HPLC–CD selectivity assay for alcohol dehydrogenases

Hamzić, Melissa; Pietruszka, Jörg; Sandkuhl, Diana

doi:10.1002/chir.21025

Cited by 9 publications

(3 citation statements)

References 42 publications

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“…The ee of the phenyl ester was determined using the same conditions as the methyl ester [ t R ( S ) = 16.32 min, t R ( R ) = 18.07 min]. Because the enantiomers of the p NP ester could not be separated by chiral HPLC, the ee was determined by measuring the g factor (dissymmetry factor) with achiral HPLC with CD detector [54]–[56]. Column: Hyperclone ODS C18, conditions: 90∶10, CH 3 CN:H 2 O, 0.5 ml/min, 220 nm, t R (ibuprofen) = 2.7 min, t R ( p NP ester) = 4.5 min.…”

Section: Methodsmentioning

confidence: 99%

The Metagenome-Derived Enzymes LipS and LipT Increase the Diversity of Known Lipases

et al. 2012

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Triacylglycerol lipases (EC 3.1.1.3) catalyze both hydrolysis and synthesis reactions with a broad spectrum of substrates rendering them especially suitable for many biotechnological applications. Most lipases used today originate from mesophilic organisms and are susceptible to thermal denaturation whereas only few possess high thermotolerance. Here, we report on the identification and characterization of two novel thermostable bacterial lipases identified by functional metagenomic screenings. Metagenomic libraries were constructed from enrichment cultures maintained at 65 to 75°C and screened resulting in the identification of initially 10 clones with lipolytic activities. Subsequently, two ORFs were identified encoding lipases, LipS and LipT. Comparative sequence analyses suggested that both enzymes are members of novel lipase families. LipS is a 30.2 kDa protein and revealed a half-life of 48 h at 70°C. The lipT gene encoded for a multimeric enzyme with a half-life of 3 h at 70°C. LipS had an optimum temperature at 70°C and LipT at 75°C. Both enzymes catalyzed hydrolysis of long-chain (C12 and C14) fatty acid esters and additionally hydrolyzed a number of industry-relevant substrates. LipS was highly specific for (R)-ibuprofen-phenyl ester with an enantiomeric excess (ee) of 99%. Furthermore, LipS was able to synthesize 1-propyl laurate and 1-tetradecyl myristate at 70°C with rates similar to those of the lipase CalB from Candida antarctica. LipS represents the first example of a thermostable metagenome-derived lipase with significant synthesis activities. Its X-ray structure was solved with a resolution of 1.99 Å revealing an unusually compact lid structure.

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Section: Methodsmentioning

confidence: 99%

The Metagenome-Derived Enzymes LipS and LipT Increase the Diversity of Known Lipases

et al. 2012

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“…Introduction of a trimethylsilyl (TMS) group, which can be attached and also removed easily, to the alkyne unit was sufficient to produce enantiopure ( R )‐3‐butin‐2‐ol ( ee >99 % compared to 60 % ee without TMS) . In our previous studies we additionally observed that compared to the desilylated compound the ADH‐catalyzed reduction of phenyl‐3‐(trimethylsilyl)‐2‐propynone provides the desired enantiopure alcohol in a more reasonable time. Because the synthesis of the TMS‐attached acetylene 16 is also more straightforward, it was performed starting from methyl 2‐bromoacetate ( 17 ) and 3‐(trifluoromethyl)‐phenol ( 15 ) as available starting materials (Scheme ).…”

Section: Figurementioning

confidence: 96%

“…[37] Introduction of at rimethylsilyl (TMS) group,w hich can be attached and also removed easily, [38] to the alkyne unit was sufficient to produce enantiopure (R)-3-butin-2-ol (ee > 99 % compared to 60 % ee without TMS). [37] In our previous studies [40] we additionally observed that compared to the desilylat-Scheme3.Preparativescale reaction of RasADH with cofactor recycling for the synthesis of alcohol (R)-6. Figure 2.…”

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confidence: 99%