Protein engineering applications of industrially exploitable enzymes: <i>Geobacillus stearothermophilus</i> LDH and <i>Candida methylica</i> FDH

“…For example, GOx and HRP catalyze redox transformations in sensing, catalysis, and waste-water remediation and they serve as cascade dyads. 17–23 In contrast, LDH catalyzes dehydrogenation reactions for the production of enantiopure (−) lactate and 2-hydroxy acids that are precursors to many pharmaceutical drugs 24,25 and for sensing of lactate. 26 Hydrolases like AP are used widely for waste remediation, metal recovery, 27 and determination of surfactants, 28 whereas lipases are employed for the production of pharmaceutical drugs, cosmetics, leather, detergents, foods, perfumery, medical diagnostics and other synthetic materials.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2017

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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.

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“…It has been previously shown that the N-terminal His tag does not influence the activity of cmFDH. 45,51 On the other hand, multi-histidine residues have a non-specific, yet considerable, high affinity to a variety of metal surfaces including gold. To avoid the unpredictable properties of poly-His affinity tag on newly introduced specific gold surface recognition ability, both the peptide-fused and control constructs were specifically designed by introducing a protease cleavage site at the end of poly-His tag region.…”

Section: Genetically Engineered Fdh With the Gold Surface Recognitionmentioning

confidence: 99%

“…[47][48][49] FDHs have been successfully isolated and produced from many different species, but methanol-consuming yeast species, such as Candida methylica (cm), have received the most attention due to the enhanced stability and relatively high activity of their isolated NAD + -dependent FDHs. 45,50,51 In this study, the authors genetically engineered NAD + -dependent FDH from C. methylica to couple with gold-binding peptide as a novel enzyme with chimeric properties. The resulting fusion enzyme was demonstrated to retain the catalytic activity both in solution-and surface-immobilised forms while gaining additional self-organisation ability verified on a variety of gold electrode surfaces.…”

Section: Direct Bioelectrocatalysis At the Interfaces By Genetically mentioning

confidence: 99%

“…43 Furthermore, FDHs have been used for sensing applications to detect formate quantitatively as an important fermentation product of aerobic and anaerobic bacteria. 44,45 The capability to transfer electron pairs between specific substrates and NAD + molecules offers another interesting potential for FDHs as power bioelectronic devices. 46 The industrial importance of NAD + -dependent FDH has led researchers to develop different kinds of immobilisation strategies through a variety of support materials to efficiently enable the desired use of the enzyme.…”

Section: Direct Bioelectrocatalysis At the Interfaces By Genetically mentioning

confidence: 99%

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Direct bioelectrocatalysis at the interfaces by genetically engineered dehydrogenase

Yucesoy

Karaca

Çetinel³

et al. 2015

Bioinspired, Biomimetic and Nanobiomaterials

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There is an emerging interest in developing bio-functionalisation routes serving as platforms for assembling diverse enzymes onto material surfaces. Specifically, the fabrication of next-generation, laboratory-on-a-chip-based sensing and energy-harvesting systems requires controlled orientation and organisation of the proteins at the inorganic interfaces. Herein, the authors take the initial steps towards designing multifunctional, enzyme-based platforms by genetically integrating the engineered materialselective peptide tags for tethering redox enzymes onto electrode surfaces. The authors engineered a fusion protein that genetically conjugates gold-binding peptide to formate dehydrogenase derived from Candida methylica. The expressed proteins were tested for both enzyme activity and self-directed gold-surface functionalisation ability. Their findings demonstrate the successful self-immobilisation of the engineered enzyme onto different gold electrodes while retaining the catalytic activity.Building on the functionalisation by the peptides, a fusion enzyme-integrated circuit-based biosensor system was designed. The catalytic conversion of the formate by the engineered dehydrogenase was successfully monitored on the electrode surface at subsequent intervals. The engineered peptide-mediated self-integrated electrode systems can be extended to develop a wide range of biosensing and energy-harvesting platforms using different combinations of materials and biomolecules. This paper contains supporting information that will be made available online once the issue is published. In the meantime, if you wish to get a copy of the supplementary file, please contact the Journals Editor, Sarah Brown, at sarah.brown@icepublishing.com.

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Protein engineering applications of industrially exploitable enzymes: Geobacillus stearothermophilus LDH and Candida methylica FDH

Cited by 28 publications

References 29 publications

Protein Engineering Applications on Industrially Important Enzymes: Candida methylica FDH as a Case Study

Protein Engineering Applications on Industrially Important Enzymes: Candida methylica FDH as a Case Study

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

Direct bioelectrocatalysis at the interfaces by genetically engineered dehydrogenase

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