February 15-18, 2009 Washington State Convention Center Seattle ...

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CLONING, EXPRESSION AND FUNCTIONAL CHRACTERIZATION OF AGARASE FROM
MARINE BACTERIA STRAIN AG4
Chulhong Oh, Youngduk Lee, Ilson Whang, Ji-Hee Lim, Se Jae Kim and Jehee Lee
Department of Marine Biotechnology
College of Ocean Science
Cheju National University
Jeju, 690-756, Republic of Korea
Agar is a polysaccharide which linked D-galactose and L-3,6-anhydro-galactose residues. The ability of degradation of agar or
agarose to oligosaccharides makes it widely used in food, cosmetic and medical industries. Agarase is produced mainly by bacteria.
Alpha-agarase cleave alpha-1,3-anhydro-L-galactosidic bonds and beta-agarase cleave the beta-1,4-galactosidic bonds. In
recent years, studies have demonstrated that the oligomer forms derive from agar or agarose exhibits variety physiological and
biological activities. The extent of these activities is also correlated with the degree of polymerization and the galactosyl groups
on the AOS and NAOS. Neoagarobiose (N2) was reported to process moisturizing effect on skin and and whitening effects on
melanoma cells (Kobayashi et al. 1997). Neoagarotetrose (N4), derived from prophyran, was reported to be utilize in vitro by
intestinal bacteria, which stimulated the growth of Bacteroides, as well as Eubacterium and Lactobacillus.
In this study, we identified agarase gene by PCR and sequencing from agarase producing marine bacteria strain. The size was
870 bp (290 aa) including 63 bp (21 aa) signal sequence and only 2 bp (2 aa) different compare to Pseudoalteromonas sp. KJ
2-4 alpha-agarase gene. Molecular weight and isoelectric point was 33 kDa and 5.9 respectively. The sequence showed GH16_
beta agarase domain. The mature sequence was cloned into pMal c2x expression vector and transformed to E. coli BL21 (DE3).
Finally the maltose binding protein-agarase fusion protein expression was induced by adding 0.5 mM IPTG. Inducted cell was
lysised by ultrasonicator and divided soluble and insoluble protein by centrifugation. Expression level was checked by SDS-
PAGE and activity test. Recombinant protein was purified with pMAL protein fusion and purification system. Characterization
and analysis of purified protein was carried out for optimum temperature, optimum pH and teen layer chromatography. The
optimum temperature and pH were 45 ℃ and 6.0. We made neoagaro-oligosaccharide with purified agarase. The product
showed activity about anti-oxidant and whitening effect.