Purification and Characterization of Exo-α- maltotetraohydrolase Produced by Mariana Isolate MS300
Hideki KOBAYASHI*, Yoshihiro TAKAKI, Akira INOUE, Hideto TAKAMI, and Koki HORIKOSHI
The DEEPSTAR group, Japan Marine Science and Technology Center, 2-15 Natsushima- cho, Yokosuka 237, Japan
There are various environments on earth. Some bacteria adapt its surroundings and produce useful enzymes such as amylase, protease, cellulase and so on. We expect that the microbes which produce novel enzymes will be isolated from mud or water of the ocean which has been never taken. Deep-sea is one of such environments and has the different ecosystem from the land, because of no sunlight, low temperature and high hydraulic pressure. We had a chance to collect six deep-sea mud samples from the Challenger Deep of Mariana Trench at the depth of about 11,000 m. It is the deepest point in the world. We have isolated various microorganisms from the mud samples (1). Some of them were the amylase or protease producers.
Among the amylase producers the strain MS300 showed a large halo around the colony on Marine agar medium containing 0.2% of starch azure. The MS300 produced two major amylases, amylase A and B whose molecular weights were estimated to 58,000 Da from SDS-PAGE and isoelectric points were about 7.1 and 7.8, respectively. Amylase A and B produced only maltotetraose from potato starch. Both amylases could not react onα-, β-, γ-cyclodextrin. Therefore, we concluded that amylase is exo-maltotetraohydrolase (G4-amylase). This G4-amylase was rare enzyme, because only two Pseudomonas stutzeri strains have been reported as the producer of this enzyme (2, 3).
The optimum temperatures of these amylases reaction were 40-45℃ and the optimum pHs of reaction were 6.8-7.0. The pH stability of this G4-amylases were pH 6.5 and 9.0 for 30 min at 40℃. The G4-amylases were stable under 40℃ (pH 7.4) for 1.0 h. The isoelectric points, optimum pHs, pH and temperature stability of MS300's amylases were different from P. stutzeri's G4-amylase.
The effect of high hydrostatic pressure on MS300 and their enzymes were investigated. There was no effect on enzyme activity under 100 MPa. Strain MS300 produced more amylase under high pressure than under atmosphere. Under 30 MPa and 60 MPa, MS300 produced amylase about 1.3 and 1.4 times as much as under 0.1 MPa. Furthermore MS300 could grow under 75 MPa in the Marine broth. These data suggested that MS300 would be active in the deep-sea environment.
We could isolate strain MS300 which produced novel type of the G4-amylase.
