Abstract
A multifunctional enzyme is one that performs multiple physiological functions, thus benefiting the organism. Characterization of multifunctional enzymes is important for researchers to understand how organisms adapt to different environmental challenges. In the present study, we report the discovery of a novel multifunctional enzyme Amy63 produced by marine bacterium Vibrio alginolyticus 63. Remarkably, Amy63 possesses amylase, agarase and carrageenase activities. Amy63 is a substrate promiscuous α-amylase, with the substrate priority order of starch, carrageenan and agar. Amy63 maintains considerable amylase, carrageenase and agarase activities and stabilities at wide temperature and pH ranges, and optimum activities are detected at temperature of 60 °C and pH of 6.0, respectively. Moreover, the heteroexpression of Amy63 dramatically enhances the ability of E. coli to degrade starch, carrageenan and agar. Motif searching shows three continuous glycosyl hydrolase 70 (GH70) family homologs existed in Amy63 encoding sequence. Combining serial deletions and phylogenetic analysis of Amy63, the GH70 homologs are proposed as the determinants of enzyme promiscuity. Notably, such enzymes exist in all kingdoms of life, thus providing an expanded perspective on studies of multifunctional enzymes. To our knowledge, this is the first report of an amylase having additional agarase and carrageenase activities.
Highlights
The polyspecific family GH13 covering more than 30 different amylolytic and related enzyme specificities with nearly 16,000 sequences ranks among the largest of the GH families[13]. Multifunctional amylases such as maltogenic amylases (EC 3.2.1.133) constitute a special subfamily in the framework of the α -amylase family or GH-13 family because they have some unique catalytic and/or structural characteristics compared with other enzymes of the α -amylase family or GH-13 family[14]
We discovered that the agarase produced by marine bacterium strain 63 exhibited the highest activity and stability in various temperatures and pHs, even in presence of surfactants or chelating agents
In view of the high homology (99% identity) with marine bacterium Vibrio alginolyticus by the 16S rRNA gene sequencing
Summary
The polyspecific family GH13 covering more than 30 different amylolytic and related enzyme specificities with nearly 16,000 sequences ranks among the largest of the GH families[13]. Multifunctional amylases such as maltogenic amylases (EC 3.2.1.133) constitute a special subfamily in the framework of the α -amylase family or GH-13 family because they have some unique catalytic and/or structural characteristics compared with other enzymes of the α -amylase family or GH-13 family[14]. The oligosaccharide-producing multifunctional amylase (OPMA) has strong α -1, 6-transglycosylation activity in addition to its α -1, 4-hydrolytic activity on starch and some oligosaccharides[5,14]. Phylogenetic analysis of Amy[63] indicates that this kind of multifunctional amylases is widely spread in the all kingdoms of life, which profoundly broaden our knowledge about the α -amylase
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