New Thermophilic α/β Class Epoxide Hydrolases Found in Metagenomes From Hot Environments

Ferrandi, Erica Elisa; Sayer, C.; Rose, S.A. De; Guazzelli, E.; Marchesi, Carlotta; Saneei, Vahid; Isupov, Michail N.; Littlechild, Jennifer A.; Monti, Daniela

doi:10.3389/fbioe.2018.00144

Cited by 23 publications

(24 citation statements)

References 90 publications

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“…The search of novel HSDH sequences was carried out by using either the Blastp tool (https://blast.ncbi.nlm.nih.gov/) for mining into the NCBI database (https://www.ncbi.nlm.nih.gov/) or the program LAST (http://last.cbrc.jp/) for the analysis of metagenomes of samples collected in hot terrestrial environments . In both cases, only sequences of functionally characterized HSDHs (Table S1 in the Supporting Information) were used as queries during the multiple sequence alignments.…”

Section: Resultsmentioning

confidence: 99%

Insights into the Substrate Promiscuity of Novel Hydroxysteroid Dehydrogenases

Bertuletti

Ferrandi²,

Marzorati³

et al. 2020

Adv Synth Catal

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Hydroxysteroid dehydrogenases (HSDHs) are valuable biocatalysts for the regio‐ and stereoselective modification of steroids, bile acids and other steroid derivatives. In this work, we investigated the substrate promiscuity of this highly selective class of enzymes. In order to reach this goal, a preliminary search of HSDH homologues in in‐house or public available (meta)genomes was carried out. Eight novel NAD(H)‐dependent HSDHs, showing either 7α‐, 7β‐, or 12α‐HSDH activity, and including, for the first time, enzymes from extremophilic microorganisms, were identified, recombinantly produced, and characterized. Among the novel HSDHs, four highly active (up to 92 U mg−1) NAD(H)‐dependent 7β‐HSDHs showing negligible similarity towards previously described 7β‐HSDHs, were discovered. These enzymes, along with previously characterized HSDHs, were tested as biocatalysts for the stereoselective reduction of a panel of substrates including two α‐ketoesters of pharmaceutical interest and selected ketones that partially resemble the structural features of steroids. All the reactions were coupled with a suitable cofactor regeneration system. Regarding the α‐ketoesters, nearly all of the tested HSDHs showed a good activity toward the selected substrates, yielding the reduced α‐hydroxyester with up to 99% conversions and enantiomeric excesses. On the other hand, only the 7β‐HSDHs from Collinsella aerofaciens and Clostridium absonum showed appreciable activity toward more complex ketones, i. e., (±)‐trans‐1‐decalone, but with interesting as well as different selectivity.

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Section: Resultsmentioning

confidence: 99%

Insights into the Substrate Promiscuity of Novel Hydroxysteroid Dehydrogenases

Bertuletti

Ferrandi²,

Marzorati³

et al. 2020

Adv Synth Catal

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“…Upon analyzing the structures of both enzymes, some thermal stability (up to 60 • C and little at 70 • C) appears to be due to a significant amount of hydrophobic regions on the protein surface while polar patches were smaller. Ferrandi et al (2018) reported that hydrophobic interactions considered an important feature of a protein to identify its thermostability.…”

Section: Discussionmentioning

confidence: 99%

Stress-Based Production, and Characterization of Glutathione Peroxidase and Glutathione S-Transferase Enzymes From Lactobacillus plantarum

Al-Madboly

Ali

Fakharany

et al. 2020

Front. Bioeng. Biotechnol.

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More attention has been recently directed toward glutathione peroxidase and s-transferase enzymes because of the great importance they hold with respect to their applications in the pharmaceutical field. This work was conducted to optimize the production and characterize glutathione peroxidase and glutathione s-transferase produced by Lactobacillus plantarum KU720558 using Plackett-Burman and Box-Behnken statistical designs. To assess the impact of the culture conditions on the microbial production of the enzymes, colorimetric methods were used. Following data analysis, the optimum conditions that enhanced the s-transferase yield were the De Man-Rogosa-Sharp (MRS) broth as a basal medium supplemented with 0.1% urea, 0.075% H 2 O 2 , 0.5% 1-butanol, 0.0125% amino acids, and 0.05% SDS at pH 6.0 and anaerobically incubated for 24 h at 40 • C. The optimum s-transferase specific activity was 1789.5 U/mg of protein, which was ∼12 times the activity of the basal medium. For peroxidase, the best medium composition was 0.17% urea, 0.025% bile salt, 7.5% Na Cl, 0.05% H 2 O 2 , 0.05% SDS, and 2% ethanol added to the MRS broth at pH 6.0 and anaerobically incubated for 24 h at 40 • C. Furthermore, the optimum peroxidase specific activity was 612.5 U/mg of protein, indicating that its activity was 22 times higher than the activity recorded in the basal medium. After SDS-PAGE analysis, GST and GPx showed a single protein band of 25 and 18 kDa, respectively. They were able to retain their activities at an optimal temperature of 40 • C for an hour and pH range 4-7. The 3D model of both enzymes was constructed showing helical structures, sheet and loops. Protein cavities were also detected to define druggable sites. GST model had two large pockets; 185Å3 and 71 Å3 with druggability score 0.5-0.8. For GPx, the pockets were relatively Al-Madboly et al.Enzymes Production From Lactobacillus plantarum smaller, 71 Å3 and 32 Å3 with druggability score (0.65-0.66). Therefore, the present study showed that the consortium components as well as the stress-based conditions used could express both enzymes with enhanced productivity, recommending their application based on the obtained results.

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“…After using PCR to amplify identified DNA, the enzymes were cloned, and assayed for their functionality. Metagenomics has now emerged as a very effective tool to mine for non‐redundant genes, creating panels of enzymes from the same families with low sequence homologies [14,15] …”

Section: Introductionmentioning

confidence: 99%

“…Metagenomics has now emerged as a very effective tool to mine for non‐redundant genes, creating panels of enzymes from the same families with low sequence homologies. [ 14 , 15 ]…”

Section: Introductionmentioning

confidence: 99%

Discovery of New Carbonyl Reductases Using Functional Metagenomics and Applications in Biocatalysis

et al. 2021

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Enzyme discovery for use in the manufacture of chemicals, requiring high stereoselectivities, continues to be an important avenue of research. Here, a sequence directed metagenomics approach is described to identify short chain carbonyl reductases. PCR from a metagenomic template generated 37 enzymes, with an average 25% sequence identity, twelve of which showed interesting activities in initial screens. Six of the most productive enzymes were then tested against a panel of 21 substrates, including bulkier substrates that have been noted as challenging in biocatalytic reductions. Two enzymes were selected for further studies with the Wieland Miescher ketone. Notably, enzyme SDR‐17, when co‐expressed with a co‐factor recycling system produced the anti‐(4aR,5S) isomer in excellent isolated yields of 89% and 99% e.e. These results demonstrate the viability of a sequence directed metagenomics approach for the identification of multiple homologous sequences with low similarity, that can yield highly stereoselective enzymes with applicability in industrial biocatalysis.

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New Thermophilic α/β Class Epoxide Hydrolases Found in Metagenomes From Hot Environments

Cited by 23 publications

References 90 publications

Insights into the Substrate Promiscuity of Novel Hydroxysteroid Dehydrogenases

Insights into the Substrate Promiscuity of Novel Hydroxysteroid Dehydrogenases

Stress-Based Production, and Characterization of Glutathione Peroxidase and Glutathione S-Transferase Enzymes From Lactobacillus plantarum

Discovery of New Carbonyl Reductases Using Functional Metagenomics and Applications in Biocatalysis

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