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Nanoarmoring: strategies for preparation of multi-catalytic enzyme polymer conjugates and enhancement of high temperature biocatalysis.

ABSTRACT: We report a general and modular approach for the synthesis of multi enzyme-polymer conjugates (MECs) consisting of five different enzymes of diverse isoelectric points and distinct catalytic properties conjugated within a single universal polymer scaffold. The five model enzymes chosen include glucose oxidase (GOx), acid phosphatase (AP), lactate dehydrogenase (LDH), horseradish peroxidase (HRP) and lipase (Lip). Poly(acrylic acid) (PAA) is used as the model synthetic polymer scaffold that will covalently conjugate and stabilize multiple enzymes concurrently. Parallel and sequential synthetic protocols are used to synthesise MECs, 5-P and 5-S, respectively. Also, five different single enzyme-PAA conjugates (SECs) including GOx-PAA, AP-PAA, LDH-PAA, HRP-PAA and Lip-PAA are synthesized. The composition, structure and morphology of MECs and SECs are confirmed by agarose gel electrophoresis, dynamic light scattering, circular dichroism spectroscopy and transmission electron microscopy. The bioreactor comprising MEC functions as a single biocatalyst can carry out at least five different or orthogonal catalytic reactions by virtue of the five stabilized enzymes, which has never been achieved to-date. Using activity assays relevant for each of the enzymes, for example AP, the specific activity of AP at room temperature and 7.4 pH in PB is determined and set at 100%. Interestingly, MECs 5-P and 5-S show specific activities of 1800% and 600%, respectively, compared to 100% specific activity of AP at room temperature (RT). The catalytic efficiencies of 5-P and 5-S are 1.55 × 10-3 and 1.68 × 10-3, respectively, compared to 9.11 × 10-5 for AP under similar RT conditions. Similarly, AP relevant catalytic activities of 5-P and 5-S at 65 °C show 100 and 300%, respectively, relative to native AP activity at RT as the native AP is catalytically inactive at 65 °C The catalytic activity trends suggest: (1) MECs show enhanced catalytic activities compared to native enzymes under similar assay conditions and (2) 5-S is better suited for high temperature biocatalysis, while both 5-S and 5-P are suitable for room temperature biocatalysis. Initial cytotoxicity results show that these MECs are non-lethal to human cells including human embryonic kidney [HEK] cells when treated with doses of 0.01 mg mL-1 for 72 h. This cytotoxicity data is relevant for future biological applications.

SUBMITTER: Zore OV

PROVIDER: S-EPMC5796544 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Nanoarmoring: strategies for preparation of multi-catalytic enzyme polymer conjugates and enhancement of high temperature biocatalysis.

Zore Omkar V OV Pande Paritosh P Okifo Oghenenyerovwo O Basu Ashis K AK Kasi Rajeswari M RM Kumar Challa V CV

RSC advances 20170606 47

We report a general and modular approach for the synthesis of multi enzyme-polymer conjugates (MECs) consisting of five different enzymes of diverse isoelectric points and distinct catalytic properties conjugated within a single universal polymer scaffold. The five model enzymes chosen include glucose oxidase (GOx), acid phosphatase (AP), lactate dehydrogenase (LDH), horseradish peroxidase (HRP) and lipase (Lip). Poly(acrylic acid) (PAA) is used as the model synthetic polymer scaffold that will ...[more]

PMID: 29403641

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Project description:The intrinsic resilience of biofilms to environmental conditions makes them an attractive platform for biocatalysis, bioremediation, agriculture or consumer health. However, one of the main challenges in these areas is that beneficial bacteria are not necessarily good at biofilm formation. Currently, this problem is solved by genetic engineering or experimental evolution, techniques that can be costly and time consuming, require expertise in molecular biology and/or microbiology and, more importantly, are not suitable for all types of microorganisms or applications. Here we show that synthetic polymers can be used as an alternative, working as simple additives to nucleate the formation of biofilms. Using a combination of controlled radical polymerization and dynamic covalent chemistry, we prepare a set of synthetic polymers carrying mildly cationic, aromatic, heteroaromatic or aliphatic moieties. We then demonstrate that hydrophobic polymers induce clustering and promote biofilm formation in MC4100, a strain of Escherichia coli that forms biofilms poorly, with aromatic and heteroaromatic moieties leading to the best performing polymers. Moreover, we compare the effect of the polymers on MC4100 against PHL644, an E. coli strain that forms biofilms well due to a single point mutation which increases expression of the adhesin curli. In the presence of selected polymers, MC4100 can reach levels of biomass production and curli expression similar or higher than PHL644, demonstrating that synthetic polymers promote similar changes in microbial physiology than those introduced following genetic modification. Finally, we demonstrate that these polymers can be used to improve the performance of MC4100 biofilms in the biocatalytic transformation of 5-fluoroindole into 5-fluorotryptophan. Our results show that incubation with these synthetic polymers helps MC4100 match and even outperform PHL644 in this biotransformation, demonstrating that synthetic polymers can underpin the development of beneficial applications of biofilms.

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Dataset Information

Nanoarmoring: strategies for preparation of multi-catalytic enzyme polymer conjugates and enhancement of high temperature biocatalysis.

Publications

Nanoarmoring: strategies for preparation of multi-catalytic enzyme polymer conjugates and enhancement of high temperature biocatalysis.

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