Semisynthetic peptide–lipase conjugates for improved biotransformations

Romero, Óscar; Filice, Marco; Rivas, Blanca de las; Carrasco-López, César; Klett, Javier; Morreale, Antonio; Hermoso, J.A.; Guisán, José M.; Abián, Olga; Palomo, José M.

doi:10.1039/c2cc34816k

Cited by 32 publications

(42 citation statements)

References 10 publications

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“…In recent years, several strategies have been applied in order to overcome this question, such as site-directed mutagenesis, directed evolution or chemical modifications [6][7][8][9]. The immobilization technology [10,11] has been a powerful approach for altering the catalytic properties of lipases [12][13][14].…”

Section: Introductionmentioning

confidence: 99%

Changes on enantioselectivity of a genetically modified thermophilic lipase by site-directed oriented immobilization

Godoy

Romero

Rivas

et al. 2013

Journal of Molecular Catalysis B: Enzymatic

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Section: Introductionmentioning

confidence: 99%

Changes on enantioselectivity of a genetically modified thermophilic lipase by site-directed oriented immobilization

Godoy

Romero

Rivas

et al. 2013

Journal of Molecular Catalysis B: Enzymatic

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“…Aldehyde-activated Sepharose or Lewatit VP OC105 (Ald or Ald-Lew105) were prepared as previously described [33]. 3,5-O-diacetylated thymidine 1 was prepared as previously described [34].…”

Section: Methodsmentioning

confidence: 99%

Covalent Immobilization of Candida rugosa Lipase at Alkaline pH and Their Application in the Regioselective Deprotection of Per-O-acetylated Thymidine

Rivero

Palomo

2016

Catalysts

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Abstract:Lipase from Candida rugosa (CRL) was stabilized at alkaline pH to overcome the inactivation problem and was immobilized for the first time by multipoint covalent attachment on different aldehyde-activated matrices. PEG was used as a stabilizing agent on the activity of CRL. At these conditions, CRL maintained 50% activity at pH 10 after 17 h incubation in the presence of 40% (w/v) of PEG, whereas the enzyme without additive was instantaneously inactive after incubation at pH 10. Thus, this enzyme was covalently immobilized at alkaline pH on three aldehyde-activated supports: aldehyde-activated Sepharose, aldehyde-activated Lewatit105 and heterofunctional aldehyde-activated EDA-Sepharose in high overall yields. Heterogeneous stable CRL catalysts at high temperature and solvent were obtained. The aldehyde-activated Sepharose-CRL preparation maintained 70% activity at 50˝C or 30% (v/v) acetonitrile after 22 h and exhibited high regioselectivity in the deprotection process of per-O-acetylated thymidine, producing the 3 1 -OH-5 1 -OAc-thymidine in 91% yield at pH 5.

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“…1). Additionally, this kind of modification can also alter the catalytic properties of enzymes for the creation of novel active and selective biocatalysts (Díaz-Rodríguez & Davis, 2011; Romero et al, 2012). …”

Section: Introductionmentioning

confidence: 99%

Novel enzyme-polymer conjugates for biotechnological applications

Romero

Rivero

Guisán

et al. 2013

PeerJ

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In the present research, a rapid, simple and efficient chemoselective method for the site-directed incorporation of tailor-made polymers into protein to create biocatalysts with excellent properties for pharmaceutical industrial purpose has been performed. First we focused on the protein engineering of the Geobacillus thermocatenulatus lipase 2 (BTL2) to replace the two cysteines (Cys65, Cys296) in the wild type enzyme (BTL-WT) by two serines. Then, by similar mode, a unique cysteine was introduced in the lid area of the protein. For the site-directed polymer incorporation, a set of different tailor-made thiol-ionic-polymers were synthesized and the protein cysteine was previously activated with 2,2-dithiodipyridine (2-PDS) to allow the disulfide exchange. The protected BTL variants were specifically modified with the different polymers in excellent yields, creating a small library of new biocatalysts. Different and important changes in the catalytic properties, possible caused by structural changes in the lid region, were observed. The different modified biocatalysts were tested in the synthesis of intermediates of antiviral and antitumor drugs, like nucleoside analogues and derivatives of phenylglutaric acid. In the hydrolysis of per-acetylated thymidine, the best biocatalyst was the BTL*-193-DextCOOH , where the activity was increased in 3-fold and the regioselectivity was improved, reaching a yield of 92% of 3’-O-acetyl-thymidine. In the case of the asymmetric hydrolysis of dimethyl phenylglutarate, the best result was found with BTL*-193-DextNH2-6000, where the enzyme activity was increased more than 5-fold and the enantiomeric excess was >99%.

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Semisynthetic peptide–lipase conjugates for improved biotransformations

Abstract: An efficient chemoselective method for the creation of semisynthetic lipases by site-specific incorporation of tailor-made peptides on the lipase-lid site was developed. These new enzymes showed excellent improved specificity and regio-or enantioselectivity in different biotransformations.

Cited by 32 publications

References 10 publications

Changes on enantioselectivity of a genetically modified thermophilic lipase by site-directed oriented immobilization

Changes on enantioselectivity of a genetically modified thermophilic lipase by site-directed oriented immobilization

Covalent Immobilization of Candida rugosa Lipase at Alkaline pH and Their Application in the Regioselective Deprotection of Per-O-acetylated Thymidine

Novel enzyme-polymer conjugates for biotechnological applications

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