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

IDENTIFICATION, CLONING AND EXPRESSION OF AGARASE FROM MARINE
BACTERIA STRAIN AG21
Youngduk Lee, Chulhong Oh, Ilson Whang, 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 an important gelatinous substance present in the cell wall of some marine red algae (Rhodophyceaea). It is mainly
composed of agarose and agaropectin. Agarose is a hydrophilic hetero polysaccharide, which consists of a linear chain of alternately
arranged 3-O-linked β-D-galacto-pyronose and 4-O-linked 3,6-anhydro-α-L-galacto pyronose residues. Agarases are
the natural enzymes of certain agarolytic organisms found mostly in marine habitats that hydrolyze the agarose. Also, agarase is
the first enzyme in the agar catabolic pathway. Availability of agarases basically in the marine environment, which is consistent
with the fact that agar, being a product of marine algae is available to utilize some marine organisms as a convenient carbon
and energy source.
We screened agarase producing marine bacteria from Jeju sea. We isolated genomic DNA from the bacteria and analyzed 16s
rRNA sequence by PCR and sequencing. The sequence was 99% (1376/1384bp) similar to Saccharophagus degradans 2-40.
We amplified patial sequence from the strain by PCR. The detection of agarase full sequence, was carried out long and accurate
polymerase chain reaction (LA-PCR). The ORF was detected as 1908 bp (636 aa) and nucleotide sequence was showed 88%
similarity to Saccharophagus sp. agarase.
We tested optimum temperature, optimum pH and thermostability. Optimum temperature and pH were 55°C and 7.5 respectively.
The purified agarase was stable at 40°C for 30 hours. Agar degrading pattern by purified agarase was identified with
thin layer chromatography.
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