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KMID : 0545120140240020254
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Journal of Microbiology and Biotechnology
2014 Volume.24 No. 2 p.254 ~ p.263
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Gene Cloning and Characterization of an α-Amylase from Alteromonas macleodii B7 for Enteromorpha Polysaccharide Degradation
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Xuefeng Han
Bokun Lin
Ganji Ru
Zhibiao Zhang
Yan Liu
Zhong Hu
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Abstract
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Enteromorpha polysaccharides (EP) extracted from green algae have displayed a wide variety of biological activities. However, their high molecular weight leads to a high viscosity and low solubility, and therefore, greatly restrains their application. To solve this problem, bacteria from the surface of Enteromorpha were screened, and an Alteromonas macleodii strain B7 was found to be able to decrease the molecular weight of EP in culture media. Proteins harvested from the supernatant of the A. macleodii B7 culture were subjected to native gel electrophoresis, and a band corresponding to the Enteromorpha polysaccharide lyase (EPL) was detected by activity staining. The enzyme identity was subsequently confirmed by MALDI-TOF/TOF mass spectrometry as the putative α-amylase reported in A. macleodii ATCC 27126. The amylase gene (amySTU) from A. macleodii B7 was cloned into Escherichia coli, resulting in highlevel expression of the recombinant enzyme with EP-degrading activity. AmySTU was found to be cold-adapted; however, its optimal enzyme activity was detected at 40oC. The α-amylase was highly stable over a broad pH range (5.5-10) with the optimal pH at 7.5-8.0. The highest enzyme activity was detected when NaCl concentration was 2%, which dropped by 50% when the NaCl concentration was increased to 16%, showing an excellent nature of halotolerance. Furthermore, the amylase activity was not significantly affected by tested surfactants or the presence of some organic solvents. Therefore, the A. macleodii strain B7 and its α-amylase can be useful in lowering EP molecular weight and in starch processing.
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KEYWORD
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Enteromorpha polysaccharides, Alteromonas macleodii, Identification, cold-adapted α-amylase, Organic solvent tolerance, Characterization
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