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Sortase‐mediated ligation (SML) has emerged as a powerful and versatile methodology for site‐specific protein conjugation, functionalization/labeling, immobilization, and design of biohybrid molecules and systems. However, the broader application of SML faces several challenges, such as limited activity and stability, dependence on calcium ions, and reversible reactions caused by nucleophilic side‐products. Over the past decade, protein engineering campaigns and particularly directed evolution, have been extensively employed to overcome sortase limitations, thereby expanding the potential application of SML in multiple directions, including therapeutics, biorthogonal chemistry, biomaterials, and biosensors. This review provides an overview of achieved advancements in sortase engineering and highlights recent progress in utilizing SML in combination with other state‐of‐the‐art chemical and biological methodologies. The aim is to encourage scientists to employ sortases in their conjugation experiments. [ABSTRACT FROM AUTHOR]

Sortase‐mediated ligation (SML) has emerged as a powerful and versatile methodology for site‐specific protein conjugation, functionalization/labeling, immobilization, and design of biohybrid molecules and systems. However, the broader application of SML faces several challenges, such as limited activity and stability, dependence on calcium ions, and reversible reactions caused by nucleophilic side‐products. Over the past decade, protein engineering campaigns and particularly directed evolution, have been extensively employed to overcome sortase limitations, thereby expanding the potential application of SML in multiple directions, including therapeutics, biorthogonal chemistry, biomaterials, and biosensors. This review provides an overview of achieved advancements in sortase engineering and highlights recent progress in utilizing SML in combination with other state‐of‐the‐art chemical and biological methodologies. The aim is to encourage scientists to employ sortases in their conjugation experiments. [ABSTRACT FROM AUTHOR]

Circular dichroism (CD) and deconvolution were used to study the structural integrity of a "plugged" and an "open" FhuA transmembrane channel protein in the presence of varied concentrations of tetrahydrofuran (THF), ethanol (EtOH) and chloroform/methanol (C/M). FhuA is an Escherichia coli outer membrane protein (78.9 kDa) consisting of 22 β-sheets and an internal globular cork domain which acts as an iron transporter. FhuA and the deletion variant FhuA Δ1-159 showed comparable and remarkable resistance in the presence of THF (≤40 vol%) and EtOH (≤10 vol%). In C/M, significant differences in structural resistance were observed (FhuA stable ≤10 vol%; FhuA Δ1-159 ≤1 vol%). Deconvolution of CD-spectra for FhuA and FhuA Δ1-159 yielded β-sheet contents of 61 % (FhuA) and 58% (FhuA Δ1-159). Interestingly, FhuA and FhuA Δ1-159 had comparable β-sheet contents in the presence and absence of all three organic cosolvents. Additionally, precipitated FhuA and FhuA Δ1-159 (in 40 vol% C/M or 65 vol% THF) redissolved by supplementing the detergent n-octyl-oligo-oxyethylene (oPOE). [ABSTRACT FROM AUTHOR]

The psychrophilic protease subtilisin S41 from the Antarctic bacillus TA41, and two variants with two and seven amino acid substitutions were studied using molecular dynamics simulation at 283 and 363 K. The analysis of protein dynamics revealed that the average global flexibility of both variants was slightly higher than wild type at both 283 and 363 K. Essential dynamics analysis evidenced that the most relevant collective motions, especially at 363 K, differ in distribution and intensity for each protein variant. At high temperature and for the thermo labile wild type, an amplification of a subset of the low-temperature largest collective motions was observed. On the other hand, the two thermostable variants showed a rather different pattern of essential motions at 363 K from those at 283 K. These results support the hypothesis that the introduced amino acid substitutions, rather than improving the global stability of the variants by increasing its rigidity, lead to a change on the principal fluxional modes allowing the protein to explore a different subset of conformations. A better understanding of this process can open alternative strategies to increase the enzyme stability in addition to increasing the rigidity of the protein scaffold. [ABSTRACT FROM AUTHOR]

Abstract: Arginine deiminase (ADI; EC 3.5.3.6), an arginine-degrading enzyme, has been studied as a potential anti-tumor drug for the treatment of arginine-auxotrophic tumors, such as hepatocellular carcinomas (HCCs) and melanomas. Studies with human lymphatic leukemia cell lines further suggest that ADI is a potential anti-angiogenic agent and is effective in the treatment of leukemia. For instance ADI-PEG-20, patented by Pheonix Pharmacologic Inc., is currently in clinical trials for the treatment of HCC (Phase II/III) and melanoma (Phase I/II). This review summarizes results on recombinant expression, structural analysis, PEG (polyethylene glycerol) modification, in vivo anti-cancer activities, and clinical studies of ADI. Discussions on heterogeneous expression of ADI, directed evolution for improving enzymatic properties, and HSA-fusion for increased in vivo activity conclude this review. [Copyright &y& Elsevier]

Electrochemistry of redox proteins is a broadly applicable technology with important applications in biosensors, biofuel cells and chemical syntheses. Escalating attention in this area is driven by remarkable progress in designing efficient interfaces for transferring electrons between electrode surfaces and redox proteins. Research in interface design is slowly shifting from modifying electrode surfaces towards the engineering of redox proteins. Protein engineering, which encompasses rational design, directed evolution and combined methods, offers many powerful methods and strategies for improving the electron transfer properties of redox proteins. [Copyright &y& Elsevier]

Widespread use of plant protection agents in agriculture is a major cause of pollution. Apart from active ingredients, the environmental impact of auxiliary synthetic polymers should be minimized if they are highly persistent. An alternative to synthetic polymers is the use of natural polysaccharides, which are abundant and biodegradable. In this study, we explore pectin microgels functionalized with anchor peptides (P‐MAPs) to be used as an alternative biobased pesticide delivery system. Using copper as the active ingredient, P‐MAPs effectively prevented infection of grapevine plants with downy mildew under semi‐field conditions on par with commercial copper pesticides. By using anchor peptides, the microgels tightly bind to the leaf surface, exhibiting excellent rain fastness and prolonged fungicidal activity. Finally, P‐MAPs are shown to be easily degradable by enzymes found in nature, demonstrating their negligible long‐term impact on the environment. [ABSTRACT FROM AUTHOR]

Widespread use of plant protection agents in agriculture is a major cause of pollution. Apart from active ingredients, the environmental impact of auxiliary synthetic polymers should be minimized if they are highly persistent. An alternative to synthetic polymers is the use of natural polysaccharides, which are abundant and biodegradable. In this study, we explore pectin microgels functionalized with anchor peptides (P‐MAPs) to be used as an alternative biobased pesticide delivery system. Using copper as the active ingredient, P‐MAPs effectively prevented infection of grapevine plants with downy mildew under semi‐field conditions on par with commercial copper pesticides. By using anchor peptides, the microgels tightly bind to the leaf surface, exhibiting excellent rain fastness and prolonged fungicidal activity. Finally, P‐MAPs are shown to be easily degradable by enzymes found in nature, demonstrating their negligible long‐term impact on the environment. [ABSTRACT FROM AUTHOR]

Material-binding peptides (MBPs) have emerged as a diverse and innovation-enabling class of peptides in applications such as plant-/human health, immobilization of catalysts, bioactive coatings, accelerated polymer degradation and analytics for micro-/nanoplastics quantification. Progress has been fuelled by recent advancements in protein engineering methodologies and advances in computational and analytical methodologies, which allow the design of, for instance, material-specific MBPs with fine-tuned binding strength for numerous demands in material science applications. A genetic or chemical conjugation of second (biological, chemical or physical property-changing) functionality to MBPs empowers the design of advanced (hybrid) materials, bioactive coatings and analytical tools. In this review, we provide a comprehensive overview comprising naturally occurring MBPs and their function in nature, binding properties of short man-made MBPs (<20 amino acids) mainly obtained from phage-display libraries, and medium-sized binding peptides (20–100 amino acids) that have been reported to bind to metals, polymers or other industrially produced materials. The goal of this review is to provide an in-depth understanding of molecular interactions between materials and material-specific binding peptides, and thereby empower the use of MBPs in material science applications. Protein engineering methodologies and selected examples to tailor MBPs toward applications in agriculture with a focus on plant health, biocatalysis, medicine and environmental monitoring serve as examples of the transformative power of MBPs for various industrial applications. An emphasis will be given to MBPs' role in detecting and quantifying microplastics in high throughput, distinguishing microplastics from other environmental particles, and thereby assisting to close an analytical gap in food safety and monitoring of environmental plastic pollution. In essence, this review aims to provide an overview among researchers from diverse disciplines in respect to material-(specific) binding of MBPs, protein engineering methodologies to tailor their properties to application demands, re-engineering for material science applications using MBPs, and thereby inspire researchers to employ MBPs in their research. [ABSTRACT FROM AUTHOR]

The effect of halide substitution in Grubbs-Hoveyda II catalysts (GHII catalysts) embedded in the engineered β-barrel protein nitrobindin (NB4exp) on metathesis activity in aqueous media was studied. Maleimide tagged dibromido and diiodido derivates of the GHII catalyst were synthesized and covalently conjugated to NB4exp. The biohybrid catalysts were characterized spectroscopically confirming the structural integrity. When the two chloride substituents at ruthenium center were exchanged against bromide and iodide, the diiodo derivative was found to show significantly higher catalytic activity in ring-closing metathesis of α,ω-diolefins, whereas the dibromido derivative was less efficient when compared with the parent dichlorido catalyst. Using the diiodido catalyst, high turnover numbers of up to 75 were observed for ring-closing metathesis (RCM) yielding unsaturated six- and seven-membered N-heterocycles. The effect of halide substitution in Grubbs-Hoveyda II catalysts (GHII catalysts) embedded in the engineered β-barrel protein expanded nitrobindin NB4exp on ring closing olefin metathesis activity in aqueous media was studied. [Display omitted] • Halide-substituted Grubbs-Hoveyda catalysts conjugated to expanded nitrobindin. • Structural integrity of the obtained biohybrid catalyst was confirmed spectroscopically. • Substitution by iodide improved the turnover number of biohybrid catalyst. • Unsaturated N -heterocycles were obtained by ring-closing metathesis in water. [ABSTRACT FROM AUTHOR]

Ein typischer Bestandteil von Polymerabfällen ist Polystyrol (PS), das in zahlreichen Anwendungen eingesetzt wird, aufgrund seiner hydrophoben Eigenschaften in der Umwelt aber nur langsam abgebaut wird. Um die Reaktivität von Polystyrol zu erhöhen, müssen polare Gruppen eingeführt werden. In dieser Arbeit werden biohybride Katalysatoren auf Grundlage des modifizerten Ankerpeptids LCI_F16C vorgestellt, die sich an Polystyrol‐Mikropartikel anheften können und benzylische C−H‐Bindungen in Polystyrol‐Mikropartikeln mit handelsüblichem Oxon als Oxidationsmittel hydroxylieren. Eine dichte Oberflächenbedeckung von PS mit LCI‐Peptid erfolgt durch Bildung von Monoschichten innerhalb von Minuten in wässrigen Lösungen bei Raumtemperatur. Der katalytisch aktive Cobalt‐Cofaktor Co‐L1 oder Co‐L2 mit einem modifizierten NNNN‐makrozyklischen TACD‐Liganden (TACD=1,4,7,10‐Tetraazacyclododecan) ist über einen Maleimid‐Linker kovalent an das Ankerpeptid LCI gebunden. Im Vergleich zu den freien Cofaktoren wurde eine 12‐ bis 15‐fache Verbesserung der katalytischen Aktivität mit Biohybrid‐Katalysatoren auf Basis von LCI_F16C beobachtet. [ABSTRACT FROM AUTHOR]

A typical component of polymer waste is polystyrene (PS) used in numerous applications, but degraded only slowly in the environment due to its hydrophobic properties. To increase the reactivity of polystyrene, polar groups need to be introduced. Here, biohybrid catalysts based on the engineered anchor peptide LCI_F16C are presented, which are capable of attaching to polystyrene microparticles and hydroxylating benzylic C−H bonds in polystyrene microparticles using commercially available oxone as oxidant. LCI peptides achieve a dense surface coverage of PS through monolayer formation within minutes in aqueous solutions at ambient temperature. The catalytically active cobalt cofactor Co‐L1 or Co‐L2 with a modified NNNN macrocyclic TACD ligand (TACD=1,4,7,10‐tetraazacyclododecane) is covalently bound to the anchor peptide LCI through a maleimide linker. Compared to the free cofactors, a 12‐ to 15‐fold improvement in catalytic activity using biohybrid catalysts based on LCI_F16C was observed. [ABSTRACT FROM AUTHOR]

The development of efficient and environmentally friendly plant protection systems is one of the major challenges for a sustainable agriculture of the future. Citrus canker caused by the pathogen Xanthomonas citri subsp. citri (X. citri) affects all cultivated citrus species worldwide and is responsible for enormous economic losses and restrictions in international trade. Currently used commercial copper‐based formulations are the origin of contamination for soil and groundwater, affecting local ecosystems and human health. A copper‐free sustainable microgel‐based plant protection system able to efficiently combat X. citri is developed. Microgels decorated with anchor peptides exhibit strong non‐covalent attachment to the surface of orange leaves and have the ability to release hexyl gallate, which inhibits the growth and spreading of pathogens. The tailored design of the microgel network allows high loadings of hexyl gallate (up to 40 wt.%) and a controlled release of gallates from microgels that provide long‐term protection. The antibacterial activity of the hexyl gallate‐loaded microgels is demonstrated by various in vitro assays as well as on orange plants in greenhouse settings. The experimental results suggest that the developed sustainable microgel‐based plant protection system for citrus trees allows efficient abatement of X. citri and reduces environmental pollution caused by copper formulations. [ABSTRACT FROM AUTHOR]

Citrus canker is an infectious bacterial disease and one of the major threats to the orange juice industry, a multibillion-dollar market that generates hundreds of thousands of jobs worldwide. This disease is caused by the Gram-negative bacterium Xanthomonas citri subsp. citri. In Brazil, the largest producer and exporter of concentrate orange juice, the control of citrus canker is exerted by integrated management practices, in which cupric solutions are intensively used in the orchards to refrain bacterial spreading. Copper ions accumulate and are as heavy metals toxic to the environment. Therefore, the aim of the present work was to evaluate bifunctional fusion proteins (BiFuProts) as novel and bio-/peptide-based alternatives to copper formulations to control citrus canker. BiFuProts are composed of an anchor peptide able to bind to citrus leaves, and an antimicrobial "killer" peptide to protect against bacterial infections of plants. The selected BiFuProt (Mel-CgDEF) was bactericidal against X. citri at 125 μg mL−1, targeting the bacterial cytoplasmic membrane within the first minutes of contact. The results in the greenhouse assays proved that Mel-CgDEF at 250 μg mL−1 provided protection against X. citri infection on the leaves, significantly reducing the number of lesions by area when compared with the controls. Overall, the present work showed that the BiFuProt Mel-CgDEF is a biobased and biodegradable possible alternative for substitute cupric formulations. Key points: • The bifunctional fusion protein Mel-CgDEF was effective against Xanthomonas citri. • Mel-CgDEF action mechanism was the disruption of the cytoplasmic membrane. • Mel-CgDEF protected citrus leaves against citrus canker disease. [ABSTRACT FROM AUTHOR]

Artificial metalloenzymes have emerged as biohybrid catalysts that allow to combine the reactivity of a metal catalyst with the flexibility of protein scaffolds. This work reports the artificial metalloenzymes based on the β‐barrel protein nitrobindin NB4, in which a cofactor [CoIIX(Me3TACD‐Mal)]+X− (X=Cl, Br; Me3TACD=N,N',N''‐trimethyl‐1,4,7,10‐tetraazacyclododecane, Mal=CH2CH2CH2NC4H2O2) was covalently anchored via a Michael addition reaction. These biohybrid catalysts showed higher efficiency than the free cobalt complexes for the oxidation of benzylic C(sp3)−H bonds in aqueous media. Using commercially available oxone (2KHSO5 ⋅ KHSO4 ⋅ K2SO4) as oxidant, a total turnover number of up to 220 and 97 % ketone selectivity were achieved for tetralin. As catalytically active intermediate, a mononuclear terminal cobalt(IV)‐oxo species [Co(IV)=O]2+ was generated by reacting the cobalt(II) cofactor with oxone in aqueous solution and characterized by ESI‐TOF MS. [ABSTRACT FROM AUTHOR]

Citrus canker is an infectious bacterial disease and one of the major threats to the orange juice industry, a multibillion-dollar market that generates hundreds of thousands of jobs worldwide. This disease is caused by the Gram-negative bacterium Xanthomonas citri subsp. citri. In Brazil, the largest producer and exporter of concentrate orange juice, the control of citrus canker is exerted by integrated management practices, in which cupric solutions are intensively used in the orchards to refrain bacterial spreading. Copper ions accumulate and are as heavy metals toxic to the environment. Therefore, the aim of the present work was to evaluate bifunctional fusion proteins (BiFuProts) as novel and bio-/peptide-based alternatives to copper formulations to control citrus canker. BiFuProts are composed of an anchor peptide able to bind to citrus leaves, and an antimicrobial “killer” peptide to protect against bacterial infections of plants. The selected BiFuProt (Mel-CgDEF) was bactericidal against X. citri at 125 μg mL−1, targeting the bacterial cytoplasmic membrane within the first minutes of contact. The results in the greenhouse assays proved that Mel-CgDEF at 250 μg mL−1 provided protection against X. citri infection on the leaves, significantly reducing the number of lesions by area when compared with the controls. Overall, the present work showed that the BiFuProt Mel-CgDEF is a biobased and biodegradable possible alternative for substitute cupric formulations. Key points: • The bifunctional fusion protein Mel-CgDEF was effective against Xanthomonas citri. • Mel-CgDEF action mechanism was the disruption of the cytoplasmic membrane. • Mel-CgDEF protected citrus leaves against citrus canker disease. [ABSTRACT FROM AUTHOR]

Enzymatic catalysis in mild conditions plays a pivotal role in green chemistry, and the mining and the directed evolution of novel enzymes are crucial for the utilization of efficient biocatalysts. Glycosyltransferases (GTs) hold great promise in the synthesis of many valuable glycosylated products; however the narrow substrate scope and low catalytic efficiency of GTs often limit their broader applications. Phylogenetic mining and directed evolution of GTs are well-established methodologies to generate powerful enzymes. The combination of these established methodologies, termed PhyloDirect, integrates the identification of an excellent performing enzyme with its engineering by directed evolution to rapidly match the demands in biocatalysis. Herein, we report and validate a combined phylogeny-based directed evolution (PhyloDirect) approach to improve the performance of novel GT BarGT-3. A single GT high-throughput screening system based on UDP-glucose recycling was established. Through one-round of directed evolution, a promising variant I63P/H108G was obtained with significantly improved catalytic efficiency (28-fold for acetaminophen) and increased conversions toward a broad range of synthetically valuable substrates (phenols, coumarins, and flavonoids, up to 5-fold). The success of this PhyloDirect approach offers new opportunities for the development and application of efficient biocatalysts in green chemistry and other industrial processes. [ABSTRACT FROM AUTHOR]

Secretion of proteins into the extracellular space has great advantages for the production of recombinant proteins. Type 1 secretion systems (T1SS) are attractive candidates to be optimized for biotechnological applications, as they have a relatively simple architecture compared to other classes of secretion systems. A paradigm of T1SS is the hemolysin A type 1 secretion system (HlyA T1SS) from Escherichia coli harboring only three membrane proteins, which makes the plasmid-based expression of the system easy. Although for decades the HlyA T1SS has been successfully applied for secretion of a long list of heterologous proteins from different origins as well as peptides, but its utility at commercial scales is still limited mainly due to low secretion titers of the system. To address this drawback, we engineered the inner membrane complex of the system, consisting of HlyB and HlyD proteins, following KnowVolution strategy. The applied KnowVolution campaign in this study provided a novel HlyB variant containing four substitutions (T36L/F216W/S290C/V421I) with up to 2.5-fold improved secretion for two hydrolases, a lipase and a cutinase. Key points: • An improvement in protein secretion via the use of T1SS • Reaching almost 400 mg/L of soluble lipase into the supernatant • A step forward to making E. coli cells more competitive for applying as a secretion host [ABSTRACT FROM AUTHOR]

The combinatorial complexity of the protein sequence space presents a significant challenge for recombination experiments targeting beneficial positions. To overcome these difficulties, a machine learning (ML) approach was employed, which was trained on a limited literature dataset and combined with iterative generation and experimental data implementation. The PyPEF method was utilized to identify existing variants and predict recombinant variants targeting the substrate channel of P450 CYP116B3. Through molecular dynamics simulations, eight multiple-substituted improved variants were successfully validated. Specifically, the RMSF of variant A86T/T91H/M108S/A109M/T111P was decreased from 3.06 Å (wild type) to 1.07 Å. Additionally, the average RMSF of the variant A86T/T91P/M108V/A109M/T111P decreased to 1.41 Å, compared to the wild type's 1.53 Å. Of particular significance was the prediction that the variant A86T/T91H/M108G/A109M/T111P exhibited an activity approximately 15 times higher than that of the wild type. Furthermore, during the selection of the regression model, PLS and MLP regressions were compared. The effect of data size and data relevance on the two regression approaches has been summarized. The aforementioned conclusions provide evidence for the feasibility of the strategy that combines ML with experimental approaches. This integrated strategy proves effective in exploring potential variations within the protein sequence space. Furthermore, this method facilitates a deeper understanding of the substrate channel in P450 CYP116B3. [ABSTRACT FROM AUTHOR]

Oxidases are of interest to chemical and pharmaceutical industries because they catalyze highly selective oxidations. However, oxidases found in nature often need to be re‐engineered for synthetic applications. Herein, we developed a versatile and robust flow cytometry‐based screening platform "FlOxi" for directed oxidase evolution. FlOxi utilizes hydrogen peroxide produced by oxidases expressed in E. coli to oxidize Fe2+ to Fe3+ (Fenton reaction). Fe3+ mediates the immobilization of a His6‐tagged eGFP (eGFPHis) on the E. coli cell surface, ensuring the identification of beneficial oxidase variants by flow cytometry. FlOxi was validated with two oxidases—a galactose oxidase (GalOx) and a D‐amino acid oxidase (D‐AAO)—yielding a GalOx variant (T521A) with a 4.4‐fold lower Km value and a D‐AAO variant (L86M/G14/A48/T205) with a 4.2‐fold higher kcat than their wildtypes. Thus, FlOxi can be used for the evolution of hydrogen peroxide‐producing oxidases and applied for non‐fluorescent substrates. [ABSTRACT FROM AUTHOR]

Oxidasen sind bedeutsam für die chemische und pharmazeutische Industrie, da sie hochselektive Oxidationen katalysieren. Allerdings müssen natürlich vorkommende Oxidasen für synthetische Anwendungen häufig mittels Gelenkter Evolution im Labor maßgeschneidert werden. Hierfür haben wir die breit anwendbare und robuste Durchmusterungs‐Plattform "FlOxi" entwickelt. FlOxi ist eine Durchflusszytometriemethode und nutzt Wasserstoffperoxid als Reaktionssnebenprodukt von in E. coli exprimierten Oxidasen, um Fe2+ zu Fe3+ zu oxidieren (Fenton‐Reaktion). Fe3+ vermittelt dabei die Immobilisierung von His6‐"markiertem" eGFP (eGFPHis) auf der E. coli‐Zelloberfläche, wodurch die Identifizierung von aktiven Oxidase‐Varianten mittels Durchflusszytometrie ermöglicht wird. FlOxi wurde mit zwei Oxidasen – einer Galaktose‐Oxidase (GalOx) und einer D‐Aminosäure‐Oxidase (D‐AAO) – validiert. Hierbei wurde in einer Gelenkten Evolutionsrunde eine GalOx‐Variante (T521A) mit einem 4.4‐fach niedrigeren Km‐Wert und eine D‐AAO‐Variante (L86M/G14/A48/T205) mit einem 4.2‐fach höheren kcat‐Wert aufgefunden. FlOxi kann universell für die Gelenkte Evolution von Wasserstoffperoxid‐produzierenden Oxidasen, auch für nicht‐fluoreszierende Substrate, eingesetzt werden. [ABSTRACT FROM AUTHOR]

The implementation of biocatalytic reactions in biotechnological processes requires enzymes with suitable properties. Thus, optimization of existing or identification of novel enzymes is needed. For enzyme optimization, various mutagenesis approaches are used yielding libraries with a large number of protein variants. Additionally, novel enzymes can be identified from metagenomic libraries, which contain bacterial clones expressing DNA isolated from different habitats. Activity-based screening of such libraries is needed to identify enzyme candidates of interest. Here, we provide a protocol for screening of regio- and enantioselective lipases stable under industrially relevant conditions. [ABSTRACT FROM AUTHOR]

Alkaptonuria (AKU) is a rare inborn error of metabolism caused by a defective homogentisate 1,2-dioxygenase (HGD), an enzyme involved in the tyrosine degradation pathway. Loss of HGD function leads to the accumulation of homogentisic acid (HGA) in connective body tissues in a process called ochronosis, which results on the long term in an early-onset and severe osteoarthropathy. HGD's quaternary structure is known to be easily disrupted by missense mutations, which makes them an interesting target for novel treatment strategies that aim to rescue enzyme activity. However, only prediction models are available providing information on a structural basis. Therefore, an E. coli based whole-cell screening was developed to evaluate HGD missense variants in 96-well microtiter plates. The screening principle is based on HGD's ability to convert the oxidation sensitive HGA into maleylacetoacetate. More precisely, catalytic activity could be deduced from pyomelanin absorbance measurements, derived from the auto-oxidation of remaining HGA. Optimized screening conditions comprised several E. coli expression strains, varied expression temperatures and varied substrate concentrations. In addition, plate uniformity, signal variability and spatial uniformity were investigated and optimized. Finally, eight HGD missense variants were generated via site-directed mutagenesis and evaluated with the developed high-throughput screening (HTS) assay. For the HTS assay, quality parameters passed the minimum acceptance criterion for Z' values > 0.4 and single window values > 2. We found that activity percentages versus wildtype HGD were 70.37 ± 3.08% (for M368V), 68.78 ± 6.40% (for E42A), 58.15 ± 1.16% (for A122V), 69.07 ± 2.26% (for Y62C), 35.26 ± 1.90% (for G161R), 35.86 ± 1.14% (for P230S), 23.43 ± 4.63% (for G115R) and 19.57 ± 11.00% (for G361R). To conclude, a robust, simple, and cost-effective HTS system was developed to reliably evaluate and distinguish human HGD missense variants by their HGA consumption ability. This HGA quantification assay may lay the foundation for the development of novel treatment strategies for missense variants in AKU. [ABSTRACT FROM AUTHOR]

Bacterial phytases have attracted industrial interest as animal feed supplement due to their high activity and sufficient thermostability (required for feed pelleting). We devised an approach named KeySIDE, an iterative Key-residues interrogation of the wild type with Substitutions Identified in Directed Evolution for improving Yersinia mollaretii phytase (Ymphytase) thermostability by combining key beneficial substitutions and elucidating their individual roles. Directed evolution yielded in a discovery of nine positions in Ymphytase and combined iteratively to identify key positions. The 'best' combination (M6: T77K, Q154H, G187S, and K289Q) resulted in significantly improved thermal resistance; the residual activity improved from 35 % (wild type) to 89 % (M6) at 58 °C and 20-min incubation. Melting temperature increased by 3 °C in M6 without a loss of specific activity. Molecular dynamics simulation studies revealed reduced flexibility in the loops located next to helices (B, F, and K) which possess substitutions (Helix-B: T77K, Helix-F: G187S, and Helix-K: K289E/Q). Reduced flexibility in the loops might be caused by strengthened hydrogen bonding network (e.g., G187S and K289E/K289Q) and a salt bridge (T77K). Our results demonstrate a promising approach to design phytases in food research, and we hope that the KeySIDE might become an attractive approach for understanding of structure-function relationships of enzymes. [ABSTRACT FROM AUTHOR]

Directed evolution has matured to a routinely applied algorithm to tailor enzyme properties to meet the demands in various applications. In order to free directed enzyme evolution from methodological restraints and to efficiently explore its potential, many different strategies have been used in directed evolution campaigns. Analysis of directed evolution campaigns reveals that traditional approaches, in which several iterative rounds of diversity generation and screening are performed, are gradually replaced by strategies which require less time, less screening efforts, and generate a molecular understanding of the targeted properties. In this review, conceptual advances in knowledge generating directed evolution strategies are summarized, compared to each other and to traditional directed evolution strategies. Finally, a ‘KnowVolution’ (knowledge gaining directed evolution) termed strategy is proposed. [ABSTRACT FROM AUTHOR]

Understanding the mechanisms of modulators' action on enzymes is crucial for optimizing and designing pharmaceutical substances. The acute inflammatory response, in particular, is regulated mainly by a disintegrin and metalloproteinase (ADAM) 17. ADAM17 processes several disease mediators such as TNFα and APP, releasing their soluble ectodomains (shedding). A malfunction of this process leads to a disturbed inflammatory response. Chemical protease inhibitors such as TAPI-1 were used in the past to inhibit ADAM17 proteolytic activity. However, due to ADAM17′s broad expression and activity profile, the development of active-site-directed ADAM17 inhibitor was discontinued. New 'exosite' (secondary substrate binding site) inhibitors with substrate selectivity raised the hope of a substrate-selective modulation as a promising approach for inflammatory disease therapy. This work aimed to develop a high-throughput screen for potential ADAM17 modulators as therapeutic drugs. By combining experimental and in silico methods (structural modeling and docking), we modeled the kinetics of ADAM17 inhibitor. The results explain ADAM17 inhibition mechanisms and give a methodology for studying selective inhibition towards the design of pharmaceutical substances with higher selectivity. [ABSTRACT FROM AUTHOR]

Wound infections originate when exogenous or endogenous bacterial pathogens can circumvent the barrier of the wound dressing and invade the wound bed. Bacterial colonization causes inflammation, stalls the healing process, and carries the risk of dissemination to other tissues. In addition, current antimicrobial dressings fail to resolve an infection once it has been established because debris of the killed bacteria rapidly accumulates on their surface and hampers the antimicrobial action. Faced with this challenge, hybrid synthetic‐natural water‐soluble macromolecules are designed that self‐assemble onto the surface of dressings to generate an antifouling brush functionalized with endolysin, a bactericidal enzyme that poses no harm for eukaryotic cells. The simultaneous action of the brush and the enzyme not only prevents the colonization of the dressing, but also enables the coating to kill planktonic bacteria with even higher efficiency than the free enzyme. Remarkably, the Kill&Repel coating could completely eradicate bacteria in a simulated infection without allowing the adhesion of residues on the surface. Thus, this strategy opens a revolutionary approach for protecting and treating an infected wound in a safer and more efficient manner. [ABSTRACT FROM AUTHOR]

Commercial applications of microalgae for biochemicals and fuels are hampered by their high production costs, and the use of conventional carbon supplies is a key reason. Bicarbonate has been proposed as an alternative carbon source due to its potential advantages in lower carbon supply costs, convenience for photobioreactor development, biomass harvesting, and labor and energy savings. We review recent progress in bicarbonate-based microalgae cultivation, which validated previous assumptions, suggested further advantages, and demonstrated potential to significantly reduce production cost. Future research should focus on improving production efficiency and reducing energy inputs, including optimizing photobioreactor design, comprehensive utilization of natural power, and automation in production systems. For microalgae production systems, bicarbonate is a better carbon supply than bubbled CO 2 gas in terms of carbon utilization efficiency, photobioreactor development, energy saving, and biomass harvesting. The carbon supply is the bottleneck in developing photobioreactors for low-cost microalgal biomass production, in contrast to the heterotrophic process, which is limited by oxygen supply. Recent progress suggests the potential of the bicarbonate-based integrated carbon capture and algae production system, to significantly reduce production costs and energy inputs in microalgae cultivation. Comprehensive utilization of natural power is a promising approach to further decrease energy consumption and improve the efficiency of the microalgae cultivation process in the future. [ABSTRACT FROM AUTHOR]

Highlights: [•] We improved thermostability and pH resistance (pH 2.8) of Yersinia mollaretii phytase without affecting catalytic efficiency. [•] Improved phytase variant was obtained by multi-site saturation mutagenesis of five sites identified in directed evolution. [•] Only <0.004% of the theoretical sequence space (3.35×107 variants) was sampled to achieve the improvement. [•] Current approach allows exploring key positions discovered in directed evolution campaign or by rational protein design. [ABSTRACT FROM AUTHOR]

Cytochrome P450 BM3 monooxygenase from Bacillus megaterium and its variants are promising catalysts for organic synthesis. Aiming at the identification of variants for selective hydroxylation of functionalised aromatic compounds, the double mutant F87A L188C showed remarkably improved catalytic activity towards a set of tested toluene derivatives. The apparent catalytic efficiency of this variant towards the model substrate methyl 2-methoxy-3-methylbenzoate was 63.6 s−1 M−1, which is 535-fold higher compared to that of wild-type BM3. Furthermore, the double mutant selectively catalysed the benzylic hydroxylation of numerous toluene derivatives, especially in the presence of carbonyl- or carboxyl-functions that are directly attached to the aromatic ring. Preparative scale conversion resulted in efficient production of methyl 3-(hydroxymethyl)-2-methoxybenzoate (73% yield) which proved that F87A L188C is a suitable, efficient and sustainable catalyst for the introduction of benzylic hydroxyl groups in general. [ABSTRACT FROM AUTHOR]

Efficient synthetic techniques for the diversification of natural products are incremental for drug discovery processes of the pharmaceutical industry because these complex bioactive compounds often require an adjustment of properties. Human liver P450 3A4, key player of the body's detoxification system and decisive factor of a drug's metabolic fate, is renowned for its broad substrate scope including many natural products. In this study, we investigated the synthetic potential of human P450 3A4 for the diversification of natural product classes and isolated the produced metabolites of six selected natural products at a preparative 100‐mg scale. Aided by efficient expression levels in P. pastoris, this whole‐cell biocatalyst was found to be highly effective at the intended job allowing the identification of a total of 31 authentic human metabolites, many of them for the first time. By revealing an unprecedented degree of diversification, this study extends the synthetic repertoire for efficient enzymatic natural product modification in a one‐step fashion and adds a completely new view to an old enzyme traditionally used for inhibition and toxicology studies. [ABSTRACT FROM AUTHOR]

[Display omitted] Nitric oxide (NO) continuously generated by healthy endothelium prevents platelet activation and maintains vascular homeostasis. However, when artificial surfaces, like of extracorporeal membrane oxygenator comes in contact with blood, protein adsorption and thereby platelet activation takes place, which eventually leads to thrombus formation. To overcome this, we present an antifouling microgel coating mimicking the function of enzyme glutathione peroxidase to endogenously generate NO in the blood plasma from endogenous NO-donors and maintain a physiological NO flux. Microgels are synthesized by copolymerization of highly hydrophilic N –(2–hydroxypropyl)methacrylamide (HPMA) and glycidyl methacrylate (GMA) with diselenide crosslinks. For immobilization of the microgels on hydrophobic poly(4-methylpentene) (TPX) membranes bioengineered amphiphilic anchor peptides with free thiols are used. The anchor peptide attaches to the TPX membranes by hydrophobic interactions while the free thiols are presented for crosslinking with the microgels. The hydrophilic nature of the microgel coating prevents protein adsorption while the reversible diselenide bridges make the microgels responsive to the reducing environment and lead to the formation of reactive selenols/selenolates. The generated selenols/selenolates provide an efficient and sustained NO-release from endogenous S -nitrosothiols (RSNOs) mimicking the enzymatic function of glutathione peroxidase. On exposure to the whole blood, the microgel coating inhibited platelet activation and prolonged the blood clotting time. [ABSTRACT FROM AUTHOR]

Protein re-engineering by directed evolution has become a standard approach for tailoring enzymes in many fields of science and industry. Advances in screening formats and screening systems are fueling progress and enabling novel directed evolution strategies, despite the fact that the quality of mutant libraries can still be improved significantly. Diversity generation strategies in directed enzyme evolution comprise three options: (a) focused mutagenesis (selected residues are randomized); (b) random mutagenesis (mutations are randomly introduced over the whole gene); and (c) gene recombination (stretches of genes are mixed to chimeras in a random or rational manner). Either format has both advantages and limitations depending on the targeted enzyme and property. The quality of diverse mutant libraries plays a key role in finding improved mutants. In this review, we summarize methodological advancements and novel concepts (since 2009) in diversity generation for all three formats. Advancements are discussed with respect to the state of the art in diversity generation and high-throughput screening capabilities, as well as robustness and simplicity in use. Furthermore, limitations and remaining challenges are emphasized 'to get what we aim for' through 'optimal diversity' generation. [ABSTRACT FROM AUTHOR]

ginine deiminase (ADI), an arginine-degrading enzyme, has been studied as a potential anti-cancer agent for inhibiting arginine-auxotrophic tumors, such as melanomas and hepatocellular carcinomas. Based on our preliminary results, it was noticed that the optimum pH of ADI from Pseudomonas plecoglossicida (PpADI) was 6.0, and less than 10% of the activity was retained at pH 7.4 (pH of human plasma). Additionally, the K value for wild-type ADI (WT-ADI) was 2.88 mM (pH 6.0), which is over 20 times of the serum arginine level (100-120 μM). These are two major limitations for PpADI as a potential anti-cancer drug. A highly sensitive and efficient high-throughput screening strategy based on a modified diacetylmonoxime-thiosemicarbazide method was established to isolate ADI mutants with higher activity and lower K under physiological pH. Three improved mutants was selected from 650 variants after one round of ep-PCR, among which mutant 314 (M314: A128T, H404R, I410L) exhibiting the highest activity. Interestingly, sequence alignment shows that three amino acid substitutes in M314 are coincident with corresponding residues in ADI from Mycoplasma arginini. The specific activity of M314 (9.02 U/mg) is over 20-fold higher than that of WT-ADI (0.44 U/mg) at pH 7.4, and the K value was reduced to 0.65 mM (pH 7.4). Noticeably, the pH optimum was shifted from 6.0 to 6.5 in M314. Homology model of M314 was constructed to understand the molecular basis of the improved enzymatic properties. This work could provide promising drug candidate for curing arginine-auxotrophic cancers. [ABSTRACT FROM AUTHOR]

How to explore protein sequence space efficiently and how to generate high-quality mutant libraries that allow to identify improved variants with current screening technologies are key questions for any directed protein evolution experiment. High-quality mutant libraries can be generated through improved random mutagenesis methodologies and by restricting diversity generation through computational methods to residues which have high success probabilities. Advances in mutant library design and computational tools to focus diversity generation are summarized in this minireview and discussed from an experimentalist point of view in the context of directed protein evolution. [ABSTRACT FROM AUTHOR]

Directed protein evolution is the most versatile method for studying protein structure-function relationships, and for tailoring a protein's properties to the needs of industrial applications. In this review, we performed a statistical analysis on the genetic code to study the extent and consequence of the organization of the genetic code on amino acid substitution patterns generated in directed evolution experiments. In detail, we analyzed amino acid substitution patterns caused by (a) a single nucleotide (nt) exchange at each position of all 64 codons, and (b) two subsequent nt exchanges (first and second nt, first and third nt, second and third nt). Additionally, transitions and transversions mutations were compared at the level of amino acid substitution patterns. The latter analysis showed that single nucleotide substitution in a codon generates only 39.5% of the natural diversity on the protein level with 5.2-7 amino acid substitutions per codon. Transversions generate more complex amino acid substitution patterns (increased number and chemically more diverse amino acid substitutions) than transitions. Simultaneous nt exchanges at both first and second nt of a codon generates very diverse amino acid substitution patterns, achieving 83.2% of the natural diversity. The statistical analysis described in this review sets the objectives for novel random mutagenesis methods that address the consequences of the organization of the genetic code. Random mutagenesis methods that favor transversions or introduce consecutive nt exchanges can contribute in this regard. [ABSTRACT FROM AUTHOR]

• Recombination of distantly related genes to generate chimeras is enabled by PTRec. • PTRec recombination yielded two phytase chimeras with improved thermal stability and low sequence identities. • Chimeras with low sequence identities open up opportunities for sequence claims. • DNA recombination can result in chimeras with improved and new properties. Being able to recombine more than two genes with four or more crossover points in a sequence independent manner is still a challenge in protein engineering and limits our capabilities in tailoring enzymes for industrial applications. By computational analysis employing multiple sequence alignments and homology modeling, five fragments of six phytase genes (sequence identities 31–64 %) were identified and efficiently recombined through phosphorothioate-based cloning using the PTRec method. By combinatorial recombination, functional phytase chimeras containing fragments of up to four phytases were obtained. Two variants (PTRec 74 and PTRec 77) with up to 32 % improved residual activity (90 °C, 60 min) and retained specific activities of > 1100 U/mg were identified. Both variants are composed of fragments from the phytases of Citrobacter braakii , Hafnia alvei and Yersinia mollaretii. They exhibit sequence identities of ≤ 80 % to their parental enzymes, highlighting the great potential of DNA recombination strategies to generate new enzymes with low sequences identities that offer opportunities for property right claims. [ABSTRACT FROM AUTHOR]

Biocatalysis for the synthesis of fine chemicals is highly attractive but usually requires organic (co‐)solvents (OSs). However, native enzymes often have low activity and resistance in OSs and at elevated temperatures. Herein, we report a smart salt bridge design strategy for simultaneously improving OS resistance and thermostability of the model enzyme, Bacillus subtilits Lipase A (BSLA). We combined comprehensive experimental studies of 3450 BSLA variants and molecular dynamics simulations of 36 systems. Iterative recombination of four beneficial substitutions yielded superior resistant variants with up to 7.6‐fold (D64K/D144K) improved resistance toward three OSs while exhibiting significant thermostability (thermal resistance up to 137‐fold, and half‐life up to 3.3‐fold). Molecular dynamics simulations revealed that locally refined flexibility and strengthened hydration jointly govern the highly increased resistance in OSs and at 50–100 °C. The salt bridge redesign provides protein engineers with a powerful and likely general approach to design OSs‐ and/or thermal‐resistant lipases and other α/β‐hydrolases. [ABSTRACT FROM AUTHOR]

Biocatalysis for the synthesis of fine chemicals is highly attractive but usually requires organic (co‐)solvents (OSs). However, native enzymes often have low activity and resistance in OSs and at elevated temperatures. Herein, we report a smart salt bridge design strategy for simultaneously improving OS resistance and thermostability of the model enzyme, Bacillus subtilits Lipase A (BSLA). We combined comprehensive experimental studies of 3450 BSLA variants and molecular dynamics simulations of 36 systems. Iterative recombination of four beneficial substitutions yielded superior resistant variants with up to 7.6‐fold (D64K/D144K) improved resistance toward three OSs while exhibiting significant thermostability (thermal resistance up to 137‐fold, and half‐life up to 3.3‐fold). Molecular dynamics simulations revealed that locally refined flexibility and strengthened hydration jointly govern the highly increased resistance in OSs and at 50–100 °C. The salt bridge redesign provides protein engineers with a powerful and likely general approach to design OSs‐ and/or thermal‐resistant lipases and other α/β‐hydrolases. [ABSTRACT FROM AUTHOR]

Biocatalysis in ionic liquids (ILs) has gained enormous attention for the production of biodiesel, sugar esters, and pharmaceuticals. However, hydrophilic IL interaction with enzymes often results in reduced activity or even inactivation. In this report, we prove that intrinsic lipase stability and preservation of hydration shells of Bacillus subtilis lipase A (BSLA) are two synergistic design principles to retain enzymatic activity in ILs. After in silico screening of nine beneficial amino acid positions by the CompassR rule (in total, 172 variants), we rationally designed two variants, to be constructed by site-directed mutagenesis, and three libraries by site-saturation mutagenesis. With minimal experiment effort, we identified three all-around variants towards four [BMIM]-based ILs. Remarkably, the variant M1a F17S/V54K/D64N/D91E/G155N had 6.7-fold higher resistance against 40% (v/v) [BMIM]Cl, 5.6-fold in 80% (v/v) [BMIM]Br, 5.0-fold in 30% (v/v) [BMIM][TfO], and 2.7-fold in 10% (v/v) [BMIM]I compared to wild-type BSLA, respectively, while showing 1.9-fold improvement in specific activity. Computational analysis of molecular dynamics and thermodynamic stability analysis of the variants revealed the molecular basis for the resistant variants M1a and M1b as the synergistic enhancement of protein stability (ΔΔGfold ranging from −4.26 to −4.80 kcal mol−1) and increased hydration shells around the substitutions in the four ILs (up to 1.7-fold). These design principles and the gained molecular knowledge not only open the door to direct experimentalists for rationally designing promising IL-resistant enzymes, but also provide new insights into enzymatic catalysis in ILs. [ABSTRACT FROM AUTHOR]

We report a facile and reversible method to immobilize a broad range of His6-tagged proteins on the E. coli cell surface through Fe(III) – metal complexes. A His6-tagged eGFP and four His6-tagged enzymes were successfully immobilized on the cell surface. Additionally, a hydrogel sheath around E. coli cells was generated by immobilized His6-tagged HRP. [ABSTRACT FROM AUTHOR]

Semi-rational redesign of the substrate binding pocket and access tunnels of prodigiosin ligase PigC enhanced the catalytic efficiency in the synthesis of pyrrolic anti-cancer agents more than 45 times. A molecular understanding was gained on residues V333 and T334 relevant to substrate binding and translocation of small pyrroles through PigC access tunnels. [ABSTRACT FROM AUTHOR]

The CompassR (computer‐assisted recombination) rule enables, among beneficial substitutions, the identification of those that can be recombined in directed evolution. Herein, a recombination strategy is systematically investigated to minimize experimental efforts and maximize possible improvements. In total, 15 beneficial substitutions from Bacillus subtilis lipase A (BSLA), which improves resistance to the organic cosolvent 1,4‐dioxane (DOX), were studied to compare two recombination strategies, the two‐gene recombination process (2GenReP) and the in silico guided recombination process (InSiReP), employing CompassR. Remarkably, both strategies yielded a highly DOX‐resistant variant, M4 (I12R/Y49R/E65H/N98R/K122E/L124K), with up to 14.6‐fold improvement after screening of about 270 clones. M4 has a remarkably enhanced resistance in 60 % (v/v) acetone (6.0‐fold), 30 % (v/v) ethanol (2.1‐fold), and 60 % (v/v) methanol (2.4‐fold) compared with wild‐type BSLA. Molecular dynamics simulations revealed that attracting water molecules by charged surface substitutions is the main driver for increasing the DOX resistance of BSLA M4. Both strategies and obtained molecular knowledge can likely be used to improve the properties of other enzymes with a similar α/β‐hydrolase fold. [ABSTRACT FROM AUTHOR]

• Semi-rational engineering on surface residues of CGTase for enhanced OSs resistance. • A variant G539I/R146F/D147N with enhanced OSs resistance and thermostability was obtained. • Strengthened structural compactness and rigidity of CGTase is conducive to its OSs resistance. Cyclodextrin glycosyltransferase (CGTase) is a preferable biocatalyst for production of cyclodextrin and phenols glycosylated derivatives due to its cost-effective donors and broad range of acceptors. Organic solvents (OSs) are often beneficial for solubilizing hydrophobic substrates and product specificity, but often harmful towards CGTases. Herein, using Paenibacillus macerans CGTase (Pm CGTase) as a template and DMSO as a screening solvent, semi-rational engineering was performed at 18 surface residues by a developed high throughput screening method. Using site saturation mutagenesis and iterative saturation mutagenesis, 5 OSs-resistant variants (G539I, G539V, G539I/R146F, G539V/R146A, G539I/R146F/D147N) were obtained. Compared with WT, the best variant G539I/R146F/D147N showed a 79.3 % enhanced residual activity at 30 % DMSO and a 60 % increased t 1/2 value at both 40 and 50 °C. In addition, a decreased K m value and an increased k cat value led to 41.9 % higher catalytic efficiency of G539I/R146F/D147N than WT in 15 % DMSO. This variant was also applied to glycosylation of sophoricoside in 20 % DMSO, leading to 26.2 % increased conversion. Molecular dynamics demonstrates that its enhanced OSs resistance may be attributed to the strengthened structural compactness and rigidity. Our results provide guidance for engineering OSs resistance of Pm CGTase, which would further improve its application potential. [Display omitted] [ABSTRACT FROM AUTHOR]

Enzymatic oxidative decarboxylation is an up‐and‐coming reaction yet lacking efficient screening methods for the directed evolution of decarboxylases. Here, we describe a simple photoclick assay for the detection of decarboxylation products and its application in a proof‐of‐principle directed evolution study on the decarboxylase OleT. The assay was compatible with two frequently used OleT operation modes (directly using hydrogen peroxide as the enzyme's co‐substrate or using a reductase partner) and the screening of saturation mutagenesis libraries identified two enzyme variants shifting the enzyme's substrate preference from long chain fatty acids toward styrene derivatives. Overall, this photoclick assay holds promise to speed‐up the directed evolution of OleT and other decarboxylases. [ABSTRACT FROM AUTHOR]

Photoactive biological systems modify the optical properties of their chromophores, known as spectral tuning. Determining the molecular origin of spectral tuning is instrumental for understanding the function and developing applications of these biomolecules. Spectral tuning in flavin-binding fluorescent proteins (FbFPs), an emerging class of fluorescent reporters, is limited by their dependency on protein-bound flavins, whose structure and hence electronic properties cannot be altered by mutation. A blue-shifted variant of the plantderived improved light, oxygen, voltage FbFP has been created by introducing a lysine within the flavin-binding pocket, but the molecular basis of this shift remains unconfirmed. We here structurally characterize the blue-shifted improved light, oxygen, voltage variant and construct a new blue-shifted CagFbFP protein by introducing an analogous mutation. X-ray structures of both proteins reveal displacement of the lysine away from the chromophore and opening up of the structure as instrumental for the blue shift. Site saturation mutagenesis and high-throughput screening yielded a redshifted variant, and structural analysis revealed that the lysine side chain of the blue-shifted variant is stabilized close to the flavin by a secondary mutation, accounting for the red shift. Thus, a single additional mutation in a blue-shifted variant is sufficient to generate a red-shifted FbFP. Using spectroscopy, X-ray crystallography, and quantum mechanics molecular mechanics calculations, we provide a firm structural and functional understanding of spectral tuning in FbFPs. We also show that the identified blue- and red-shifted variants allow for two-color microscopy based on spectral separation. In summary, the generated blue- and red-shifted variants represent promising new tools for application in life sciences. [ABSTRACT FROM AUTHOR]

Enzymes are nature's catalysts and have great potential for sustainable processes from renewable resources. The adaptation of enzymes to the requirements of industrial processes is a prerequisite to harness their benefits in large-scale transformations for the synthesis of fine chemicals, food products, and pharmaceuticals. Immobilisation of wild-type or engineered enzymes using natural and synthetic carriers has been extensively employed to improve catalytic performance, ensure recovery and reuse, and thereby promote their use in industrial processes. Over the past three decades, significant progress has been achieved in the design and application of immobilised enzymes. Microgels as containers for protein immobilisation are advancing into a promising alternative as reflected by a growing body of literature that documents their use in sustainable catalytic processes. Microgels are crosslinked submicron-sized colloidal polymer networks and in this review, we summarise the progress in the synthesis and applications of enzyme-loaded microgels (μ-Gelzymes). We start by exploring the different approaches used for enzyme immobilisation on or within microgels and give representative examples for the use of μ-Gelzymes in different applications. Subsequently, the potential of μ-Gelzymes in achieving sustainable catalysis is discussed from a green chemistry perspective. Finally, we draw future directions for further improvement of biocatalytic μ-Gelzymes as an emerging interdisciplinary research field of interactive soft matter. [ABSTRACT FROM AUTHOR]

Cytochrome P450s are an important group of enzymes catalyzing hydroxylation, and epoxidations reactions. In this work we describe the characterization of the CinA–CinC fusion enzyme system of a previously reported P450 using genetically fused heme (CinA) and FMN (CinC) enzyme domains from Citrobacter braaki. We observed that mixing individually inactivated heme (-) with FMN (-) domain in the CinA-10aa linker - CinC fusion constructs results in recovered activity and the formation of (2S)-2β-hydroxy,1,8-cineole (174 µM), a similar amount when compared to the fully functional fusion protein (176 µM). We also studied the effect of the fusion linker length in the activity complementation assay. Our results suggests an intermolecular interaction between heme and FMN parts from different CinA–CinC fusion protein similar to proposed mechanisms for P450 BM3 on the other hand, linker length plays a crucial influence on the activity of the fusion constructs. However, complementation assays show that inactive constructs with shorter linker lengths have functional subunits, and that the lack of activity might be due to incorrect interaction between fused enzymes. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus sortase A (SaSrtA) is widely used for site‐specific protein modifications, but it lacks the robustness for performing bioconjugation reactions at elevated temperatures or in presence of denaturing agents. Loop engineering and subsequent head‐to‐tail backbone cyclization of SaSrtA yielded the cyclized variant CyM6 that has a 7.5 °C increased melting temperature and up to 4.6‐fold increased resistance towards denaturants when compared to the parent rM4. CyM6 gained up to 2.6‐fold (vs. parent rM4) yield of conjugate in ligation of peptide and primary amine under denaturing conditions. [ABSTRACT FROM AUTHOR]