2022
DOI: 10.1038/s41589-022-01071-x
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Functional metagenomic screening identifies an unexpected β-glucuronidase

Abstract: The abundance of recorded protein sequence data stands in contrast to the small number of experimentally verified functional annotation. Here we screened a millionmembered metagenomic library at ultrahigh throughput in microfluidic droplets for βglucuronidase activity. We identified SN243, a genuine β-glucuronidase with little homology to previously studied enzymes of this type, as a glycoside hydrolase (GH) 3 family member. This GH family contains only one recently added β-glucuronidase, showing that a functi… Show more

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Cited by 29 publications
(52 citation statements)
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“…The values determined for k cat (0.4 s -1 ) and K M (21.3 mM) differ only minimally from the kinetic data obtained in microtiter plate (k cat = 0.4 s -1 , K M = 23.0 mM). 22 Comparison of the datasets from all twelve detection points (Figures S5-S12, Supporting Information), which were obtained from three different sets of droplets (each feeding four detection points) proved reliable reproducibility of the data. Mean values and their standard deviation averaged from the twelve datasets for the hydrolysis of p NP-β-Xyl resulted in k cat = 0.4 ± 0.04 s -1 and K M = 21.4 ± 2.2 mM with the highest relative difference of 10% in k cat and 15% in K M .…”
Section: Resultsmentioning
confidence: 70%
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“…The values determined for k cat (0.4 s -1 ) and K M (21.3 mM) differ only minimally from the kinetic data obtained in microtiter plate (k cat = 0.4 s -1 , K M = 23.0 mM). 22 Comparison of the datasets from all twelve detection points (Figures S5-S12, Supporting Information), which were obtained from three different sets of droplets (each feeding four detection points) proved reliable reproducibility of the data. Mean values and their standard deviation averaged from the twelve datasets for the hydrolysis of p NP-β-Xyl resulted in k cat = 0.4 ± 0.04 s -1 and K M = 21.4 ± 2.2 mM with the highest relative difference of 10% in k cat and 15% in K M .…”
Section: Resultsmentioning
confidence: 70%
“…SN243 was expressed and purified as described in Neun et al 22 Kinetics were determined in 100 mM Tris pH 8.0, 150 mM NaCl at room temperature. For kinetic measurements in droplets, the following concentrations were used: injection of 500 μM p NP-β-D-glucuronide ( p NP-β-GlcA) into 5 nM SN243; 100 mM p NP-β-D-galacturonide ( p NP-β-GalA) into 25 nM SN243; 400 mM p NP-β-D-glucopyranoside ( p NP-β-Glc) or p NP-β-D-xylopyranoside ( p NP-β-Xyl) into 1 μM SN243; 400 mM p NP-β-D-galactopyranoside ( p NP-β-Gal) or p NP-α-L-arabinofuranoside ( p NP-α-Ara f ) into 10 μM SN243.…”
Section: Methodsmentioning
confidence: 99%
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