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KMID : 0545119930030010031
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Journal of Microbiology and Biotechnology
1993 Volume.3 No. 1 p.31 ~ p.38
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Enzymatic Characteristics of an Extracellular Agarase of Cytophaga sp. KY-1 and Molecular Cloning of the Agarase gene
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Kim, Young Ho
Kim, Youn Sook/Lee, Jae Ran/Lee, Eun Kyung/Seu, Jung Hwn
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Abstract
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A bacterial strain KY-1 isolated from sewage was able to produce an extracellular agarase(agarose4-glycanohydrolase, EC 3.2.1.81). The strain KY-1 was identified as Cytophaga fermentans subsp. agarovorans based on its morphological and physiological characteristics. The agarase was purified by ammonium sulfate precipitation followed by DEAE-Sephadex A-50, Bio-Gel P-100, and CM-Cellulose column chromatography. The molecular weight of the purified enzyme was 24 kDa by SDS-polyacrylamide gel electrophoresis. The optimum temperature and pH for the enzyme activity were 30℃ and 7.5, respectively. The enzyme activity was significantly inhibited in the presence of 0.1 mM HgCl_2, whereas it was elevated 3 times by MnSO_4 at 1 mM concentration. The Km value and Vmax were 16.67㎎/㎖ and 3.77unit/ml·min. The agarase gene was cloned into Escherichia coli MC1061 using the plasmid vector pBR322. A 1.4 Kb DNA fragment of PstI-digested chromosomal DNA of C. fermentans KY-1 was inserted into the PstI site of pBR322, expressed in the E. coli, and up to 60% of the total enzyme was extracellularly secreted. Enzymatic properties of the extracellular agarases produced by both the transformant and the donor were very similar in terms of optimal pH and temperature.
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