Purification and characterization of Clostridium thermocellum xylanase from recombinant Escherichia coli

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Title
Purification and characterization of Clostridium thermocellum xylanase from recombinant Escherichia coli
Author(s)
Bon Joon Koo; Hwa Gyun Oh; Ki Haeng Cho; Chang Kun Yang; Kyung Hwa Jung; Dai Young Ryu
Bibliographic Citation
Journal of Microbiology and Biotechnology, vol. 6, no. 6, pp. 414-419
Publication Year
1996
Abstract
The xylnX gene encoding a xylanase from Clostridium thermocellum ATCC27405 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-Sepharose column chromatography. Molecular wieght of the xylanase was estimated to be 53 kDa by gel filtration. The pI value was determined to be pH 8.8. The N-terminal sequence of the enzyme protein was Asp-Asp-Asn-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 to be 2.63 mg/ml and the Vmax value was 0.47 μmole/min. The xylanase had a pH optimum for its activity at pH 5.4 and a temperature optimum at 60°C. 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 ATCC27405 is an endo-acting enzyme.
Keyword
clostridium thermocellumxylanase
ISSN
1017-7825
Publisher
Korea Soc-Assoc-Inst
Type
Article
Appears in Collections:
1. Journal Articles > Journal Articles
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