Intense PEGylation of Enzyme Surfaces

Moreno-Pérez, Sonia; Orrego, Alejandro H.; Romero‐Fernández, María; Trobo-Maseda, Lara; Martins-DeOliveira, S.; Munilla, Roberto; Fernández‐Lorente, Gloria; Guisán, José M.

doi:10.1016/bs.mie.2016.02.016

Cited by 31 publications

(11 citation statements)

References 16 publications

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“…Finally, the stability of TLL in anhydrous hexane is quite low for practical application of these interesting processes. High stabilizations of TLL adsorbed on hydrophobic agarose gels (by coating with polymers) have been reported for thermal inactivation of TLL derivatives in aqueous media [ 69 ]. Similar protocols could be tested for stabilization of immobilized TLL in anhydrous media.…”

Section: Discussionmentioning

confidence: 99%

Modulation of the regioselectivity of Thermomyces lanuginosus lipase via biocatalyst engineering for the Ethanolysis of oil in fully anhydrous medium

Silveira

Moreno-Pérez

Basso³

et al. 2017

BMC Biotechnol

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BackgroundEnzymatic ethanolysis of oils (for example, high oleic sunflower oil containing 90% of oleic acid) may yield two different reaction products depending on the regioselectivity of the immobilized lipase biocatalyst. Some lipase biocatalysts exhibit a 1,3-regioselectivity and they produced 2 mols of fatty acid ethyl ester plus 1 mol of sn2-monoacylglycerol (2-MAG) per mol of triglyceride without the release of glycerol. Other lipase biocatalysts are completely non-regioselective releasing 3 mols of fatty acid ethyl ester and 1 mol of glycerol per mol of triglyceride. Lipase from Thermomyces lanuginosus (TLL) adsorbed on hydrophobic supports is a very interesting biocatalyst for the ethanolysis of oil. Modulation of TLL regioselectivity in anhydrous medium was intended via two strategies of TLL immobilization: a. - interfacial adsorption on different hydrophobic supports and b.- interfacial adsorption on a given hydrophobic support under different experimental conditions.ResultsImmobilization of TLL on supports containing divinylbenezene moieties yielded excellent 1,3-regioselective biocatalysts but immobilization of TLL on supports containing octadecyl groups yielded non-regioselective biocatalysts.On the other hand, TLL immobilized on Purolite C18 at pH 8.5 and 30 °C in the presence of traces of CTAB yielded a biocatalyst with a perfect 1,3-regioselectivity and a very interesting activity: 2.5 μmols of oil ethanolyzed per min per gram of immobilized derivative. This activity is 10-fold higher than the one of commercial Lipozyme TL IM. Immobilization of the same enzyme on the same support, but at pH 7.0 and 25 °C, led to a biocatalyst which can hydrolyze all ester bonds in TG backbone.ConclusionsActivity and regioselectivity of TLL in anhydrous media can be easily modulated via Biocatalysis Engineering producing very active immobilized derivatives able to catalyze the ethanolysis of triolein. When the biocatalyst was 1,3-regioselective a 33% of 2-monoolein was obtained and it may be a very interesting surfactant. When biocatalyst catalyzed the ethanolysis of the 3 positions during the reaction process, a 99% of ethyl oleate was obtained and it may be a very interesting drug-solvent and surfactant. The absence of acyl migrations under identical reaction conditions is clearly observed and hence the different activities and regioselectivities seem to be due to the different catalytic properties of different derivatives of TLL.

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Section: Discussionmentioning

confidence: 99%

Modulation of the regioselectivity of Thermomyces lanuginosus lipase via biocatalyst engineering for the Ethanolysis of oil in fully anhydrous medium

Silveira

Moreno-Pérez

Basso³

et al. 2017

BMC Biotechnol

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“…These derivatives were very active and highly stabilized. Additionally, immobilization of the enzyme through the amino termini allows the region with the highest density of lysine residues to remain unaltered, thus permitting further stabilization protocols with polymers (Moreno-Pérez et al, 2016).…”

Section: Introductionmentioning

confidence: 99%

Capture of enzyme aggregates by covalent immobilization on solid supports. Relevant stabilization of enzymes by aggregation

García-García

Fernández‐Lorente

Guisán

2021

Journal of Biotechnology

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Amino oxidase of P. sativum (AO) was stabilized (200 fold) by immobilization tools  30% PEG promotes the formation of soluble bimolecular enzyme aggregates  The aggregate is ¡ §fixed¡¨ by multipoint covalent immobilization (MCI) on solid supports  Enzyme aggregation protects AO from distortions promoted by MCI  Enzyme aggregation promotes a 40 fold increase of thermal stability of AO

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“…Upon improvement of the recombinant expression of HsTP, we undertook efforts to optimize its PEGylation. PEG has been increasingly used in the field of biologics during the recent decades as a time extension strategy aiming at the improvement of the thermodynamic stability (Santos et al, 2019;Moreno-Pérez et al, 2016;immunogenicity (Gefen et al, 2013) and the pharmacokinetic (PK) properties of therapeutic proteins (Harris et al, 2001;Hamidi et al, 2006). Initial efforts to conjugate HsTP 199 with methoxy-5-kDa-PEG-succinimidyl-succinate (mPEG 5kDa ) targeting primary amines on the surface (primarily lysines and N'-terminus-amino group) resulted in an apparent heterogeneous mixture of three distinct PEGylated enzyme species (Figures 4A,B) indicative of an inefficient PEGylation reaction.…”

Section: Pegylation Optimization Of Hstpmentioning

confidence: 99%

Engineering of the Recombinant Expression and PEGylation Efficiency of the Therapeutic Enzyme Human Thymidine Phosphorylase

Karamitros

Somody

Agnello

et al. 2021

Front. Bioeng. Biotechnol.

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Human thymidine phosphorylase (HsTP) is an enzyme with important implications in the field of rare metabolic diseases. Defective mutations of HsTP lead to mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), a disease with a high unmet medical need that is associated with severe neurological and gastrointestinal complications. Current efforts focus on the development of an enzyme replacement therapy (ERT) using the Escherichia coli ortholog (EcTP). However, bacterial enzymes are counter-indicated for human therapeutic applications because they are recognized as foreign by the human immune system, thereby eliciting adverse immune responses and raising significant safety and efficacy risks. Thus, it is critical to utilize the HsTP enzyme as starting scaffold for pre-clinical drug development, thus de-risking the safety concerns associated with the use of bacterial enzymes. However, HsTP expresses very poorly in E. coli, whereas its PEGylation, a crucial chemical modification for achieving long serum persistence of therapeutic enzymes, is highly inefficient and negatively affects its catalytic activity. Here we focused on the engineering of the recombinant expression profile of HsTP in E. coli cells, as well as on the optimization of its PEGylation efficiency aiming at the development of an alternative therapeutic approach for MNGIE. We show that phylogenetic and structural analysis of proteins can provide important insights for the rational design of N’-terminus-truncation constructs which exhibit significantly improved recombinant expression levels. In addition, we developed and implemented a criteria-driven rational surface engineering strategy for the substitution of arginine-to-lysine and lysine-to-arginine residues to achieve more efficient, homogeneous and reproducible PEGylation without negatively affecting the enzymatic catalytic activity upon PEGylation. Collectively, our proposed strategies provide an effective way to optimize enzyme PEGylation and E. coli recombinant expression and are likely applicable for other proteins and enzymes.

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Intense PEGylation of Enzyme Surfaces

Cited by 31 publications

References 16 publications

Modulation of the regioselectivity of Thermomyces lanuginosus lipase via biocatalyst engineering for the Ethanolysis of oil in fully anhydrous medium

Modulation of the regioselectivity of Thermomyces lanuginosus lipase via biocatalyst engineering for the Ethanolysis of oil in fully anhydrous medium

Capture of enzyme aggregates by covalent immobilization on solid supports. Relevant stabilization of enzymes by aggregation

Engineering of the Recombinant Expression and PEGylation Efficiency of the Therapeutic Enzyme Human Thymidine Phosphorylase

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