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Novel enzyme-polymer conjugates for biotechnological applications.

ABSTRACT: 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%.

SUBMITTER: Romero O

PROVIDER: S-EPMC3628993 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Novel enzyme-polymer conjugates for biotechnological applications.

Romero Oscar O Rivero Cintia W CW Guisan Jose M JM Palomo Jose M JM

PeerJ 20130212

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 introdu ...[more]

PMID: 23638362

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Project description:The development of multifunctional hybrid biomaterials is an important area of focus in tissue engineering, drug delivery, biocatalysis, and biosensing applications. Combining bioconjugation methodology with ice templating technique, we show here the development of a new class of multifunctional and biocatalytic scaffold-like spongy material fabricated from an aqueous solution of enzyme-polymer surfactant (enzyme-PS) core-shell conjugates, and polyethyleneimine (PEI) coated silica/silk nanoparticles. The generality of this process is demonstrated by the fabrication of biocatalytic sponges comprising PEI coated nanoparticles and core-shell conjugates of alkaline phosphatase (ALP-PS), or glucose oxidase (GOx-PS), and horseradish peroxidase (HRP-PS). We show that ALP-PS conjugate driven biocatalytic transformations can be simply achieved by saturating the highly porous, and manoeuvrable sponges with the p-nitrophenyl phosphate substrate solution. Subsequently, the compressible and elastic property of the sponge can be utilized for the extrusion of the product, p-nitrophenol, by applying controlled and normal mechanical stress. Further, the sponges can be washed and recycled upto ten times, with approximately 67% retention of initial biocatalytic activity. Interestingly, the ALP-PS conjugate based sponges exhibit mechanoresponsive catalytic behaviour; the amount of product obtained over 25 minutes of reaction time can be increased by approx. 8 times by compressing-decompressing the sponge after every 15 seconds. This is attributed to the change in mass transfer and diffusion of the substrate within the porous channels of the sponge. We also highlight the importance of bioconjugation of enzymes for fabricating such sponges; our results show that, whilst the native enzymes either denature or are leached away during the fabrication/biocatalytic usage, their enzyme-PS conjugate counterparts integrate efficiently to form sturdy, robust, highly catalytic, and recyclable sponge material.

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Novel enzyme-polymer conjugates for biotechnological applications.

Publications

Novel enzyme-polymer conjugates for biotechnological applications.

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