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d- Allulose is one of the most common rare sugars with low calorie and is extensively applied in the food, cosmetic, and pharmaceutical fields. Here, a novel in vitro multienzyme cascade route (MCAS) was designed to produce d- allulose from sucrose. The MCAS route comprised three modules (sucrose phosphorolysis, phosphorylation–dephosphorylation, and ATP regeneration), including five enzymes (sucrose phosphorylase, fructokinase, d- allulose 6-phosphate 3-epimerase, d- allulose 6-phosphatephosphatase, and polyphosphate kinase). As a rate-limiting enzyme, d- allulose 6-phosphate 3-epimerase from Pantoea sp. was semi-rationally engineered, and variant Y143F led to 88.8% yield of d- allulose from 10 mM sucrose using one-pot strategy. ATP consumption was reduced to 5% of the sucrose concentration after introducing ATP-regeneration system. At elevated sucrose concentration of 100 mM by two-step strategy, a d- allulose yield of 70.4% was achieved with a space-time yield of 1.06 g L−1·h−1. This study provides an efficient and cost-effective approach for producing d- allulose from sucrose. [Display omitted] [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]

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]

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]

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]

Abstract Accumulation of microplastic in the environment and food chain will be a grand challenge for our society. Polyurethanes are widely used synthetic polymers in medical (e.g. catheters) and industrial products (especially as foams). Polyurethane is not abundant in nature and only a few microbial strains (fungi and bacteria) and enzymes (polyurethaneases and cutinases) have been reported to efficiently degrade polyurethane. Notably, in nature a long period of time (from 50 to >100 years depending on the literature) is required for degradation of plastics. Material binding peptides (e.g. anchor peptides) bind strongly to polymers such as polypropylene, polyethylene terephthalate, and polyurethane and can target specifically polymers. In this study we report the fusion of the anchor peptide Tachystatin A2 to the bacterial cutinase Tcur1278 which accelerated the degradation of polyester-polyurethane nanoparticles by a factor of 6.6 in comparison to wild-type Tcur1278. Additionally, degradation half-lives of polyester-polyurethane nanoparticles were reduced from 41.8 h to 6.2 h (6.7-fold) in a diluted polyester-polyurethane suspension (0.04% w/v). Graphical abstract Unlabelled Image Highlights • Material binding peptides such as anchor peptides strongly bind synthetic polymers. • The anchor peptide Tachystatin A2 served as adhesion promoter for cutinase Tcur1278. • Tachystatin A2 enhanced enzymatic degradation of polyester-polyurethane by 6.6-fold. • Tachystatin A2 reduced half-life of polyester-polyurethane nanoparticles by 6.7-fold. • Prominent anchoring effect of adhesion promoter in highly diluted nanoparticles. [ABSTRACT FROM AUTHOR]

Polypropylene is one of the widest spread commodity polymers in plastic industry with an estimated global consumption of 62.4 million tons in 2020. Surface modification of polypropylene is required for its application as textile fibers, packaging material or filtration membranes. Modification of polypropylene is challenging due to absent functional surface groups. An anchor-peptide-based toolbox for green and versatile polypropylene functionalization was developed. Fusion proteins composed of enhanced green fluorescent protein (EGFP) and anchor peptides (e.g. cecropin A or LCI) were designed and applied to polypropylene surfaces. Resulting protein coatings of EGFP-LCI were characterized by fluorescence and scanning force microscopy. The fusion protein EGFP-LCI formed densely packed monolayers of 4.1 ± 0.2 nm thickness. A microtiter plate-based fluorescence assay was developed to analyze the coating in presence of surfactants. Washing of EGFP-LCI coated polypropylene with 10 mM non-ionic surfactant (Triton X-100) did not detach the protein film, whereas EGFP was removed completely. Anchor peptides promote binding to polypropylene by simple dip-coating at room temperature in water. The high coating density (0.8 pmol/cm 2 ) as well as the number and diversity of provided functional groups offer a viable alternative to conventional modification strategies of functionalizing polypropylene. LCI's role as broadly applicable adhesion promoter was demonstrated by equipping polypropylene with the fluorescent dye ThioGlo-1 via the anchor peptide LCI. [ABSTRACT FROM AUTHOR]

The research on protein-polymer conjugates by grafting from the surface of proteins has gained significant interest in the last decade. While there are many studies with globular proteins, membrane proteins have remained untouched to the best of our knowledge. In this study, we established the conjugate formation with a class of transmembrane proteins and grow polymer chains from the ferric hydroxamate uptake protein component A (FhuA; a β-barrel transmembrane protein of Escherichia coli ). As the lysine residues of naturally occurring FhuA are distributed over the whole protein, FhuA was reengineered to have up to 11 lysines, distributed symmetrically in a rim on the membrane exposed side (outside) of the protein channel and exclusively above the hydrophobic region. Reengineering of FhuA ensures a polymer growth only on the outside of the β-barrel and prevents blockage of the channel as a result of the polymerization. A water-soluble initiator for controlled radical polymerization (CRP) was consecutively linked to the lysine residues of FhuA and N -isopropylacrylamide (NIPAAm) polymerized under copper-mediated CRP conditions. The conjugate formation was analyzed by using MALDI-ToF mass spectrometry, SDS-PAGE, circular dichroism spectroscopy, analytical ultracentrifugation, dynamic light scattering, transmission electron microscopy and size exclusion chromatography. Such conjugates combine the specific functions of the transmembrane proteins, like maintaining membrane potential gradients or translocation of substrates with the unique properties of synthetic polymers such as temperature and pH stimuli handles. FhuA-PNIPAAm conjugates will serve as functional nanosized building blocks for applications in targeted drug delivery, self-assembly systems, functional membranes and transmembrane protein gated nanoreactors. [ABSTRACT FROM AUTHOR]

Cytochrome P450s are heme-thiolate enzymes that have been broadly applied in pharmaceutical and biosynthesis because of their efficient oxidation at inert carbons. Extensive engineering campaigns are applied to P450s to explore new non-natural substrates and reactions; however, achieving high coupling efficiency is one of the main challenges. The undesirable uncoupling reactions result in the extra consumption of expensive cofactor NAD(P)H, and lead to the accumulation of reactive oxygen species and the inactivation of enzymes and organisms. Using protein engineering methods, these limitations can be overcome by engineering and fine-tuning P450s. A systemic perspective of the enzyme structure and the catalytic mechanism is essential for P450 engineering campaigns for higher coupling efficiency. This review provide an overview on factors contributing to uncoupling and protein engineering approaches to minimize uncoupling and thereby generating efficient and robust P450s for industrials use. Contributing uncoupling factors are classified into three main groups: i) substrate binding pocket; ii) ligand access tunnel(s); and iii) electron transfer pathway(s). Finally, we draw future directions for combinations of effective state-of-the-art technologies and available software/online tools for P450s engineering campaigns. • Achieving high coupling efficiency of cytochrome P450s is a quite challenge calling for solutions. • Uncoupling reasons of P450s based on structures and molecular mechanisms are deciphered. • The deep view for improving coupling efficiency is reviewed. • State-of-the-art technologies and software for P450 engineering campaign are summarized. [ABSTRACT FROM AUTHOR]

Phytases are hydrolytic enzymes capable of a stepwise phosphate release from phytate which is the main phosphorous storage in seeds, cereals and legumes. Limitations such as low enzyme activity or incomplete phytate hydrolysis to inositol are a great challenge in phytase applications in food and feed. Herein we report a phytase blend of two enzymes with additive effects on phytate (InsP6) hydrolysis and its application in the enzymatic phosphorous recovery process. Blending the fast 6-phytase rPhyXT52 with the 3-phytase from Debaryomyces castellii , which is capable of fully hydrolyzing InsP6, we achieved rapid phosphate release with higher yields compared to the individual enzymes and a rapid disappearance of InsP6–3 intermediates, monitored by HPLC. NMR data suggest a nearly complete phytate hydrolysis to inositol and phosphate. The blend was applied for phosphate mobilization from phytate-rich biomass, such as deoiled seeds. For this emerging application, an up to 43% increased phosphate mobilization yield was achieved when using 1000 U of the blend per kg biomass compared to using only the E. coli phytase. Even so, the time of enzyme treatment was decreased by more than half (6 h instead of 16 h) when using 4000 U of blend, we reached a 78–90% reduction of the total phosphorous content in the explored deoiled seeds. In summary, the phytase blend of Dc phyt/rPhyXT52 was proven very efficient to obtain inositol phosphate depleted meal which has its potential application in animal feeding and is concomitant with the production of green phosphate from renewable resources. [Display omitted] • Enzymatic phosphorous mobilization from biomass such as deoiled seeds. • Complete phytate hydrolysis with a blend of a 3-phytase and a 6-phytase. • Phytase blend of Dc phyt / rPhyXT52 lead to inositol phosphate depleted meal. • Reduction of the total phosphorous in feed. • Phosphate recycling from renewable resources in circular P bioeconomy. [ABSTRACT FROM AUTHOR]