Pub Date : 2022-05-10DOI: 10.3389/fctls.2022.900554
A. S. de Miranda, C. D. Milagre, F. Hollmann
Alcohol dehydrogenases (ADHs) have become important catalysts for stereoselective oxidation and reduction reactions of alcohols, aldehydes and ketones. The aim of this contribution is to provide the reader with a timely update on the state-of-the-art of ADH-catalysis. Mechanistic basics are presented together with practical information about the use of ADHs. Current concepts of ADH engineering and ADH reactions are critically discussed. Finally, this contribution highlights some prominent examples and future-pointing concepts.
{"title":"Alcohol Dehydrogenases as Catalysts in Organic Synthesis","authors":"A. S. de Miranda, C. D. Milagre, F. Hollmann","doi":"10.3389/fctls.2022.900554","DOIUrl":"https://doi.org/10.3389/fctls.2022.900554","url":null,"abstract":"Alcohol dehydrogenases (ADHs) have become important catalysts for stereoselective oxidation and reduction reactions of alcohols, aldehydes and ketones. The aim of this contribution is to provide the reader with a timely update on the state-of-the-art of ADH-catalysis. Mechanistic basics are presented together with practical information about the use of ADHs. Current concepts of ADH engineering and ADH reactions are critically discussed. Finally, this contribution highlights some prominent examples and future-pointing concepts.","PeriodicalId":73071,"journal":{"name":"Frontiers in catalysis","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-05-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"48137485","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2022-04-26DOI: 10.3389/fctls.2022.887458
Carolin Bertelmann, M. Mock, R. Koch, A. Schmid, B. Bühler
The implementation of biocatalytic steroid hydroxylation processes at an industrial scale still suffers from low conversion rates. In this study, we selected variants of the self-sufficient cytochrome P450 monooxygenase BM3 from Bacillus megaterium (BM3) for the hydroxylation of testosterone either at the 2β- or 15β-position. Recombinant Escherichia coli cells were used as biocatalysts to provide a protective environment for recombinant enzymes and to ensure continuous cofactor recycling via glucose catabolism. However, only low initial whole-cell testosterone conversion rates were observed for resting cells. Results obtained with different biocatalyst formats (permeabilized cells, cell-free extracts, whole cells) indicated a limitation in substrate uptake, most likely due to the hydrophilic character of the outer membrane of E. coli. Thus, we co-expressed nine genes encoding hydrophobic outer membrane proteins potentially facilitating steroid uptake. Indeed, the application of four candidates led to increased initial testosterone hydroxylation rates. Respective whole-cell biocatalysts even exceeded activities obtained with permeabilized cells or cell-free extracts. The highest activity of 34 U gCDW −1 was obtained for a strain containing the hydrophobic outer membrane protein AlkL from Pseudomonas putida GPo1 and the BM3 variant KSA14m. Overall, we show that the straightforward application of hydrophobic outer membrane pores can boost whole-cell steroid conversion rates and thus be game-changing with regard to industrial steroid production efficiency.
{"title":"Hydrophobic Outer Membrane Pores Boost Testosterone Hydroxylation by Cytochrome P450 BM3 Containing Cells","authors":"Carolin Bertelmann, M. Mock, R. Koch, A. Schmid, B. Bühler","doi":"10.3389/fctls.2022.887458","DOIUrl":"https://doi.org/10.3389/fctls.2022.887458","url":null,"abstract":"The implementation of biocatalytic steroid hydroxylation processes at an industrial scale still suffers from low conversion rates. In this study, we selected variants of the self-sufficient cytochrome P450 monooxygenase BM3 from Bacillus megaterium (BM3) for the hydroxylation of testosterone either at the 2β- or 15β-position. Recombinant Escherichia coli cells were used as biocatalysts to provide a protective environment for recombinant enzymes and to ensure continuous cofactor recycling via glucose catabolism. However, only low initial whole-cell testosterone conversion rates were observed for resting cells. Results obtained with different biocatalyst formats (permeabilized cells, cell-free extracts, whole cells) indicated a limitation in substrate uptake, most likely due to the hydrophilic character of the outer membrane of E. coli. Thus, we co-expressed nine genes encoding hydrophobic outer membrane proteins potentially facilitating steroid uptake. Indeed, the application of four candidates led to increased initial testosterone hydroxylation rates. Respective whole-cell biocatalysts even exceeded activities obtained with permeabilized cells or cell-free extracts. The highest activity of 34 U gCDW −1 was obtained for a strain containing the hydrophobic outer membrane protein AlkL from Pseudomonas putida GPo1 and the BM3 variant KSA14m. Overall, we show that the straightforward application of hydrophobic outer membrane pores can boost whole-cell steroid conversion rates and thus be game-changing with regard to industrial steroid production efficiency.","PeriodicalId":73071,"journal":{"name":"Frontiers in catalysis","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-04-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"45188223","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2022-04-08DOI: 10.3389/fctls.2022.883263
M. Dolz, Ivan Mateljak, D. Méndez-Sánchez, Israel Sánchez-Moreno, Patricia Gomez de Santos, Javier Viña‐Gonzalez, M. Alcalde
At present, the end-of-life management of thermoset composite epoxy resins is limited to incineration and landfill storage, highlighting the demand for the development of more sustainable measures. Due to their broad spectrum of C-H oxyfunctionalization reactions, fungal unspecific peroxygenases (UPOs) are becoming important biotechnological tools in organic synthesis while their potential use in biodegradation processes should not be underestimated. Here, we present a colorimetric screening assay aimed at engineering UPOs for the degradation of epoxy resins. We based our study on Hexflow® RTM-6, a commercial epoxy resin used extensively in the aeronautics sector. UPO mutants from the short and long families were initially benchmarked by GC/MS to determine their potential N-dealkylation activity on N,N-bis(2-hydroxypropyl)-p-toluidine (NNBT), the main structural scaffold of Hexflow® RTM-6. A reliable high-throughput colorimetric screening method was developed to quantify the lactaldehyde released by UPO attack on the tertiary amine of NNBT. Based on an evolved UPO from Psathyrella aberdarensis that was expressed by yeast, a small subset of mutant libraries with different mutational loadings was constructed and screened for NNBT N-dealkylation, thereby establishing a directed evolution platform as a vehicle to engineer UPO composite degrading variants.
{"title":"Colorimetric High-Throughput Screening Assay to Engineer Fungal Peroxygenases for the Degradation of Thermoset Composite Epoxy Resins","authors":"M. Dolz, Ivan Mateljak, D. Méndez-Sánchez, Israel Sánchez-Moreno, Patricia Gomez de Santos, Javier Viña‐Gonzalez, M. Alcalde","doi":"10.3389/fctls.2022.883263","DOIUrl":"https://doi.org/10.3389/fctls.2022.883263","url":null,"abstract":"At present, the end-of-life management of thermoset composite epoxy resins is limited to incineration and landfill storage, highlighting the demand for the development of more sustainable measures. Due to their broad spectrum of C-H oxyfunctionalization reactions, fungal unspecific peroxygenases (UPOs) are becoming important biotechnological tools in organic synthesis while their potential use in biodegradation processes should not be underestimated. Here, we present a colorimetric screening assay aimed at engineering UPOs for the degradation of epoxy resins. We based our study on Hexflow® RTM-6, a commercial epoxy resin used extensively in the aeronautics sector. UPO mutants from the short and long families were initially benchmarked by GC/MS to determine their potential N-dealkylation activity on N,N-bis(2-hydroxypropyl)-p-toluidine (NNBT), the main structural scaffold of Hexflow® RTM-6. A reliable high-throughput colorimetric screening method was developed to quantify the lactaldehyde released by UPO attack on the tertiary amine of NNBT. Based on an evolved UPO from Psathyrella aberdarensis that was expressed by yeast, a small subset of mutant libraries with different mutational loadings was constructed and screened for NNBT N-dealkylation, thereby establishing a directed evolution platform as a vehicle to engineer UPO composite degrading variants.","PeriodicalId":73071,"journal":{"name":"Frontiers in catalysis","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-04-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"47125898","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2022-04-06DOI: 10.3389/fctls.2022.839072
Xincong Lv, F. L. Lam, Xijun Hu
Solar energy transformation over semiconductor-based photocatalysis is an ideal solution to environmental problems and future sustainability. Layered bismuth oxyhalides (BiOX, X = Cl, Br or I) are very attractive and promising photocatalysts in the environment fields. This review summarizes recent advances on the design of BiOX to enhance energy converting efficiency. Especially, the emerging techniques to enhance the photocatalytic behaviors of BiOX are discussed, including non-metal/metal doping, heterojunction engineering, carbon interfacing, coupling with noble metals, defect engineering, and morphology tuning. The application of BiOX composites in wastewater remediation is also reviewed in terms of organic photocatalytic oxidation and heavy metal ion photocatalytic reduction. Finally, the future chances and challenges of BiOX photocatalysts for practical application are summarized. In all, this review well underlies the innovative preparation of BiOX products for environment-related purposes.
{"title":"A Review on Bismuth Oxyhalide (BiOX, X=Cl, Br, I) Based Photocatalysts for Wastewater Remediation","authors":"Xincong Lv, F. L. Lam, Xijun Hu","doi":"10.3389/fctls.2022.839072","DOIUrl":"https://doi.org/10.3389/fctls.2022.839072","url":null,"abstract":"Solar energy transformation over semiconductor-based photocatalysis is an ideal solution to environmental problems and future sustainability. Layered bismuth oxyhalides (BiOX, X = Cl, Br or I) are very attractive and promising photocatalysts in the environment fields. This review summarizes recent advances on the design of BiOX to enhance energy converting efficiency. Especially, the emerging techniques to enhance the photocatalytic behaviors of BiOX are discussed, including non-metal/metal doping, heterojunction engineering, carbon interfacing, coupling with noble metals, defect engineering, and morphology tuning. The application of BiOX composites in wastewater remediation is also reviewed in terms of organic photocatalytic oxidation and heavy metal ion photocatalytic reduction. Finally, the future chances and challenges of BiOX photocatalysts for practical application are summarized. In all, this review well underlies the innovative preparation of BiOX products for environment-related purposes.","PeriodicalId":73071,"journal":{"name":"Frontiers in catalysis","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-04-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49512488","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2022-03-30DOI: 10.3389/fctls.2022.806316
Yanlong Xie, Jiao Wang, Fengguo Ren, H. Shuai, G. Du
The composite photocatalyst can be constructed by using natural porous minerals with a wide range of sources, low prices and stable chemical properties as the titanium dioxide catalyst carrier, which can not only reduce the cost of catalyst preparation and application, but also effectively improve the dispersion, recyclability and catalytic performance of the catalyst. In recent years, a large number of scientific researchers have conducted a lot of research on the preparation and performance of porous mineral-supported composite catalytic materials. This paper describes the catalytic mechanism of titanium dioxide, as well as the research and application progress of various nonmetallic mineral materials supported by titanium dioxide, and prospects the development trend in the future.
{"title":"Nonmetallic Mineral as the Carrier of TiO2 Photocatalyst: A Review","authors":"Yanlong Xie, Jiao Wang, Fengguo Ren, H. Shuai, G. Du","doi":"10.3389/fctls.2022.806316","DOIUrl":"https://doi.org/10.3389/fctls.2022.806316","url":null,"abstract":"The composite photocatalyst can be constructed by using natural porous minerals with a wide range of sources, low prices and stable chemical properties as the titanium dioxide catalyst carrier, which can not only reduce the cost of catalyst preparation and application, but also effectively improve the dispersion, recyclability and catalytic performance of the catalyst. In recent years, a large number of scientific researchers have conducted a lot of research on the preparation and performance of porous mineral-supported composite catalytic materials. This paper describes the catalytic mechanism of titanium dioxide, as well as the research and application progress of various nonmetallic mineral materials supported by titanium dioxide, and prospects the development trend in the future.","PeriodicalId":73071,"journal":{"name":"Frontiers in catalysis","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-03-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"45799514","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2022-03-21DOI: 10.3389/fctls.2022.858706
Bastien O. Burek, A. Dawood, F. Hollmann, A. Liese, D. Holtmann
Enzyme catalysis, made tremendous progress over the last years in identification of new enzymes and new enzymatic reactivity’s as well as optimization of existing enzymes. However, the performance of the resulting processes is often still limited, e.g., in regard of productivity, realized product concentrations and the stability of the enzymes. Different topics (like limited specific activity, unfavourable kinetics or limited enzyme stability) can be addressed via enzyme engineering. On the other hand, there is also a long list of topics that are not addressable by enzyme engineering. Here typical examples are unfavourable reaction thermodynamics, selectivity in multistep reactions or low water solubility. These challenges can only be addressed through an adaption of the reaction system. The procedures of process intensification (PI) represent a good approach to reach most suitable systems. The general objective of PI is to achieve significant benefits in terms of capital and operating costs as well as product quality, waste, and process safety by applying innovative principles. The aim of the review is to show the current capabilities and future potentials of PI in enzyme catalysis focused on enzymes of the class of oxidoreductases. The focus of the paper is on alternative methods of energy input, innovative reactor concepts and reaction media with improved properties.
{"title":"Process Intensification as Game Changer in Enzyme Catalysis","authors":"Bastien O. Burek, A. Dawood, F. Hollmann, A. Liese, D. Holtmann","doi":"10.3389/fctls.2022.858706","DOIUrl":"https://doi.org/10.3389/fctls.2022.858706","url":null,"abstract":"Enzyme catalysis, made tremendous progress over the last years in identification of new enzymes and new enzymatic reactivity’s as well as optimization of existing enzymes. However, the performance of the resulting processes is often still limited, e.g., in regard of productivity, realized product concentrations and the stability of the enzymes. Different topics (like limited specific activity, unfavourable kinetics or limited enzyme stability) can be addressed via enzyme engineering. On the other hand, there is also a long list of topics that are not addressable by enzyme engineering. Here typical examples are unfavourable reaction thermodynamics, selectivity in multistep reactions or low water solubility. These challenges can only be addressed through an adaption of the reaction system. The procedures of process intensification (PI) represent a good approach to reach most suitable systems. The general objective of PI is to achieve significant benefits in terms of capital and operating costs as well as product quality, waste, and process safety by applying innovative principles. The aim of the review is to show the current capabilities and future potentials of PI in enzyme catalysis focused on enzymes of the class of oxidoreductases. The focus of the paper is on alternative methods of energy input, innovative reactor concepts and reaction media with improved properties.","PeriodicalId":73071,"journal":{"name":"Frontiers in catalysis","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49006668","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2022-03-16DOI: 10.3389/fctls.2022.835919
T. Gerlach, Jendrik Schain, Simone Söltl, Morten M. C. H. van Schie, F. Hilgers, N. Bitzenhofer, T. Drepper, D. Rother
Genetically encoded photosensitizers are able to produce reactive oxygen species upon illumination and are exploited in a wide range of applications, especially in the medical field. In this work, we envisioned to further apply these genetically encoded photosensitizers for the light-dependent control of single enzymes in multi-step biocatalysis. One of the challenges in the application of several enzymes in a cascade is the unwanted cross-reactivity of these biocatalysts on reaction intermediates when all enzymes are simultaneously present in the reaction. As one strategy to address this issue, we investigated whether the introduction of genetically encoded photosensitizers as fusion tags would allow the selective inactivation of enzymes after successful transformation by simply turning on light. We tested five different photosensitizers as molecular biological fusion tags to inactivate the pyruvate decarboxylase variant E469G/W543H from Acetobacter pasteurianus. Dimeric photosensitizer tags, like the flavin-binding fluorescent proteins from Bacillus subtilis and Pseudomonas putida showed the tendency to form insoluble protein aggregates in combination with the tetrameric carboligase. Enzyme activity was, to some extent, retained in these aggregates, but the handling of the insoluble aggregates proved to be unfeasible. Monomeric photosensitizer tags appeared to be much more suitable when fused to the tetrameric enzyme. In the dark, the singlet oxygen photosensitizing protein (SOPP3)-tagged carboligase retained 79% of its activity as compared to the unfused enzyme. Upon blue light exposure, the SOPP3 tag showed the best specific inactivation and enabled complete inactivation of the carboligase within 30 min. SOPP3 is thus seen as a promising photosensitizer tag to be applied in future multi-step enzyme cascades to overcome the challenge of cross-reactivity.
{"title":"Photo-Regulation of Enzyme Activity: The Inactivation of a Carboligase with Genetically Encoded Photosensitizer Fusion Tags","authors":"T. Gerlach, Jendrik Schain, Simone Söltl, Morten M. C. H. van Schie, F. Hilgers, N. Bitzenhofer, T. Drepper, D. Rother","doi":"10.3389/fctls.2022.835919","DOIUrl":"https://doi.org/10.3389/fctls.2022.835919","url":null,"abstract":"Genetically encoded photosensitizers are able to produce reactive oxygen species upon illumination and are exploited in a wide range of applications, especially in the medical field. In this work, we envisioned to further apply these genetically encoded photosensitizers for the light-dependent control of single enzymes in multi-step biocatalysis. One of the challenges in the application of several enzymes in a cascade is the unwanted cross-reactivity of these biocatalysts on reaction intermediates when all enzymes are simultaneously present in the reaction. As one strategy to address this issue, we investigated whether the introduction of genetically encoded photosensitizers as fusion tags would allow the selective inactivation of enzymes after successful transformation by simply turning on light. We tested five different photosensitizers as molecular biological fusion tags to inactivate the pyruvate decarboxylase variant E469G/W543H from Acetobacter pasteurianus. Dimeric photosensitizer tags, like the flavin-binding fluorescent proteins from Bacillus subtilis and Pseudomonas putida showed the tendency to form insoluble protein aggregates in combination with the tetrameric carboligase. Enzyme activity was, to some extent, retained in these aggregates, but the handling of the insoluble aggregates proved to be unfeasible. Monomeric photosensitizer tags appeared to be much more suitable when fused to the tetrameric enzyme. In the dark, the singlet oxygen photosensitizing protein (SOPP3)-tagged carboligase retained 79% of its activity as compared to the unfused enzyme. Upon blue light exposure, the SOPP3 tag showed the best specific inactivation and enabled complete inactivation of the carboligase within 30 min. SOPP3 is thus seen as a promising photosensitizer tag to be applied in future multi-step enzyme cascades to overcome the challenge of cross-reactivity.","PeriodicalId":73071,"journal":{"name":"Frontiers in catalysis","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-03-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"47633291","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2022-02-03DOI: 10.3389/fctls.2021.810779
A. Ivanova, F. Falcioni
Variability in the glycosylation profile of therapeutic monoclonal antibodies (mAbs), due to recombinant production technologies, leads to inconsistencies in effector functions and pharmacokinetic properties, both batch-to-batch and within single batches. It also poses regulatory concerns over the effectiveness of commercially available formulations. In vitro chemoenzymatic glycoengineering of variants displaying a homogeneous glycan profile is a trending strategy for ensuring consistent, controlled, and enhanced therapeutic performance, but reported successes are largely limited to small-scale applications. The major challenges for the industrial-scale introduction of the technique stem from the need for activated sugar donors, which can participate in undesired side reactions, and from the economic cost of the additional enzymatic steps and purification stages. While recent developments within the area address some of these obstacles, it appears that more effort is required in order to access the untapped potential of biocatalysis to enable the robust production of therapeutically superior constructs.
{"title":"Challenges and Opportunities for the Large-Scale Chemoenzymatic Glycoengineering of Therapeutic N-Glycosylated Monoclonal Antibodies","authors":"A. Ivanova, F. Falcioni","doi":"10.3389/fctls.2021.810779","DOIUrl":"https://doi.org/10.3389/fctls.2021.810779","url":null,"abstract":"Variability in the glycosylation profile of therapeutic monoclonal antibodies (mAbs), due to recombinant production technologies, leads to inconsistencies in effector functions and pharmacokinetic properties, both batch-to-batch and within single batches. It also poses regulatory concerns over the effectiveness of commercially available formulations. In vitro chemoenzymatic glycoengineering of variants displaying a homogeneous glycan profile is a trending strategy for ensuring consistent, controlled, and enhanced therapeutic performance, but reported successes are largely limited to small-scale applications. The major challenges for the industrial-scale introduction of the technique stem from the need for activated sugar donors, which can participate in undesired side reactions, and from the economic cost of the additional enzymatic steps and purification stages. While recent developments within the area address some of these obstacles, it appears that more effort is required in order to access the untapped potential of biocatalysis to enable the robust production of therapeutically superior constructs.","PeriodicalId":73071,"journal":{"name":"Frontiers in catalysis","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-02-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46658605","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2022-02-01DOI: 10.3389/fctls.2021.803850
Nikolaos Kaloudis, P. Zygouri, Nikolaos Chalmpes, Konstantinos Spyrou, D. Gournis, Ioannis V. Pavlidis
Graphite oxide (GO) has been used for the immobilization of several classes of enzymes, exhibiting very interesting properties as an immobilization matrix. However, the effect the nanomaterial has on the enzyme cannot be predicted. Herein, the effect GO has on the catalytic behavior of several (S)-selective amine transaminases [(S)-ATAs] has been investigated. These enzymes were the focus of this work as they are homodimers with pyridoxal 5′-phosphate in their active site, significantly more complex systems than other enzymes previously studied. Addition of GO (up to 0.1 mg/ml) in the reaction medium leads to activation (up to 50% improved activity) for most enzymes studied, while they maintain their temperature profile (they perform better between 40 and 45°C) and their stability. However, the effect is not universal and there are enzymes that are negatively influenced by the presence of the nanomaterial. More profound is the effect on the (S)-ATA from Chromobacterium violaceum which loses almost 50% of its activity in the presence of 0.1 mg/ml GO, while the stability was significantly decreased, losing its activity after 2 h incubation at 40°C, in the presence of 25 μg/ml GO. This negative effect seems to rise from minor secondary structure alterations; namely, a loss of α-helices and subsequent increase in random coil (∼3% in the presence of 25 μg/ml GO). We hypothesize that the effect the GO has on (S)-ATAs is correlated to the surface chemistry of the enzymes; the less negatively-charged enzymes are deactivated from the interaction with GO. This insight will aid the rationalization of ATA immobilization onto carbon-based nanomaterials.
{"title":"Effect of Graphite Oxide on the Catalytic Behavior of (S)-Selective Amine Transaminases","authors":"Nikolaos Kaloudis, P. Zygouri, Nikolaos Chalmpes, Konstantinos Spyrou, D. Gournis, Ioannis V. Pavlidis","doi":"10.3389/fctls.2021.803850","DOIUrl":"https://doi.org/10.3389/fctls.2021.803850","url":null,"abstract":"Graphite oxide (GO) has been used for the immobilization of several classes of enzymes, exhibiting very interesting properties as an immobilization matrix. However, the effect the nanomaterial has on the enzyme cannot be predicted. Herein, the effect GO has on the catalytic behavior of several (S)-selective amine transaminases [(S)-ATAs] has been investigated. These enzymes were the focus of this work as they are homodimers with pyridoxal 5′-phosphate in their active site, significantly more complex systems than other enzymes previously studied. Addition of GO (up to 0.1 mg/ml) in the reaction medium leads to activation (up to 50% improved activity) for most enzymes studied, while they maintain their temperature profile (they perform better between 40 and 45°C) and their stability. However, the effect is not universal and there are enzymes that are negatively influenced by the presence of the nanomaterial. More profound is the effect on the (S)-ATA from Chromobacterium violaceum which loses almost 50% of its activity in the presence of 0.1 mg/ml GO, while the stability was significantly decreased, losing its activity after 2 h incubation at 40°C, in the presence of 25 μg/ml GO. This negative effect seems to rise from minor secondary structure alterations; namely, a loss of α-helices and subsequent increase in random coil (∼3% in the presence of 25 μg/ml GO). We hypothesize that the effect the GO has on (S)-ATAs is correlated to the surface chemistry of the enzymes; the less negatively-charged enzymes are deactivated from the interaction with GO. This insight will aid the rationalization of ATA immobilization onto carbon-based nanomaterials.","PeriodicalId":73071,"journal":{"name":"Frontiers in catalysis","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42929644","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2022-01-21DOI: 10.3389/fctls.2021.780474
A. Tüllinghoff, Magdalena B. Uhl, Friederike E H Nintzel, A. Schmid, B. Bühler, J. Toepel
Photosynthesis-driven whole-cell biocatalysis has great potential to contribute to a sustainable bio-economy since phototrophic cells use light as the only energy source. It has yet to be shown that phototrophic microorganisms, such as cyanobacteria, can combine the supply of high heterologous enzyme levels with allocation of sufficient reduction equivalents to enable efficient light-driven redox biocatalysis. Here, we demonstrated that the heterologous expression of an NADPH-dependent Baeyer–Villiger monooxygenase (BVMO) gene from Acidovorax sp. CHX100 turns Synechocystis sp. PCC6803 into an efficient oxyfunctionalization biocatalyst, deriving electrons and O2 from photosynthetic water oxidation. Several expression systems were systematically tested, and a PnrsB-(Ni2+)–controlled expression based on a replicative plasmid yielded the highest intracellular enzyme concentration and activities of up to 60.9 ± 1.0 U gCDW −1. Detailed analysis of reaction parameters, side reactions, and biocatalyst durability revealed—on the one hand—a high in vivo BVMO activity in the range of 6 ± 2 U mgBVMO −1 and—on the other hand—an impairment of biocatalyst performance by product toxicity and by-product inhibition. Scale-up of the reaction to 2-L fed-batch photo-bioreactors resulted in the stabilization of the bioconversion over several hours with a maximal specific activity of 30.0 ± 0.3 U g CDW −1, a maximal volumetric productivity of 0.21 ± 0.1 gL−1 h−1, and the formation of 1.3 ± 0.1 gL−1 of ε-caprolactone. Process simulations based on determined kinetic data revealed that photosynthesis-driven cyclohexanone oxidation on a 2-L scale under high-light conditions was kinetically controlled and not subject to a limitation by photosynthesis.
光合作用驱动的全细胞生物催化具有巨大的潜力，有助于可持续的生物经济，因为光营养细胞使用光作为唯一的能量来源。目前还没有研究表明，光养微生物，如蓝藻，可以将高异源酶水平的供应与足够的还原等效物的分配相结合，以实现高效的光驱动氧化还原生物催化。在这里，我们证明了来自Acidovorax sp. CHX100的nadph依赖性Baeyer-Villiger单加氧酶(BVMO)基因的异源表达将Synechocystis sp. PCC6803转化为有效的氧官能化生物催化剂，从光合作用的水氧化中获得电子和O2。系统测试了几种表达系统，基于复制质粒的PnrsB-(Ni2+)控制表达产生了最高的细胞内酶浓度和活性，高达60.9±1.0 U gCDW−1。对反应参数、副反应和生物催化剂耐久性的详细分析表明，一方面，BVMO在体内的活性在6±2 μ mgBVMO−1范围内，另一方面，由于产物毒性和副产物抑制作用，生物催化剂的性能受到损害。将反应扩大到2 l进料间歇式光生物反应器后，生物转化在数小时内稳定下来，最大比活性为30.0±0.3 U g CDW−1，最大体积生产力为0.21±0.1 gL−1 h−1，ε-己内酯的生成为1.3±0.1 gL−1。基于确定的动力学数据的过程模拟表明，在强光条件下，光合作用驱动的2-L环己酮氧化是动力学控制的，不受光合作用的限制。
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