic space of the recombinant E. coli cells. The crude enzyme was obtained by treating the E. coli cells with lysozyme, and purified by DEAE-Sepharode column chromatography. Molecular weight of the xylanase was estimated to be 53 kDa by gel filteration. The pI value was determined to be pH 8.8. The N-terminal sequence of the enzyme protein was Asp-Asp-Asn-Ala-Asn-Leu-Val-Ser-Asn which was considered to be the sequence of that of the mature form protein. The Km value of the enzyme for oat spelt xylan was calculated be 2.63 mg/ml and the V_max value was 0.47 ??mole/min. The xylanase had a pH optimum for its activity at pH 5.4 and a temperature optimum at 60℃. The enzyme hydrolyzed xylan into xylooligosaccharides which were composed mainly of xylobiose (40%) and xyloltriose (12%) after 5 hour reaction. This result indicates that the xylanase from C. thermocellum ATSS27405 is an endo-acting enzyme.
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