Identification and Characterization of Carboxyl Esterases of Gill Chamber-Associated Microbiota in the Deep-Sea Shrimp Rimicaris exoculata by Using Functional Metagenomics

Abstract

iThe shrimp Rimicaris exoculata dominates the fauna in deep-sea hydrothermal vent sites along the Mid-Atlantic Ridge (depth, 2,320 m). Here, we identified and biochemically characterized three carboxyl esterases from microbial communities inhabiting the R. exoculata gill that were isolated by naive screens of a gill chamber metagenomic library. These proteins exhibit low to moderate identity to known esterase sequences (<52%) and to each other (11.9 to 63.7%) and appear to have originated from unknown species or from genera of Proteobacteria related to Thiothrix/Leucothrix (MGS-RG1/RG2) and to the Rhodobacteraceae group (MGS-RG3). A library of 131 esters and 31 additional esterase/lipase preparations was used to evaluate the activity profiles of these enzymes. All 3 of these enzymes had greater esterase than lipase activity and exhibited specific activities with ester substrates (<356 U mg ؊1 ) in the range of similar enzymes. MGS-RG3 was inhibited by salts and pressure and had a low optimal temperature (30°C), and its substrate profile clustered within a group of low-activity and substrate-restricted marine enzymes. In contrast, MGS-RG1 and MGS-RG2 were most active at 45 to 50°C and were salt activated and barotolerant. They also exhibited wider substrate profiles that were close to those of highly active promiscuous enzymes from a marine hydrothermal vent (MGS-RG2) and from a cold brackish lake (MGS-RG1). The data presented are discussed in the context of promoting the examination of enzyme activities of taxa found in habitats that have been neglected for enzyme prospecting; the enzymes found in these taxa may reflect distinct habitat-specific adaptations and may constitute new sources of rare reaction specificities. Metagenomics provides a means for the discovery of entirely new enzymes in microorganisms and their communities without the need to culture these microorganisms as individual species, which is technically very difficult (1-5). Although the discovery of new enzyme activities has progressed considerably (6), it has not managed to go beyond the effective identification of enzymatic activities at a rather limited number of environmental sites. While an extensive search in the specialized literature and public databases indicated that microbial communities from approximately 1,800 different sites worldwide have been examined for their genomic content, only in approximately 200 of those locations (11% of the total) have new active clones or enzymes been identified and/or partially characterized. Thus, only a tiny fraction of the Earth's biosphere has been explored for the purpose of enzyme discovery, despite the fact that natural microbial diversity has been recognized to be the major source of new microbes (7). Such diversity also contains novel genes and functions (8) whose activities remain mostly unknown but whose potential to contribute to an innovation-based economy is recognized (9, 10).One of the habitats less explored to date in terms of examining enzyme repertoires is the deep-sea realm, where...

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