Who's Who? Allocation of Carbonyl Reductase Isoenzymes from <i>Candida parapsilosis</i> by Combining Bio‐ and Computational Chemistry

Jakoblinnert, Andre; Bocola, Marco; Bhattacharjee, Monideepa; Steinsiek, Sonja; Bönitz-Dulat, Mara; Schwaneberg, Ulrich; Ansorge‐Schumacher, Marion B.

doi:10.1002/cbic.201200023

Cited by 26 publications

(21 citation statements)

References 27 publications

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“…The enzymew as first described, purified, andc haracterizedb yP eters and co-workers. [11] Thereby,itwas demonstrated that the enzyme preparation used previously actually consisted of two different ADHs, CPCR1 and CPCR2, the latter being mainly responsible for the broad substrate spectrum described in previouss tudies. [11] Thereby,itwas demonstrated that the enzyme preparation used previously actually consisted of two different ADHs, CPCR1 and CPCR2, the latter being mainly responsible for the broad substrate spectrum described in previouss tudies.…”

Section: Introductionmentioning

confidence: 80%

“…[3b, [6][7] Later,t he enzymew as genetically located, cloned, and expressed in Escherichia coli. [11] Thereby,itwas demonstrated that the enzyme preparation used previously actually consisted of two different ADHs, CPCR1 and CPCR2, the latter being mainly responsible for the broad substrate spectrum described in previouss tudies. It became obvious that the same enzyme had independently been described and applied under ad ifferent name before.…”

Section: Introductionmentioning

confidence: 80%

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Investigation of Structural Determinants for the Substrate Specificity in the Zinc‐Dependent Alcohol Dehydrogenase CPCR2 from Candida parapsilosis

et al. 2015

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Zinc-dependent alcohol dehydrogenases (ADHs) are a class of enzymes applied in different biocatalytic processes ranging from lab to industrial scale. However, one drawback is the limited substrate range, necessitating a whole array of different ADHs for the relevant substrate classes. In this study, we investigated structural determinants of the substrate spectrum in the zinc-dependent ADH carbonyl reductase 2 from Candida parapsilosis (CPCR2), combining methods of mutational analysis with in silico substrate docking. Assigned active site residues were genetically randomized, and the resulting mutant libraries were screened with a selection of challenging carbonyl substrates. Three variants (C57A, W116K, and L119M) with improved activities toward different substrates were detected at neighboring positions in the active site. Thus, all possible combinations of the mutations were generated and characterized for their substrate specificity, yielding several improved variants. The most interesting were a C57A variant, with a 27-fold increase in specific activity for 4'-acetamidoacetophenone, and the double mutant CPCR2 B16-(C57A, L119M), with a 45-fold improvement in the kcat ⋅KM (-1) value. The obtained variants were further investigated by in silico docking experiments. The results indicate that the mentioned residues are structural determinants of the substrate specificity of CPCR2, being major players in the definition of the active site. Comparison of these results with closely related enzymes suggests that these might even be transferred to other ADHs.

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Section: Introductionmentioning

confidence: 80%

Section: Introductionmentioning

confidence: 80%

Investigation of Structural Determinants for the Substrate Specificity in the Zinc‐Dependent Alcohol Dehydrogenase CPCR2 from Candida parapsilosis

et al. 2015

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“…The likeability of the latter has for example been demonstrated for the carbonyl reductase activity of Candida parapsilosis described by Kula and coworkers in 1995 [20]. Only in 2011 it became clear, that the CPCR preparation actually contained two enzymes where one is mainly responsible for the characteristics that were initially described [21].…”

Section: Kred-activity Of Scleroderma Citrinummentioning

confidence: 88%

Magic Mushrooms: Screening for Novel Biocatalysts in the Phylum Basidiomycota

Loderer¹,

Ansorge‐Schumacher²

2016

ABB

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The ascending application of enzymes in organic synthesis creates a growing demand for novel biocatalysts. The applied methods for their identification range from microbial enrichment cultures over metagenome screenings to solely computational methods. In this communication, we demonstrate a straightforward screening approach for the detection of novel biocatalysts in fungi belonging to the phylum Basidiomycota. It basically relies on mincing of the whole fruit bodies of freshly collected mushrooms with subsequent direct screening. Suitability was demonstrated with eight different mushrooms which were investigated for carbonyl reductase activity on sterically demanding carbonyl compounds. The results indicate the presence of potentially useful carbonyl reductases (KREDs) in all tested fungi. Closer characterization of the preparation from pigskin poison puffball (Scleroderma citrinum) showed the presence of KRED exhibiting a broad substrate range. Thus, applicability of this low-tech screening approach could be verified in this study.

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“…19 Such silicones are mostly generated by the treatment of “standard” silicone with UV/ozone20 or plasma discharge followed by silanization21 or by incorporation of hydrophilic moieties into the matrix 22. A simple method for the incorporation of hydrophilic compounds involves vinylic groups because these enable direct cross‐linking during hydrosilylation,23 although this requires preliminary modification of the hydrophilic components 24…”

Section: Introductionmentioning

confidence: 99%

“…The hydrophilization of silicone for the preparation of silCoat biocatalysts was performed with carbonyl reductase from Candida parapsilosis (CPCR2)24 as a model enzyme and poly(ethylene glycol) (PEG) as an environmentally benign and enzyme‐friendly hydrophilizer 23. 25–27 CPCR2 is a synthetically interesting enzyme owing to its broad substrate range and high enantioselectivity26 requiring a distinctly aqueous environment for appropriate activity.…”

Section: Introductionmentioning

confidence: 99%

Hydrophilized Silicone Matrix for the Preparation of Stable Carbonyl Reductase Immobilizates

2013

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Industrial use of enzymes requires high activity, stability, and manageability under technical conditions. Therefore, most biocatalysts are immobilized before use, often by physical adsorption onto a solid carrier. However, a major limitation for the technical use of such immobilizates is the insufficient operational stability under process conditions resulting from enzyme leaching and carrier disintegration. Our group demonstrated recently that for lipase‐catalyzed esterifications these limitations can be largely overcome by the formation of silCoat biocatalysts, composite materials of carrier‐bound enzymes and silicone. Here, we report the successful transformation of this method to the immobilization of carbonyl reductase (CR), an enzyme representing oxidoreductases as a completely different class of biocatalysts. The silCoat biocatalysts cannot be transferred directly to CR because they require an overall hydrophilic surrounding, which opposes the hydrophobicity of silicone. Consequently, the hydrophilization of the material is necessary to achieve reasonable enzyme activity. This was realized by the rational modification of the precursors for silicone formation and applied to increase the leaching stability and mechanical strength of the carrier‐bound CR. The resulting HYsilCoat CR had a significantly longer half‐life than that obtained through the uncoated preparation; the mechanical stability of an alumina carrier was increased by a factor of 120. This enables the consideration of mechanically unstable but environmentally friendly and/or cheap material as an enzyme carrier for industrial use.

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Who's Who? Allocation of Carbonyl Reductase Isoenzymes from Candida parapsilosis by Combining Bio‐ and Computational Chemistry

Cited by 26 publications

References 27 publications

Investigation of Structural Determinants for the Substrate Specificity in the Zinc‐Dependent Alcohol Dehydrogenase CPCR2 from Candida parapsilosis

Investigation of Structural Determinants for the Substrate Specificity in the Zinc‐Dependent Alcohol Dehydrogenase CPCR2 from Candida parapsilosis

Magic Mushrooms: Screening for Novel Biocatalysts in the Phylum Basidiomycota

Hydrophilized Silicone Matrix for the Preparation of Stable Carbonyl Reductase Immobilizates

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