Directed Evolution of a Bacterial Laccase (CueO) for Enzymatic Biofuel Cells

Zhang, Lingling; Cui, Haiyang; Zhi, Zou; Garakani, Tayebeh Mirzaei; Novoa‐Henriquez, Catalina; Jooyeh, Bahareh; Schwaneberg, Ulrich

doi:10.1002/anie.201814069

Cited by 62 publications

(33 citation statements)

References 44 publications

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“…This is because most of the fungal laccases show an optimum pH of 3–5.5 over phenolic substrates and they rapidly lose their activities at pH > 7 (Xu 1997; Xu et al 1998). In consequence, fungal laccases with optimum pH and high activity at neutral or alkaline conditions are highly desirable, especially for applications in specific industrial processes (Madzak et al 2006; Torres-Salas et al 2013; Schwaneberg et al 2018).…”

Section: Discussionmentioning

confidence: 99%

The first fungal laccase with an alkaline pH optimum obtained by directed evolution and its application in indigo dye decolorization

et al. 2019

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Engineering of fungal laccases with optimum catalytic activity at alkaline pH has been a long-lasting challenge. In this study, a mutant library containing 3000 clones was obtained by error-prone PCR to adapt the optimum pH of a fungal laccase Lcc9 from the basidiomycete Coprinopsis cinerea. After three rounds of functional screening, a mutant with three amino acid changes (E116K, N229D, I393T) named PIE5 was selected. PIE5 showed an optimum pH of 8.5 and 8.0 against guaiacol and 2,6-DMP when expressed in Pichia pastoris, representing the first fungal laccase that possesses an optimum pH at an alkaline condition. Site directed mutagenesis disclosed that N229D contributed the most to the optimum pH increment. A single N229D mutation caused an increase in optimum pH by 1.5 units. When used in indigo dye decolorization, PIE5 efficiently decolorized 87.1 ± 1.1% and 90.9 ± 0.3% indigo dye at the optimum conditions of pH 7.0–7.5 and 60 °C, and with either methyl 3,5-dimethoxy-4-hydroxybenzoate or 2,2′-azino-bis(3-ethylbenzothazoline-6-sulfonate) as the mediator. In comparison, the commercially available fungal laccase TvLac from Trametes villosa decolorized 84.3 ± 1.8% of indigo dye under its optimum conditions (opt. pH 5.0 and 60 °C). The properties of an alkaline-dependent activity and the high indigo dye decolorization ability (1.3-fold better than the parental Lcc9) make the new fungal laccase PIE5 an alternative for specific industrial applications.

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Section: Discussionmentioning

confidence: 99%

The first fungal laccase with an alkaline pH optimum obtained by directed evolution and its application in indigo dye decolorization

et al. 2019

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“…Laccase (benzenediol: oxygen oxidoreductase, EC 1.10.3.2), belonging to a group of enzymes called multicopper blue oxidasehas been noted to exhibit a wide substrate specificity [1]. It has been applied in various sectors, such as biomedical [2], dye degradation [3], paper industries for delignification [4][5], bioremediation [6], in biosensors [7], as melanin degraders in the cosmetic industry [8], as an enzymatic biofuel [9] and used in juice clarification [10]. Furthermore, laccase is a key biological mediator and the best alternative for chemical mediators; thus, it is regarded as a green enzyme in dye degradation, which is a new era for dye degradation [11].…”

Section: Introductionmentioning

confidence: 99%

Tree bark scrape fungus: A potential source of laccase for application in bioremediation of non-textile dyes

Sayyed

Bhamare²,

Sapna

et al. 2020

PLoS ONE

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Although laccase has been recognized as a wonder molecule and green enzyme, the use of low yielding fungal strains, poor production, purification, and low enzyme kinetics have hampered its large-scale application. Thus,this study aims to select high yielding fungal strains and optimize the production, purification, and kinetics of laccase of Aspergillus sp. HB_RZ4. The results obtained indicated that Aspergillus sp. HB_RZ4 produced a significantly large amount of laccase under meso-acidophilic shaking conditions in a medium containing glucose and yeast extract. A 25 μM CuSO 4 was observed to enhance the enzyme yield. The enzyme was best purified on a Sephadex G-100 column. The purified enzyme resembled laccase of A. flavus. The kinetics of the purified enzyme revealed high substrate specificity and good velocity of reaction,using ABTS as a substrate. The enzyme was observed to be stable over various pH values and temperatures. The peptide structure of the purified enzyme was found to resemble laccase of A. kawachii IFO 4308. The fungus was observed to decolorize various dyes independent of the requirement of a laccase mediator system. Aspergillus sp. HB_RZ4 was observed to be a potent natural producer of laccase, and it decolorized the dyes even in the absence of a laccase mediator system. Thus, it can be used for bioremediation of effluent that contains non-textile dyes.

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“…The physical parameters viz. pH (2)(3)(4)(5)(6)(7)(8)(9)(10), temperature (20- were optimized for optimum production of laccase [30].…”

Section: Optimization Of Variables For Growth and Laccase Productionmentioning

confidence: 99%

“…Laccase (benzenediol: oxygen oxidoreductase, EC 1.10.3.2) belonging to a group of an enzyme called a multicopper blue oxidase (MCBO) exhibits wide substrate specificity ranging from phenolic to non-phenolic compounds [1]. It finds applications in various sectors such as biomedical [2], dye degradation [3] (paper industries for delignification [4][5], bioremediation [6], in biosensors [7] in cosmetic industry as melanin degraders [8] and as a enzymatic biofuel [9]. It is the major ligninolytic enzyme used in juice clarification [10].…”

Section: Introductionmentioning

confidence: 99%

Tree bark scrape fungus: A potential source of laccase for application in bioremediation of non-textile dyes

Bhamare¹,

Sayyed

Marraiki

et al. 2020

Preprint

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Although laccase has been recognized as a wonder molecule, and green enzyme, the use of low yielding fungal strains, poor production, purification, and low enzyme kinetics have hampered its larger-scale applications. Hence the present research was aimed to select high yielding fungal strains and to optimize the production, purification, and kinetics of laccase of Aspergillus sp. HB_RZ4. Aspergillus sp. HB_RZ4 produced a copious amount of laccase on under meso-acidophillic shaking conditions in a medium containing glucose and yeast extract. A 25 µM of CuSO 4 enhanced the enzyme yield. The enzyme was best purified on Sephadex G-100 column. Purified enzyme resembled with the laccase of A. flavus. Kinetics of purified enzyme revealed the high substrate specificity and good velocity of reaction with ABTS as substrate. The enzyme was stable over a wide range of pH and temperature. The peptide structure of the purified enzyme resembled with the laccase of A. kawachii IFO 4308. The fungus decolorized various dyes independent of the requirement of a laccase mediator system (LMS). Aspergillus sp. HB_RZ4 came out as a potent natural producer of laccase, it decolorized the dyes even in absence of LMS and thus can be used for bioremediation.

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Directed Evolution of a Bacterial Laccase (CueO) for Enzymatic Biofuel Cells

Cited by 62 publications

References 44 publications

The first fungal laccase with an alkaline pH optimum obtained by directed evolution and its application in indigo dye decolorization

The first fungal laccase with an alkaline pH optimum obtained by directed evolution and its application in indigo dye decolorization

Tree bark scrape fungus: A potential source of laccase for application in bioremediation of non-textile dyes

Tree bark scrape fungus: A potential source of laccase for application in bioremediation of non-textile dyes

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