Biochemical studies on a versatile esterase that is most catalytically active with polyaromatic esters

et al. 2015

Appl Environ Microbiol

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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...

Section: Figsupporting

confidence: 86%

“…These chemicals included 11 model esters (7 pNP esters and 4 triacylglycerols) (Fig. 4), as well as a battery of 120 structurally different esters (3,6,27,28,37,53) (Fig. 5).…”

Section: Metagenome Library Construction and Screening For Carboxyl Ementioning

confidence: 99%

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

Alcaide

Tchigvintsev

et al. 2015

Appl Environ Microbiol

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“…This result implies that the enzymes with the lipase phenotype (most active against longer insoluble triglycerides such as triolein) were 83‐fold less abundant in this experiment than were those with an esterase (most active towards shorter triglycerides such as tributyrin) character. Additionally, among the common substrates used for the esterase/lipase screen, the methods using pH indicators resulted in a higher incidence rate (1:29) (Martínez‐Martínez et al ., ), followed, to a lesser extent, by methods based on the utilization of indoxyl acetate (1:700) (Alcaide et al ., ), nitrocefin [3‐(2, 4 dinitrostyrl)‐(6R,7R‐7‐(2‐thienylacetamido)‐ceph‐3‐em‐4‐carboxylic acid, E‐isomer)] (1:10 000) (Rashamuse et al ., ), poly(DL‐lactic acid) (1:13 334) (Akutsu‐Shigeno et al ., ; Okamura et al ., ), tributyrin (1:15 478), α‐naphthyl acetate (1:19 925), polyethylene terephthalate (1:21 400) (Sulaiman et al ., ), triolein/olive oil and rhodamine B (1:22 061) (Glogauer et al ., ), Tween‐20 and CaCl 2 (1:26 496) (Heravi et al ., ; Okamura et al ., ), methyl and ethyl ferulate (1:26 496) (Vieites et al ., ), 5‐bromo‐4‐chloro‐3‐indolylcaprylate (1:50 000) (Li et al ., ), and tricaprylin (1:68 279) (Tirawongsaroj et al ., ), in that order (Fig. A).…”

Section: Quantifying the Success Of The Screening Protocols For Enzymmentioning

confidence: 99%

Estimating the success of enzyme bioprospecting through metagenomics: current status and future trends

Ferrer

Microbial Biotechnology

Bargiela

et al. 2015

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185

149

SummaryRecent reports have suggested that the establishment of industrially relevant enzyme collections from environmental genomes has become a routine procedure. Across the studies assessed, a mean number of approximately 44 active clones were obtained in an average size of approximately 53 000 clones tested using naïve screening protocols. This number could be significantly increased in shorter times when novel metagenome enzyme sequences obtained by direct sequencing are selected and subjected to high‐throughput expression for subsequent production and characterization. The pre‐screening of clone libraries by naïve screens followed by the pyrosequencing of the inserts allowed for a 106‐fold increase in the success rate of identifying genes encoding enzymes of interest. However, a much longer time, usually on the order of years, is needed from the time of enzyme identification to the establishment of an industrial process. If the hit frequency for the identification of enzymes performing at high turnover rates under real application conditions could be increased while still covering a high natural diversity, the very expensive and time‐consuming enzyme optimization phase would likely be significantly shortened. At this point, it is important to review the current knowledge about the success of fine‐tuned naïve‐ and sequence‐based screening protocols for enzyme selection and to describe the environments worldwide that have already been subjected to enzyme screen programmes through metagenomic tools. Here, we provide such estimations and suggest the current challenges and future actions needed before environmental enzymes can be successfully introduced into the market.

“…Most of the LIP-EST enzymes can recognize the carboxyl group on fatty acids, alkanes and, in general terms, long chain structures where the carboxyl group is located at one end. However, the process does not work as well when the carboxyl group is near a bigger, more complex, and especially less flexible, structure, such as oleanolic acid, abietic acid, or generally speaking naphtene, phenantrene or terpenoid structures (Martínez-Martínez et al 2014). Another problem to be addressed is how to perform the reaction when the substrate/substrates (the acid and/or the alcohol molecules) are nearly insoluble in water or in solvents where the LIP-EST enzymes are active.…”

Section: Biocatalysis Innovation Guided By Metagenomicsmentioning

confidence: 99%

Biodiversity for biocatalysis: A review of the α/β-hydrolase fold superfamily of esterases-lipases discovered in metagenomes

Ferrer

Bargiela

Biocatalysis and Biotransformation

et al. 2015

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Natural biodiversity undoubtedly inspires biocatalysis research and innovation.Biotransformations of interest also inspire the search for appropriate biocatalysts in nature.Indeed, natural genetic resources have been found to support the hydrolysis and synthesis of not only common but also unusual synthetic scaffolds. The emerging tool of metagenomics has the advantage of allowing straightforward identification of activity directly applicable as biocatalysis. However, new enzymes must not only have outstanding properties in terms of performance but also other properties superior to those of well-established commercial preparations in order to successfully replace the latter. Esterases (EST) and lipases (LIP) from the α/ -hydrolase fold superfamily are among the enzymes primarily used in biocatalysis. Accordingly, they have been extensively examined with metagenomics. Here we provided an updated (October 2015) overview of sequence and functional datasets of 288 EST-LIP enzymes with validated functions that have been isolated in metagenomes and (mostly partially) characterized. Through sequence, biochemical and reactivity analyses we attempted to understand the phenomenon of variability and versatility within this group of enzymes and to implement this knowledge to identify sequences encoding EST-LIP which may be useful for biocatalysis. We found that the diversity of described EST-LIP polypeptides was not dominated by a particular type of protein or highly similar clusters of proteins but rather by diverse nonredundant sequences. Purified EST-LIP exhibited a wide temperature activity range of 10-85°C, although a preferred bias for a mesophilic temperature range (35-40ºC) was observed. At least 60% of the total characterized metagenomics-derived EST-LIP showed outstanding properties in terms of stability (solvent tolerance) and reactivity (selectivity and substrate profile), which are the features of interest in biocatalysis. We hope that, in the future, the search for and utilization of sequences similar to those already encoded and characterized EST-LIP enzymes 3 from metagenomes may be of interest for promoting unresolved biotransformations in the chemical industry. Some examples are discussed in this review.4