Isolation of a novel carboxylesterase from Bacillus coagulans with high enantioselectivity toward racemic esters of 1,2-O-isopropylideneglycerol

Molinari, Francesco; Brenna, Oreste V.; Valenti, M.; Aragozzini, Fabrizio

doi:10.1016/s0141-0229(96)00066-x

Cited by 48 publications

(31 citation statements)

References 16 publications

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“…YbfF was found to be completely inactive after preincubation (5 or 10 min) at 70°C. Previously reported esterases from strains of Bacillus (23), Pseudomonas (18), and Kluyveromyces marxianus (24) also show maximum esterase activities at high temperature (60 to 70°C) but, like YbfF, were not stable at these temperatures.…”

Section: Resultsmentioning

confidence: 92%

“…This enantioselectivity could even be further enhanced when using DMF or DMSO as cosolvents in kinetic resolution experiments. Together with CesA from B. coagulans (23), YbfF displays the highest enantioselectivity in the hydrolysis of IPG-caprylate among reported esterases. However, YbfF is more active for this sub- …”

Section: Resultsmentioning

confidence: 99%

“…OPL showed, however, low enantioselectivity toward both IPG butyrate (E ϭ 2.9) and IPG caprylate (E ϭ 1.4) (21). In contrast, carboxylesterase A (CesA) from Bacillus coagulans showed similar enantioselectivity in the hydrolysis of IPG caprylate compared to YbfF, producing (S)-IPG with 94% ee (23).…”

Section: Resultsmentioning

confidence: 99%

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Discovery of an Escherichia coli Esterase with High Activity and Enantioselectivity toward 1,2- O -Isopropylideneglycerol Esters

Godinho

Reis

Tepper

et al. 2011

Appl Environ Microbiol

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Escherichia coli has been widely used as an expression host for the identification of desired biocatalysts through screening or selection assays. We have previously used E. coli in growth selection and screening assays for identification of Bacillus subtilis lipase variants (located in the periplasm) with improved activity and enantioselectivity toward 1,2-O-isopropylideneglycerol (IPG) esters. In the course of these studies, we discovered that E. coli itself exhibits significant cytoplasmic esterase activity toward IPG esters. In order to identify the enzyme (or enzymes) responsible for this esterase activity, we analyzed eight E. coli knockout strains, in which single esterase genes were deleted, for their ability to hydrolyze IPG butyrate. This approach led to the identification of esterase YbfF as the major E. coli enzyme responsible for the hydrolytic activity toward IPG esters. The gene coding for YbfF was cloned and overexpressed in E. coli, and the corresponding protein was purified and characterized for its biocatalytic performance. YbfF displays a high level of activity toward IPG butyrate and IPG caprylate and prefers the R-enantiomer of these substrates, producing the S-enantiomer of the IPG product with high enantiomeric excess (72 to 94% ee). The enantioselectivity of YbfF for IPG caprylate (E ‫؍‬ 40) could be significantly enhanced when using dimethylformamide (DMF) or dimethyl sulfoxide (DMSO) as cosolvents in kinetic resolution experiments. The enzyme also shows high enantioselectivity toward 1-phenylethyl acetate (E > 200), giving the chiral product (R)-1-phenylethanol with >99% ee. The high activity and enantioselectivity of YbfF make it an attractive enzyme for organic synthesis.Hydrolases (EC 3) represent a class of enzymes that catalyze the hydrolysis of a chemical bond and are widely employed in organic synthesis (7,8,29). Depending on the chemical bond that they act on, hydrolases are grouped into several subclasses. Esterases and other hydrolytic enzymes that belong to the broad group of carboxylic ester hydrolases (EC 3.1.1) catalyze the hydrolysis and synthesis of ester bonds (5). Their broad substrate acceptance and high stability and enantioselectivity and the fact that they do not require cofactors make them attractive biocatalysts for organic synthesis (5). For example, the carboxylesterase NP from Bacillus subtilis Thai I-8 shows very high levels of activity and stereoselectivity toward esters of nonsteroidal anti-inflammatory drugs (NSAID), such as the naproxen and ibuprofen methyl esters, demonstrating its exciting potential for selective drug synthesis (6).We are interested in developing a biocatalytic process for the kinetic resolution of racemic 1,2-O-isopropylideneglycerol (IPG) esters. In previous studies, we have used Escherichia coli in directed evolution experiments for the identification of Bacillus subtilis lipase variants (located in the periplasm) with improved activity and enantioselectivity toward IPG butyrate (substrate 1) and IPG caprylate (substrate 3) (Fig. 1) ...

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Section: Resultsmentioning

confidence: 92%

Section: Resultsmentioning

confidence: 99%

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Discovery of an Escherichia coli Esterase with High Activity and Enantioselectivity toward 1,2- O -Isopropylideneglycerol Esters

Godinho

Reis

Tepper

et al. 2011

Appl Environ Microbiol

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“…Molinari et al showed that the CesA from B. coagulans hydrolyzed IPG caprylate esters producing (S)-IPG with 94% enantiomeric excess at 23% conversion, corresponding to an E value of 43. [15,16] Although the YbfF selectivity towards substrate 3 is high enough for practical applications, further improvement might still be possible. A plausible approach would pass by promoting a second round of mutagenesis in an iterative manner, according to the iterative saturation mutagenesis approach (ISM) described by Reetz.…”

Section: Molecular Docking Of (R)-ipg-c4 (R-1) (A) (S)-ipg-c4 (S-1mentioning

confidence: 99%

An Esterase with Superior Activity and Enantioselectivity towards 1,2‐O‐Isopropylideneglycerol Esters Obtained by Protein Design

et al. 2012

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Abstract:The Escherichia coli esterase YbfF displays high activity towards 1,2-O-isopropylideneglycerol (IPG) butyrate and IPG caprylate, and prefers the R-enantiomer of these substrates, producing the Senantiomer of the IPG product in excess. To improve the potential of the enzyme for the kinetic resolution of racemic esters of IPG, an enhancement of the activity and enantioselectivity would be highly desirable. Molecular docking of the R-enantiomer of both IPG esters into the active site of YbfF allowed the identification of proximal YbfF active site residues. Four residues (25, 124, 185 and 235) were selected as targets for mutagenesis, in order to enhance YbfF activity and enantioselectivity towards IPG esters. Random mutagenesis at positions 25, 124, 185 and 235 yielded several best YbfF variants with enhanced activity and enantioselectivity towards IPG esters. The best YbfF mutant, W235I, exhibited a 2-fold higher enantioselectivity than wild-type YbfF, with an E = 38 for IPG butyrate and an E = 77 for IPG caprylate. Molecular docking experiments further support the enhanced enantioselectivity shown experimentally and the structural effects of this amino acid substitution on the active site of YbfF are provided. The engineered W235I mutant is an attractive catalyst for practical applications in the kinetic resolution of IPG esters.

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“…Therefore, they have become two of the most widely used enzymes in organic synthesis and various industrial applications such as detergent industry, oleochemical industry, pulp and paper industry [3,4,5]. Also, the modification of triglycerides for fat and oil industry, synthesis of flavor esters for food industry, resolution of racemic mixtures used for the synthesis of fine chemicals for the pharmaceutical industry can be performed with esterases [6]. It was reported that the carboxylesterase produced by Bacillus subtilis has been used in the synthesis of naproxen as a nonsteroidal anti-inflammatory drug [3] and 2-arylpropionic acids with high enantioselectivity [7].…”

Section: Introductionmentioning

confidence: 99%

Purification and characterization of a novel thermostable esterase from Thermus sp. NCCB 100425T

Akatin

2015

Turk J Bioch

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Purification and characterization of a novel thermostable esterase from Thermus sp. NCCB 100425 T [Thermus sp. NCCB 100425 T ' den yeni bir ısıya dayanıklı esterazın saflaştırılması ve karakterizasyonu] ABSTRACT Objective: The purpose of the present study was to purify and characterize an esterase from a thermophilic bacterium Thermus sp. NCCB 100425 T and to check its suitability for industrial applications. Methods: Thermus sp. NCCB 100425 T esterase was purified by using ammonium sulphate precipitation and hydrophobic interaction chromatography and then characterized biochemically. Results: The purity of the enzyme was observed as a single band on native-and SDS-PAGE. In the presence of p-nitrophenyl acetate (pNPA) as a substrate, the optimum pH and temperature of the enzyme were found to be 7.5 and 60°C, respectively. K m and V max values are calculated as 18.32 mM and 96.15 U/mg protein, respectively, with pNPA. pH stability was investigated in the range of pH 4.0-9.0 at 4°C and 60°C. After 7 days incubation, activity of pure enzyme was retained 85-90% for all pH at 4°C and 59±5.2% for pH 8.0 at 60°C. It was determined that approximately 80% of enzyme activity was retained between 30-60°C after 7 days incubation. In the presence of 10% ethanol and DMSO, the enzyme activity was retained 96±2.7% and 78±2.5%, respectively. Additionally, it was detected that some metal ions affect the enzyme activity at different ratios. T esterazı amonyum sülfat çöktürmesi ve hidrofobik etkileşim kromatografisi kullanılarak saflaştırıldı ve biyokimyasal özellikleri incelendi. Bulgular: Doğal-ve SDS-PAGE'de tek bant şeklinde görülen saf enzimin, p-nitrofenil asetat (pNPA) varlığında optimum pH ve sıcaklık değerleri sırasıyla 7,5 ve 60°C olarak belirlendi. Yine pNPA bir substrat olarak kullanıldığında K m ve V max değerlerinin 18,32 mM ve 96,15 U/ mg protein olduğu tespit edildi. pH kararlılığı pH 4,0-9,0 aralığında 4°C ve 60°C'de incelendi. 7 günlük bir inkübasyondan sonra saf enzimin aktivitesinin 4°C'de bütün pH'larda yaklaşık %85-90 oranında korunduğu, bu oranın 60°C'de pH 8,0'de ise %59±5,2 olduğu görüldü. 30-60°C'ler arasında 7 gün inkübe edilen enzim, orijinal aktivitesini %80 oranında korudu. %10 nihai konsantrasyondaki etanol ve DMSO varlığında enzim aktivitesi sırasıyla %96±2,7 ve %78±2,5 oranlarında korundu. Ayrıca, bazı metal iyonlarının enzim aktivitesini farklı oranlarda etkilediği tespit edildi. Sonuç: Bütün bu sonuçlar değerlendirildiğinde Thermus sp. NCCB 100425 T esterazının özel-likle yüksek sıcaklık-ve pH-kararlılığı nedeniyle endüstriyel ve/veya klinik uygulamalar için avantajlara sahip olabileceği açıkça görülmektedir. Thus, this study can be considered as a step to obtain an industrially suitable esterase having excellent features. ) and solvents were purchased from Merck A.G. (Darmstadt, Germany). All chemicals were analytical and microbiological grade. Conclusion Materials and Methods Chemicals Bacterial strain and crude enzyme extractionThermus sp. NCCB 100425 T bacterial strain isolated from a geotherm...

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Isolation of a novel carboxylesterase from Bacillus coagulans with high enantioselectivity toward racemic esters of 1,2-O-isopropylideneglycerol

Cited by 48 publications

References 16 publications

Discovery of an Escherichia coli Esterase with High Activity and Enantioselectivity toward 1,2- O -Isopropylideneglycerol Esters

Discovery of an Escherichia coli Esterase with High Activity and Enantioselectivity toward 1,2- O -Isopropylideneglycerol Esters

An Esterase with Superior Activity and Enantioselectivity towards 1,2‐O‐Isopropylideneglycerol Esters Obtained by Protein Design

Purification and characterization of a novel thermostable esterase from Thermus sp. NCCB 100425T

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