Identification of yttrium oxide-specific peptides for future recycling of rare earth elements from electronic scrap.

Yttrium is a heavy rare earth element (REE) that acquires remarkable characteristics when it is in oxide form and doped with other REEs. Owing to these characteristics Y2 O3 can be used in the manufacture of several products. However, a supply deficit of this mineral is expected in the coming years, contributing to its price fluctuation. Thus, developing an efficient, cost-effective, and eco-friendly process to recover Y2 O3 from secondary sources has become necessary. In this study, we used phage surface display to screen peptides with high specificity for Y2 O3 particles. After three rounds of enrichment, a phage expressing the peptide TRTGCHVPRCNTLS (DM39) from the random pVIII phage peptide library Cys4 was found to bind specifically to Y2 O3 , being 531.6-fold more efficient than the wild-type phage. The phage DM39 contains two arginines in the polar side chains, which may have contributed to the interaction between the mineral targets. Immunofluorescence assays identified that the peptide's affinity was strong for Y2 O3 and negligible to LaPO4 :Ce3+ ,Tb3+ . The identification of a peptide with high specificity and affinity for Y2 O3 provides a potentially new strategic approach to recycle this type of material from secondary sources, especially from electronic scrap.

Application of machine learning on understanding biomolecule interactions in cellular machinery.

Machine learning (ML) applications have become ubiquitous in all fields of research including protein science and engineering. Apart from protein structure and mutation prediction, scientists are focusing on knowledge gaps with respect to the molecular mechanisms involved in protein binding and interactions with other components in the experimental setups or the human body. Researchers are working on several wet-lab techniques and generating data for a better understanding of concepts and mechanics involved. The information like biomolecular structure, binding affinities, structure fluctuations and movements are enormous which can be handled and analyzed by ML. Therefore, this review highlights the significance of ML in understanding the biomolecular interactions while assisting in various fields of research such as drug discovery, nanomedicine, nanotoxicity and material science. Hence, the way ahead would be to force hand-in hand of laboratory work and computational techniques.

Quantification of Peptide-Bound Particles: A Phage Mimicking Approach via Site-Selective Immobilization on Glass.

The increasing complexity and need of high-tech materials for modern electronics raise the demand for rare earth elements. While recycling rates are still negligible for most elements, geopolitical tensions, circular economy, and the aim for a carbon-neutral society put pressure on conventional supply strategies and emphasize the need for new ideas for recycling. Our research group works on the development of phage surface display (PSD)-derived peptide-based recycling methods for electronic waste. This study focuses on LaPO4:Ce,Tb (LAP), a component of electronic waste from compact energy-saving lamps containing rare earth element-enriched fluorescent powders. While free solution-phase peptides show little to no interaction with the target material, we re-enabled the binding capability by immobilizing them on various glass supports. We shine a spotlight on the transition from phage-bound to free peptides and present the first proof of successful peptide-LAP particle interactions of previously reported PSD-derived sequences. Therefore, we introduce a method to investigate peptide-particle-interactions qualitatively and quantitatively. Additionally, a calibration curve allowed the quantification of peptide-bound particles. Combined with the quantification of the immobilized peptide on the surface, it was possible to calculate a potential dosage of peptides for future recycling processes.

Investigation of the structure and dynamics of gallium binding to high-affinity peptides elucidated by multi-scale simulation, quantum chemistry, NMR and ITC.

Gallium (as Ga3+) is a Group IIIa metal and its recovery from wastewaters has become increasingly important for its reuse. The use of peptides for recycling offers a low-cost and environmentally-friendly option but the structural characteristics of peptides likely to bind Ga3+ are largely unknown. Multiple computational methods, coupled with experimental verification via NMR and Isothermal Calorimetry (ITC), were used to establish that Ga3+ binds with high affinity to peptide sequences and to elucidate the structural characteristics that contributed. It was demonstrated that peptide pre-organisation is key to Ga3+ binding and that a favourable binding position is necessarily governed by the size and shape of the electrostatic environment as much as individual electrostatic interactions with peptide residues themselves. Given favourable conditions, Ga3+ retrieved plausible binding positions involving both charged and uncharged residues that greatly increases the range of bonding possibilities with other peptide sequences and offers insights for binding other metals. The addition of pH buffer substantially improved the affinity of Ga3+ and a structural role for a buffer component was demonstrated.

Gallium-binding peptides as a tool for the sustainable treatment of industrial waste streams.

Here we provide a proof of principle for an application-oriented concept for the peptide-based recovery of gallium in industrial wastewater, which was supported by biosorption studies with a real wastewater sample. We investigated the interaction of the gallium-binding peptides TMHHAAIAHPPH, NYLPHQSSSPSR, SQALSTSRQDLR, HTQHIQSDDHLA, and NDLQRHRLTAGP with gallium and arsenic through different experimental and computational approaches. Data obtained from isothermal titration microcalorimetry indicated a competitive influence by the presence of acetate ions with an exothermic contribution to the otherwise endothermic peptide gallium interactions. For peptide HTQHIQSDDHLA, a stabilizing influence of acetate ions on the metal peptide interaction was found. Peptide NYLPHQSSSPSR showed the highest affinity for gallium in ITC studies. Computational modeling of peptide NYLPHQSSSPSR was used to determine interaction parameters and to explain a possible binding mechanism. Furthermore, the peptides were immobilized on polystyrene beads. Thus, we created a novel and exceptionally robust peptide-based material for the biosorption of gallium from an aqueous solution. Data obtained from isothermal titration microcalorimetry indicated a competitive influence by the presence of acetate ions with an exothermic contribution to the otherwise endothermic peptide gallium interactions. For peptide HTQHIQSDDHLA, a stabilizing influence of acetate ions on the metal peptide interaction was found. Peptide NYLPHQSSSPSR showed the highest affinity for gallium in ITC studies. Computational modeling of peptide NYLPHQSSSPSR was used to determine interaction parameters and to explain a possible binding mechanism. Furthermore, the peptides were immobilized on polystyrene beads. Thus, we created a novel and exceptionally robust peptide-based material for the biosorption of gallium from an aqueous solution.

Application of Next Generation Sequencing (NGS) in Phage Displayed Peptide Selection to Support the Identification of Arsenic-Binding Motifs.

Next generation sequencing (NGS) in combination with phage surface display (PSD) are powerful tools in the newly equipped molecular biology toolbox for the identification of specific target binding biomolecules. Application of PSD led to the discovery of manifold ligands in clinical and material research. However, limitations of traditional phage display hinder the identification process. Growth-based library biases and target-unrelated peptides often result in the dominance of parasitic sequences and the collapse of library diversity. This study describes the effective enrichment of specific peptide motifs potentially binding to arsenic as proof-of-concept using the combination of PSD and NGS. Arsenic is an environmental toxin, which is applied in various semiconductors as gallium arsenide and selective recovery of this element is crucial for recycling and remediation. The development of biomolecules as specific arsenic-binding sorbents is a new approach for its recovery. Usage of NGS for all biopanning fractions allowed for evaluation of motif enrichment, in-depth insight into the selection process and the discrimination of biopanning artefacts, e.g., the amplification-induced library-wide reduction in hydrophobic amino acid proportion. Application of bioinformatics tools led to the identification of an SxHS and a carboxy-terminal QxQ motif, which are potentially involved in the binding of arsenic. To the best of our knowledge, this is the first report of PSD combined with NGS of all relevant biopanning fractions.

Bacillus safensis JG-B5T affects the fate of selenium by extracellular production of colloidally less stable selenium nanoparticles.

Understanding the impact of microorganisms on the mobility of selenium (Se) is important for predicting the fate of toxic Se in the environment and improving wastewater treatment technologies. The bacteria strain Bacillus safensis JG-B5T, isolated from soil in a uranium mining waste pile, can influence the Se speciation in the environment and engineered systems. However, the mechanism and conditions of this process remain unknown. This study found that the B. safensis JG-B5T is an obligate aerobic microorganism with an ability to reduce 70% of 2.5 mM selenite to produce red spherical biogenic elemental selenium nanoparticles (BioSeNPs). Only extracellular production of BioSeNPs was observed using transmission electron microscopy. The two-chamber reactor experiments, genome analysis and corona proteins identified on BioSeNPs suggested that the selenite reduction process was primarily mediated through membrane-associated proteins, like succinate dehydrogenase. Extracellular presence and low colloidal stability of BioSeNPs as indicated by ζ-potential measurements, render B. safensis JG-B5T an attractive candidate in wastewater treatment as it provides easy way of recovering Se while maintaining low Se discharge. As this microorganism decreases Se mobility, it will affect Se bioavailability in the environment and decreases its toxicity.

Chromatopanning for the identification of gallium binding peptides.

This study is concerned with a chromatography-based approach (Immobilized Metal Ion Affinity Chromatography) for the recovery of gallium binding peptide sequences from a recombinant phage display library. The here described methods apply the fundamental knowledge and methods of separation science and meet thereby the key requirement of the phage display technique of precise separation of target-binding bacteriophage clones from non-interacting bacteriophage during the biopanning. During the chromatopanning called process, a total of 101 bacteriophage clones were identified of which in subsequent binding experiments, phage clones expressing the peptide sequences TMHHAAIAHPPH, SQALSTSRQDLR and HTQHIQSDDHLA were characterized to bind >10 fold better to a target that presents immobilized gallium ions than control phage, displaying no peptide sequence. The performance of biopanning experiments in chromatographic systems is particularly suitable for demanding targets such as trivalent metal ions. We found, that the selection process benefits immensely from the stable immobilization of the target metal ions during the entire biopanning process as well as the complete recovery of well interacting bacteriophage clones. Among others, this was possible due to an enhanced monitoring of process conditions and fractionation of eluates.

Directed Evolution and Engineering of Gallium-Binding Phage Clones-A Preliminary Study.

The phage surface display technology is a useful tool to screen and to extend the spectrum of metal-binding protein structures provided by nature. The directed evolution approach allows identifying specific peptide ligands for metals that are less abundant in the biosphere. Such peptides are attractive molecules in resource technology. For example, gallium-binding peptides could be applied to recover gallium from low concentrated industrial wastewater. In this study, we investigated the affinity and selectivity of five bacteriophage clones displaying different gallium-binding peptides towards gallium and arsenic in independent biosorption experiments. The displayed peptides were highly selective towards Ga3+ whereby long linear peptides showed a lower affinity and specificity than those with a more rigid structure. Cysteine scanning was performed to determine the relationship between secondary peptide structure and gallium sorption. By site-directed mutagenesis, the amino acids of a preselected peptide sequence are systematically replaced by cysteines. The resulting disulphide bridge considerably reduces the flexibility of linear peptides. Subsequent biosorption experiments carried out with the mutants obtained from cysteine scanning demonstrated, depending on the position of the cysteines in the peptide, either a considerable increase in the affinity of gallium compared to arsenic or an increase in the affinity for arsenic compared to gallium. This study shows the impressive effect on peptide-target interaction based on peptide structure and amino acid position and composition via the newly established systematic cysteine scanning approach.

Draft Genome Sequence of Bacillus safensis Strain JG-B5T, Isolated from a Uranium Mining Waste Pile.

Bacillus safensis strain JG-B5T was isolated from soil of the uranium mining waste pile Haberland located near Johanngeorgenstadt, Saxony, Germany. We report here a draft genome sequence (3.7 Mb) of this bacterial strain. The high metal resistance abilities of B. safensis strain JG-B5T can be exploited for bioremediation of metal and metalloid-contaminated environments.

Peptides as biosorbents - Promising tools for resource recovery.

Despite many innovations, meeting both economic and ecological requirements remains challenging for conventional resource recovery technology. The development of highly selective peptides puts a new competitor on the market. We present an approach to identify peptides for resource recovery using Phage Surface Display. Here, we describe the development of peptides for binding of rare earth element terbium-containing solids and for removal and enrichment of the heavy metal ions of cobalt and nickel out of waste waters and leaching solutions. We identified phage displaying specific peptides with ∼100× enhanced affinity towards terbium-containing solids or ∼20× enhanced affinity towards nickel (∼3× cobalt).

Bio-recycling of metals: Recycling of technical products using biological applications.

The increasing demand of different essential metals as a consequence of the development of new technologies, especially in the so called "low carbon technologies" require the development of innovative technologies that enable an economic and environmentally friendly metal recovery from primary and secondary resources. There is serious concern that the demand of some critical elements might exceed the present supply within a few years, thus necessitating the development of novel strategies and technologies to meet the requirements of industry and society. Besides an improvement of exploitation and processing of ores, the more urgent issue of recycling of strategic metals has to be enforced. However, current recycling rates are very low due to the increasing complexity of products and the low content of certain critical elements, thus hindering an economic metal recovery. On the other hand, increasing environmental consciousness as well as limitations of classical methods require innovative recycling methodologies in order to enable a circular economy. Modern biotechnologies can contribute to solve some of the problems related to metal recycling. These approaches use natural properties of organisms, bio-compounds, and biomolecules to interact with minerals, materials, metals, or metal ions such as surface attachment, mineral dissolution, transformation, and metal complexation. Further, modern genetic approaches, e.g. realized by synthetic biology, enable the smart design of new chemicals. The article presents some recent developments in the fields of bioleaching, biosorption, bioreduction, and bioflotation, and their use for metal recovery from different waste materials. Currently only few of these developments are commercialized. Major limitations are high costs in comparison to conventional methods and low element selectivity. The article discusses future trends to overcome these barriers. Especially interdisciplinary approaches, the combination of different technologies, the inclusion of modern genetic methods, as well as the consideration of existing, yet unexplored natural resources will push innovations in these fields.

Identification of lanthanum-specific peptides for future recycling of rare earth elements from compact fluorescent lamps.

As components of electronic scrap, rare earth minerals are an interesting but little used source of raw materials that are highly important for the recycling industry. Currently, there exists no cost-efficient technology to separate rare earth minerals from an electronic scrap mixture. In this study, phage surface display has been used as a key method to develop peptides with high specificity for particular inorganic targets in electronic scrap. Lanthanum phosphate doped with cerium and terbium as part of the fluorescent phosphors of spent compact fluorescent lamps (CFL) was used as a target material of economic interest to test the suitability of the phage display method to the separation of rare earth minerals. One random pVIII phage library was screened for peptide sequences that bind specifically to the fluorescent phosphor LaPO4 :Ce3+ ,Tb3+ (LAP). The library contained at least 100 binding pVIII peptides per phage particle with a diversity of 1 × 109 different phage per library. After three rounds of enrichment, a phage clone containing the surface peptide loop RCQYPLCS was found to bind specifically to LAP. Specificity and affinity of the identified phage bound peptide was confirmed by using binding and competition assays, immunofluorescence assays, and zeta potential measurements. Binding and immunofluorescence assays identified the peptide's affinity for the fluorescent phosphor components CAT (CeMgAl11 O19 :Tb3+ ) and BAM (BaMgAl10 O17 :Eu2+ ). No affinity was found for other fluorescent phosphor components such as YOX (Y2 O3 :Eu3+ ). The binding specificity of the RCQYPLCS peptide loop was improved 3-51-fold by using alanine scanning mutagenesis. The identification of peptides with high specificity and affinity for special components in the fluorescent phosphor in CFLs provides a potentially new strategic approach to rare earth recycling. Biotechnol. Bioeng. 2017;114: 1016-1024. © 2016 Wiley Periodicals, Inc.

Identification of mineral-binding peptides that discriminate between chalcopyrite and enargite.

Innovative approaches to the separation of minerals and subsequent extraction of metals are imperative owing to the increasing mineralogical complexity of ore deposits that are difficult or even impossible to separate into slurries or solutions containing only the minerals or metals of interest. Low recovery of metal is typical for these complex deposits leading to significant losses to tailings. In addition, the minerals often contain impurities, some toxic, which are difficult and costly to control or manage during the processing of a concentrate or other mineral product. One example of this complex situation is the significant economic and environmental costs associated with diluting and processing copper concentrates containing arsenic (in the form of the mineral enargite, Cu3 AsS4 ) in the production of pure copper. To overcome these separation problems, we have utilized phage display to identify peptides that demonstrate selective recognition of enargite and the arsenic-free copper sulfide, chalcopyrite. Screening of two random peptide phage display libraries resulted in the identification of an enargite-selective peptide with the sequence MHKPTVHIKGPT and a chalcopyrite-selective peptide with the sequence RKKKCKGNCCYTPQ. Mineral-binding selectivity was demonstrated by binding studies, zeta potential determination and immunochemistry. Peptides that have the ability to discriminate between enargite and chalcopyrite provide a greener option for the separation of arsenic containing contaminants from copper concentrates. This represents the first step towards a major advance in the replacement or reduction of toxic collectors as well as reducing the level of arsenic-bearing minerals in the early stages of mineral processing. Biotechnol. Bioeng. 2017;114: 998-1005. © 2016 Wiley Periodicals, Inc.

Characterization of Three Different Unusual S-Layer Proteins from Viridibacillus arvi JG-B58 That Exhibits Two Super-Imposed S-Layer Proteins.

Genomic analyses of Viridibacillus arvi JG-B58 that was previously isolated from heavy metal contaminated environment identified three different putative surface layer (S-layer) protein genes namely slp1, slp2, and slp3. All three genes are expressed during cultivation. At least two of the V. arvi JG-B58 S-layer proteins were visualized on the surface of living cells via atomic force microscopy (AFM). These S-layer proteins form a double layer with p4 symmetry. The S-layer proteins were isolated from the cells using two different methods. Purified S-layer proteins were recrystallized on SiO2 substrates in order to study the structure of the arrays and self-assembling properties. The primary structure of all examined S-layer proteins lack some features that are typical for Bacillus or Lysinibacillus S-layers. For example, they possess no SLH domains that are usually responsible for the anchoring of the proteins to the cell wall. Further, the pI values are relatively high ranging from 7.84 to 9.25 for the matured proteins. Such features are typical for S-layer proteins of Lactobacillus species although sequence comparisons indicate a close relationship to S-layer proteins of Lysinibacillus and Bacillus strains. In comparison to the numerous descriptions of S-layers, there are only a few studies reporting the concomitant existence of two different S-layer proteins on cell surfaces. Together with the genomic data, this is the first description of a novel type of S-layer proteins showing features of Lactobacillus as well as of Bacillus-type S-layer proteins and the first study of the cell envelope of Viridibacillus arvi.

Identification of multiple putative S-layer genes partly expressed by Lysinibacillus sphaericus JG-B53.

Lysinibacillus sphaericus JG-B53 was isolated from the uranium mining waste pile Haberland near Johanngeorgenstadt, Germany. Previous studies have shown that many bacteria that have been isolated from these heavy metal contaminated environments possess surface layer (S-layer) proteins that enable the bacteria to survive by binding metals with high affinity. Conversely, essential trace elements are able to cross the filter layer and reach the interior of the cell. This is especially true of the S-layer of L. sphaericus JG-B53, which possesses outstanding recrystallization and metal-binding properties. In this study, S-layer protein gene sequences encoded in the genome of L. sphaericus JG-B53 were identified using next-generation sequencing technology followed by bioinformatic analyses. The genome of L. sphaericus JG-B53 encodes at least eight putative S-layer protein genes with distinct differences. Using mRNA analysis the expression of the putative S-layer protein genes was studied. The functional S-layer protein B53 Slp1 was identified as the dominantly expressed S-layer protein in L. sphaericus JG-B53 by mRNA studies, SDS-PAGE and N-terminal sequencing. B53 Slp1 is characterized by square lattice symmetry and a molecular mass of 116 kDa. The S-layer protein B53 Slp1 shows a high similarity to the functional S-layer protein of L. sphaericus JG-A12, which was isolated from the same uranium mining waste pile Haberland and has been described by previous research. These similarities indicate horizontal gene transfer and DNA rearrangements between these bacteria. The presence of multiple S-layer gene copies may enable the bacterial strains to quickly adapt to changing environments.

Development of functionalised polyelectrolyte capsules using filamentous Escherichia coli cells.

BACKGROUND: Escherichia coli is one of the best studied microorganisms and finds multiple applications especially as tool in the heterologous production of interesting proteins of other organisms. The heterologous expression of special surface (S-) layer proteins caused the formation of extremely long E. coli cells which leave transparent tubes when they divide into single E. coli cells. Such natural structures are of high value as bio-templates for the development of bio-inorganic composites for many applications. In this study we used genetically modified filamentous Escherichia coli cells as template for the design of polyelectrolyte tubes that can be used as carrier for functional molecules or particles. Diversity of structures of biogenic materials has the potential to be used to construct inorganic or polymeric superior hybrid materials that reflect the form of the bio-template. Such bio-inspired materials are of great interest in diverse scientific fields like Biology, Chemistry and Material Science and can find application for the construction of functional materials or the bio-inspired synthesis of inorganic nanoparticles. RESULTS: Genetically modified filamentous E. coli cells were fixed in 2% glutaraldehyde and coated with alternating six layers of the polyanion polyelectrolyte poly(sodium-4styrenesulfonate) (PSS) and polycation polyelectrolyte poly(allylamine-hydrochloride) (PAH). Afterwards we dissolved the E. coli cells with 1.2% sodium hypochlorite, thus obtaining hollow polyelectrolyte tubes of 0.7 µm in diameter and 5-50 µm in length. For functionalisation the polyelectrolyte tubes were coated with S-layer protein polymers followed by metallisation with Pd(0) particles. These assemblies were analysed with light microscopy, scanning electron microscopy, energy dispersive X-ray spectroscopy and transmission electron microscopy. CONCLUSION: The thus constructed new material offers possibilities for diverse applications like novel catalysts or metal nanowires for electrical devices. The novelty of this work is the use of filamentous E. coli templates and the use of S-layer proteins in a new material construct.

E. coli filament formation induced by heterologous S-layer expression.

Escherichia coli is a rod-shaped intestinal bacterium which has a size of 1.1-1.5 µm x 2.0-6.0 µm. The fast cell division process and the uncomplicated living conditions have turned E. coli into a widely used host in genetic engineering and into one of the best studied microorganisms of all. We used E. coli BL21(DE3) as host for heterologous expression of S-layer proteins of Lysinibacillus sphaericus JG-A12 in order to enable a fast and high efficient protein production. The S-layer expression induced in E. coli an unusual elongation of the cells, thus producing filaments of > 100 µm in length. In the stationary growth phase, E. coli filaments develop tube-like structures that contain E. coli single cells. Fluorescence microscopic analyses of S-layer expressing E. coli cells that were stained with membrane stain FM (®) 5-95 verify the membrane origin of the tubes. Analyses of DAPI stained GFP-S-layer expressing E. coli support the assumption of a disordered cell division that is induced by the huge amount of recombinant S-layer proteins. However, the underlying mechanism is still not characterized in detail. These results describe the occurrence of a novel stable cell form of E. coli as a result of a disordered cell division process. 

Heterologous expression of the surface-layer-like protein SllB induces the formation of long filaments of Escherichia coli consisting of protein-stabilized outer membrane.

Escherichia coli is one of the best studied micro-organisms and is the most widely used host in genetic engineering. The Gram-negative single cells are rod-shaped, and filaments are usually not found. Here, we describe the reproducible formation of elongated E. coli cells. During heterologous expression of the silent surface (S)-layer protein gene sllB from Lysinibacillus sphaericus JG-A12 in E. coli BL21(DE3), the cells were arranged as long chains which were surrounded by highly stable sheaths. These filaments had a length of >100 µm. In the stationary growth phase, microscopic analyses demonstrated the formation of unusually long transparent tube-like structures which were enclosing separate single cells. The tube-like structures were isolated and analysed by SDS-PAGE, infrared-spectroscopy and different microscopic methods in order to identify their unusual composition and structure. The tube-like structures were found to be like outer membranes, containing high levels of proteins and to which the recombinant S-layer proteins were attached. Despite the entire structure being indicative of a disordered cell division, the bacterial cells were highly viable and stable. To our knowledge, this is the first time that the induction of drastic morphological changes in E. coli by the expression of a foreign protein has been reported.