Cover Picture: Biotechnology Journal 8/2016

This regular issue of BTJ includes articles on biocatalysis, biochemical engineering, and bioprocess engineering. This cover page highlights the applications of biomolecules (e.g., proteins, enzymes, and DNA) in ionic liquids (ILs). The technological utility of biomolecules can be enhanced significantly by combining them with ILs. Image is provided by Magaret Sivapragasam, Muhammad Moniruzzaman, and Masahiro Goto authors of ”Recent advances in exploiting ionic liquids for biomolecules: Solubility, stability and applications“ ( http://dx.doi.org/10.1002/biot.201500603 ).     

Protein engineering and de novo designing of a biocatalyst

Proteins as a biomolecule have been recognized as a “molecule with manifold biological functions”. The functions not only include the structural, regulatory and transportation processes inside the body but also its capacity as an extremely specific catalyst for various biochemical reactions. Nature has been quite admirably using proteins as biocatalysts which are known as enzymes. Properties like higher reaction rate, good specificity, faster kinetics, production of lesser by‐products and their non‐hazardous nature make enzymes the most suitable targets for a process chemist to exploit. At the same time, limitations like a narrow range of substrates, requirement of coenzymes, lesser stability, smaller shelf‐life, along with difficulties in procuring these enzymes, make this biocatalysis field quite challenging. For exploiting a broad range of applications related to therapeutics, biosensors, biotechnology, nanotechnology etc., de novo designing of proteins is of utmost importance. Enzymes with altered, specific and modified properties might be designed by utilizing the prior knowledge of structure and function of a protein with the help of computational modeling. Various protein engineering techniques like directed evolution, rational designing and immobilization strategies etc. have already been extensively used to address some of the issues. This review aims to update the repertoire of the advancements in the field of protein engineering, which can help in laying some guiding principles about designing, modifying and altering their usage for commercial industrial purposes. This possibility of effective and novel designing of peptides and proteins might further facilitate our understanding about the structure, function and folding patterns along with their inter‐relationships. Copyright © 2016 John Wiley & Sons, Ltd.     

Enzymes from solvent-tolerant microbes: Useful biocatalysts for non-aqueous enzymology

Solvent-tolerant microbes are a newly emerging class that possesses the unique ability to thrive in the presence of organic solvents. Their enzymes adapted to mediate cellular and metabolic processes in a solvent-rich environment and are logically stable in the presence of organic solvents. Enzyme catalysis in non-aqueous/low-water media is finding increasing applications for the synthesis of industrially important products, namely peptides, esters, and other trans-esterification products. Solvent stability, however, remains a prerequisite for employing enzymes in non-aqueous systems. Enzymes, in general, get inactivated or give very low rates of reaction in non-aqueous media. Thus, early efforts, and even some recent ones, have aimed at stabilization of enzymes in organic media by immobilization, surface modifications, mutagenesis, and protein engineering. Enzymes from solvent-tolerant microbes appear to be the choicest source for studying solvent-stable enzymes because of their unique ability to survive in the presence of a range of organic solvents. These bacteria circumvent the solvent’s toxic effects by virtue of various adaptations, e.g. at the level of the cytoplasmic membrane, by degradation and transformation of solvents, and by active excretion of solvents. The recent screening of these exotic microbes has generated some naturally solvent-stable proteases, lipases, cholesterol oxidase, cholesterol esterase, cyclodextrin glucanotransferase, and other important enzymes. The unique properties of these novel biocatalysts have great potential for applications in non-aqueous enzymology for a range of industrial processes.     

Modular Structure of Biochips Based on Microstructured Deposition of Functional Nanoparticles

This paper presents the microstructured deposition of nanoparticles, which are chemically modified to be conjugated to biomolecules or dyes. In our approach, we separated the technical steps “surface chemistry” and “microstructuring” into two fully independent processes. This was realized by firstly synthesizing specific nanoparticles with tailor‐made surfaces. Thus, the surface chemistry was customized to the demands of most different kinds of specific proteins. Then, microstructured monolayer arrays of these nanoparticles were generated by photolithography, microcontact printing or spotting with a microarrayer. The overall system resulted in a nanoparticle‐based microarray for the selective binding of protein ligands providing both wide flexibility and high specificity.     

Baggasse Preservation: A Need for a Biotechnological Approach

ABSTRACT

Microarrays with biomolecules (e.g., DNA and proteins), cells, and tissues immobilized on solid substrates are important tools for biological research, including genomics, proteomics, and cell analysis. In this paper, the current state of microarray fabrication is reviewed. According to spot formation techniques, methods are categorized as “contact printing” and “non-contact printing.” Contact printing is a widely used technology, comprising methods such as contact pin printing and microstamping. These methods have many advantages, including reproducibility of printed spots and facile maintenance, as well as drawbacks, including low-throughput fabrication of arrays. Non-contact printing techniques are newer and more varied, comprising photochemistry-based methods, laser writing, electrospray deposition, and inkjet technologies. These technologies emerged from other applications and have the potential to increase microarray fabrication throughput; however, there are several challenges in applying them to microarray fabrication, including interference from satellite drops and biomolecule denaturization.     

From structure and dynamics to biomolecular functions: The ubiquitous role of solvent in biology

Biological activity requires a solvent that can provide a suitable environment, which satisfies the twin need for stability and the ability to change. Among all the solvents water plays the most important role. We review, analyze, and comment on recent works on the structure and dynamics of water around biomolecules and their role in specific biological functions. While studies in the past have focused on understanding the biomolecule–water interactions through a hydration layer; recently the attention has shifted towards understanding functions at a molecular level. Such a microscopic understanding clearly requires elucidation of detailed dynamical processes where solvent molecules play an important role. Finally, we comment on the advances made in understanding the role of water inside a biological cell.     

Surface manipulation of biomolecules for cell microarray applications

Many biological events, such as cellular communication, antigen recognition, tissue repair and DNA linear transfer, are intimately associated with biomolecule interactions at the solid-liquid interface. To facilitate the study and use of these biological events for biodevice and biomaterial applications, a sound understanding of how biomolecules behave at interfaces and a concomitant ability to manipulate biomolecules spatially and temporally at surfaces is required. This is particularly true for cell microarray applications, where a range of biological processes must be duly controlled to maximize the efficiency and throughput of these devices. Of particular interest are transfected-cell microarrays (TCMs), which significantly widen the scope of microarray genomic analysis by enabling the high-throughput analysis of gene function within living cells. This article reviews this current research focus, discussing fundamental and applied research into the spatial and temporal surface manipulation of DNA, proteins and other biomolecules and the implications of this work for TCMs.     

Mapping of ligand‐binding cavities in proteins

The complex interactions between proteins and small organic molecules (ligands) are intensively studied because they play key roles in biological processes and drug activities. Here, we present a novel approach to characterize and map the ligand‐binding cavities of proteins without direct geometric comparison of structures, based on Principal Component Analysis of cavity properties (related mainly to size, polarity, and charge). This approach can provide valuable information on the similarities and dissimilarities, of binding cavities due to mutations, between‐species differences and flexibility upon ligand‐binding. The presented results show that information on ligand‐binding cavity variations can complement information on protein similarity obtained from sequence comparisons. The predictive aspect of the method is exemplified by successful predictions of serine proteases that were not included in the model construction. The presented strategy to compare ligand‐binding cavities of related and unrelated proteins has many potential applications within protein and medicinal chemistry, for example in the characterization and mapping of “orphan structures”, selection of protein structures for docking studies in structure‐based design, and identification of proteins for selectivity screens in drug design programs. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Der „Sekundenkleber“ der Stummelfüßer

Novel insights into the “superglue” of velvet worms The biological “superglue” of velvet worms provides inspiration towards circular processing of advanced polymers. In nature, velvet worms employ a fluid, protein‐rich secretion for hunting and defense, which forms rapidly into stiff fibers. The fluid‐to‐fiber transition occurs outside the body without regulations, indicating that the “instructions” for assembly are programmed into the protein building blocks. Electrostatic interactions between oppositely charged protein domains and free ions drive protein folding, self‐organization (coacervation) and stabilization of the building blocks into nanoscale droplets. Yet, nanodroplets can be instantly transformed via simple mechanical stimulus as proteins partially unfold, merge together and form a strong network, which solidifies into a fiber. The mechanism is based on basic physico‐chemical principles. Thus, by extracting these principles, new methods of synthesizing sustainable polymer‐based materials can be developed.     

135 Arranging enzymes and receptors with nanometer-scale precision on molecular switchboards

DNA is a useful material for constructing nanoscale structures in nearly any three-dimensional (3D) shape desired. The DNA nanostructure can also be equipped with specific docking sites for proteins. Cellular processes and chemical transformations take place in several reaction steps. Multiple enzymes cooperate in specific fashion to catalyze the sequential chemical transformation steps. Such natural systems are effectively reconstructed in vitro if the individual enzymes locate in the correct relative orientations. DNA-origami structures can be used as “molecular switchboards” to arrange enzymes and other proteins with nanometer-scale precision. A new method was developed for locating the proteins by means of special “adapters” known as zinc-finger proteins based only on proteins. Zinc fingers are suitable site-selective adapters for targeting specific locations within DNA-origami structures. Several different adapters carrying different proteins can independently bind at defined locations on this type of nanostructure. A basic leucine zipper (bZIP) protein is also a candidate for the site-selective adaptor. A well-characterized bZIP protein GCN4 was chosen as an adaptor for specific addresses. Analyses by atomic force microscopy and gel electrophoreses demonstrate specific binding of GCN4 adaptor to the addresses containing the GCN4 binding sites on DNA origami. The adaptor derived from GCN4 and that form a zinc-finger protein zif268, for which we have reported previously, acted as orthogonal adaptors to the respective addresses on DNA origami. Therefore, these orthogonal adaptors would be useful to place multiple engineered proteins at different addresses on DNA origami. Especially, the homodimeric nature of GCN4 adaptor is indispensable for constructing the assembly of the naturally abundant dimeric proteins and/or enzymes to efficiently carry out chemical reactions and signal transductions in vitro on DNA origami.     

Practical application of synthetic biology principles

Synthetic biology can be defined as the “repurposing and redesign of biological systems for novel purposes or applications, ” and the field lies at the interface of several biological research areas. This broad definition can be taken to include a variety of investigative endeavors, and successful design of new biological paradigms requires integration of many scientific disciplines including (but not limited to) protein engineering, metabolic engineering, genomics, structural biology, chemical biology, systems biology, and bioinformatics. This review focuses on recent applications of synthetic biology principles in three areas: (i) the construction of artificial biomolecules and biomaterials; (ii) the synthesis of both fine and bulk chemicals (including biofuels); and (iii) the construction of “smart” biological systems that respond to the surrounding environment.     

Ionic liquids in biotransformations: from proof-of-concept to emerging deep-eutectic-solvents

Ionic liquids (ILs) have been extensively assessed in biotransformations with different purposes, for example, non-conventional (co-)solvents, performance additives, coating agents for immobilizing/stabilizing enzymes, and IL-membrane-based processes. Fuelled by their premature labelling as 'green solvents', academic research has flourished. However, in recent years environmental aspects related to ILs have been strongly addressed, stating that many ILs commonly used cannot be regarded as 'green derivatives'. Likewise, ILs costs are still a barrier for practical uses. Attempting to combine sustainability with the promising added-values of ILs, the third generation of ILs is currently under development. Likewise, deep-eutectic-solvents (DESs) appear in the horizon as an attractive and cost-effective option for using ionic solvents in biotransformations. DESs are often produced by gently warming and stirring two (bio-based and cheap) salts (e.g. choline chloride and urea). First successful uses of DES in biotransformations were reported recently. It may be expected that knowledge accumulated in (second generation) ILs and biotransformations could be turned into real applications by using these DESs, and third generation ILs, in the coming years.     

Modulating enzyme activity using ionic liquids or surfactants

One of the important strategies for modulating enzyme activity is the use of additives to affect their microenvironment and subsequently make them suitable for use in different industrial processes. Ionic liquids (ILs) have been investigated extensively in recent years as such additives. They are a class of solvents with peculiar properties and a "green" reputation in comparison to classical organic solvents. ILs as co-solvents in aqueous systems have an effect on substrate solubility, enzyme structure and on enzyme–water interactions. These effects can lead to higher reaction yields, improved selectivity, and changes in substrate specificity, and thus there is great potential for IL incorporation in biocatalysis. The use of surfactants, which are usually denaturating agents, as additives in enzymatic reactions is less reviewed in recent years. However, interesting modulations in enzyme activity in their presence have been reported. In the case of surfactants there is a more pronounced effect on the enzyme structure, as can be observed in a number of crystal structures obtained in their presence. For each additive and enzymatic process, a specific optimization process is needed and there is no one-fits-all solution. Combining ILs and surfactants in either mixed micelles or water-in-IL microemulsions for use in enzymatic reaction systems is a promising direction which may further expand the range of enzyme applications in industrial processes. While many reviews exist on the use of ILs in biocatalysis, the present review centers on systems in which ILs or surfactants were able to modulate and improve the natural activity of enzymes in aqueous systems.     

Genetic and chemical approaches for surface charge engineering of enzymes and their applicability in biocatalysis: A review

Surface charge engineering has received considerable interest from the scientific and industrial community in the last few decades. Although it was previously hypothesized that the surface charge–charge interactions were not a fundamental force to determine protein folding and stability, many studies today show that surface charge plays a key role determining protein structure and activity. This review aims to (a) highlight the value of surface charged engineering of proteins to improve enzyme stability and activity in aqueous media and in the presence of ionic liquids (ILs) and organic solvents, (b) describe the existing approaches (genetic engineering or chemical modifications) for surface charged engineering, and (c) demonstrate the applicability of these surface charged enzymes in biocatalysis. The review provides a new foundation for the scientific and research community to exploit the surface engineering of protein concept for the development of new enzymes that are more active and stable in the presence of ILs and organic solvents, thereby offering new opportunities for industrial biocatalysis. Furthermore, this review is a useful tool for researchers to decide the best available technology to improve their enzyme system/process.     

Rational engineering of enzyme stability

During the past 15 years there has been a continuous flow of reports describing proteins stabilized by the introduction of mutations. These reports span a period from pioneering rational design work on small enzymes such as T4 lysozyme and barnase to protein design, and directed evolution. Concomitantly, the purification and characterization of naturally occurring hyperstable proteins has added to our understanding of protein stability. Along the way, many strategies for rational protein stabilization have been proposed, some of which (e.g. entropic stabilization by introduction of prolines or disulfide bridges) have reasonable success rates. On the other hand, comparative studies and efforts in directed evolution have revealed that there are many mutational strategies that lead to high stability, some of which are not easy to define and rationalize. Recent developments in the field include increasing awareness of the importance of the protein surface for stability, as well as the notion that normally a very limited number of mutations can yield a large increase in stability. Another development concerns the notion that there is a fundamental difference between the "laboratory stability" of small pure proteins that unfold reversibly and completely at high temperatures and "industrial stability", which is usually governed by partial unfolding processes followed by some kind of irreversible inactivation process (e.g. aggregation). Provided that one has sufficient knowledge of the mechanism of thermal inactivation, successful and efficient rational stabilization of enzymes can be achieved.     

Nanoscale elongating control of the self‐assembled protein filament with the cysteine‐introduced building blocks

Self‐assembly of artificially designed proteins is extremely desirable for nanomaterials. Here we show a novel strategy for the creation of self‐assembling proteins, named “Nanolego.” Nanolego consists of “structural elements” of a structurally stable symmetrical homo‐oligomeric protein and “binding elements,” which are multiple heterointeraction proteins with relatively weak affinity. We have established two key technologies for Nanolego, a stabilization method and a method for terminating the self‐assembly process. The stabilization method is mediated by disulfide bonds between Cysteine‐residues incorporated into the binding elements, and the termination method uses “capping Nanolegos,” in which some of the binding elements in the Nanolego are absent for the self‐assembled ends. With these technologies, we successfully constructed timing‐controlled and size‐regulated filament‐shape complexes via Nanolego self‐assembly. The Nanolego concept and these technologies should pave the way for regulated nanoarchitecture using designed proteins.     

A robust,cost‐effective method for DNA,RNA and protein co‐extraction from soil,other complex microbiomes and pure cultures

The soil microbiome is inherently complex with high biological diversity, and spatial heterogeneity typically occurring on the submillimetre scale. To study the microbial ecology of soils, and other microbiomes, biomolecules, that is, nucleic acids and proteins, must be efficiently and reliably co‐recovered from the same biological samples. Commercial kits are currently available for the co‐extraction of DNA, RNA and proteins but none has been developed for soil samples. We present a new protocol drawing on existing phenol–chloroform‐based methods for nucleic acids co‐extraction but incorporating targeted precipitation of proteins from the phenol phase. The protocol is cost‐effective and robust, and easily implemented using reagents commonly available in laboratories. The method is estimated to be eight times cheaper than using disparate commercial kits for the isolation of DNA and/or RNA, and proteins, from soil. The method is effective, providing good quality biomolecules from a diverse range of soil types, with clay contents varying from 9.5% to 35.1%, which we successfully used for downstream, high‐throughput gene sequencing and metaproteomics. Additionally, we demonstrate that the protocol can also be easily implemented for biomolecule co‐extraction from other complex microbiome samples, including cattle slurry and microbial communities recovered from anaerobic bioreactors, as well as from Gram‐positive and Gram‐negative pure cultures.     

Uncovering the In Vivo Proxisome Using Proximity‐Tagging Methods

The development of new approaches is critical to gain further insights into biological processes that cannot be obtained by existing methods or technologies. The detection of protein–protein interaction is often challenging, especially for weak and transient interactions or for membrane proteins. Over the last decade, several proximity‐tagging methodologies have been developed to explore protein interactions in living cells. Among those, the most efficient are based on protein partner modification, such as biotinylation or pupylation. Such technologies are based on engineered variants of enzymes like peroxidases or ligases that release reactive molecules, in the presence of specific substrates, that bind surrounding proteins. Fusing a protein of interest (POI) to these enzymes allows the definition of an unbiased “proxisome,” that is, all of the proteins in interaction or in close vicinity of the POI. Here, the different proximity‐labeling tools available are described and comprehensive comparison to discuss advantages and limitations is provided.     

Patenting trends in enzyme related microfluidic applications

The miniaturization of continuous processes has been of interest in the academia and industry which is reflected by the increase in scientific publications and patent disclosures in the last decade. The aim of this study was to evaluate the patenting trends regarding enzyme related microfluidic applications in order to observe the progress of science and technology. The mapped patents have been classified as “immobilization method”, “biomolecule screening systems”, “integrated process development” and “microreactor design”. Half of the patent disclosures were filed by academia, whereas the other half was from industrial research which complies with the shift in microfluidics from academic and industrial research to commercial applications. Immobilization procedures carried out at room temperatures such as formulation of silica matrices using sol–gel technique, incorporation of novel hybrid materials, the integration of supercritical fluids and microfluidics, employing ionic liquids as wall-less microreactors, designing low cost, high performance microfluidic devices were the highlights which can pose challenges in various life science applications. The increasing trend is expected to continue and the presented state-of-the-art in enzyme related microfluidic applications have the potential to enhance industry's capabilities for designing innovative systems which would demonstrate significant economic, societal and environmental benefits.     

DNA Binding and Bending Protein-Based DNA Actuator and its Practical Realization

In the life forms, the biomolecules such as DNA and protein are interacting with each other to maintain their life activity. Simultaneously, these biomolecules form DNA–protein complexes; as a result, the life forms can adjust to the external environment and continue its life activities. Therefore, using these characteristics of the DNA recognition ability of proteins, the novel molecular devices can be established. Here, we show the application of DNA binding and bending protein for design of the DNA actuator and its practical realization. The single polypeptide chain integrated host factor 2 (scIHF2), which is a DNA binding and bending protein, was used to bind to and/or bend the specific domains of DNA. Using this protein and designing the sequences of DNA, mechanical-functionalized DNA-based biomolecular device could be fabricated. Using these mechanisms, DNA binding and bending proteins have great potentials for establishing the DNA actuator for nanobiotechnology and nanotechnology applications.