The bioseparation needs for tomorrow

Will we replace oil with wheat or corn as a feedstock for producing natural plastic? The success of biotechnology for bulk product manufacturing will heavily depend on engineering solutions in the downstream processes in which separation and purification have a crucial role with respect to commercial development. Development of efficient bioseparation methods is important for a broad range of business areas including pharmaceuticals, nutrition and health products, bio-based materials and crop protection chemicals. Depending on the value of the end product and the scale of production, the processing required varies significantly. Key factors that have an impact on the choice of separation strategy include process throughput, particle size of the product and impurities and the desired end-product concentration. The development of efficient, economical and selective separation methods will be required for successful commercialization of bioprocesses. Despite this well-recognized need, there are relatively few available methods for commercial implementations. Development of novel mechanical systems for selective separation of solid and liquid mixtures must become a top priority for current research investment to reduce the reliance on expensive chromatographic and thermal separation methods.     

纳豆激酶集成化分离技术

综述了纳豆激酶的分离纯化技术的研究现状和发展趋势。通过对常规分离技术的分析,重点讨论了集成化分离技术的应用及其优势,包括集成化双水相分配技术、扩张床吸附技术以及耐盐性混合模式吸附技术等分离方法。并指出集成化分离技术在生产纳豆激酶以及其他活性蛋白方面,具有广阔的应用前景。     

Hydrophobic peptide tags as tools in bioseparation

Hydrophobic interactions are highly selective, and differences in surface hydrophobicities between proteins can be used as an efficient handle to facilitate protein isolation. Aromatic amino acid residues are of particular importance for molecular recognition because they have a key role in several biological functions. The hydrophobicity of a protein can easily be altered with minor genetic modifications, such as site-directed mutagenesis or fusions of hydrophobic peptide tags. An important advantage of hydrophobic peptide tags over traditional affinity tags is the possibility of exploring simple and inexpensive bioseparation materials. Recent results demonstrate the potential of hydrophobic interaction chromatography and aqueous two-phase systems as tools to study relative hydrophobicities of recombinant proteins with only minor alterations. This review focuses on hydrophobic peptide tags as fusion partners, which can be used as important tools in bioseparation.     

Ecosystem Ecology and Evolutionary Biology,a New Frontier for Experiments and Models

One of the most important scientific problems about which we are profoundly ignorant is how ecosystem processes change as populations evolve. These changes in ecosystem processes are propelled by evolutionary changes in organism traits and in turn may exert additional selection pressures on the evolving populations. Understanding these feedbacks between ecosystem and evolutionary processes is a challenge for evolutionary and ecosystem theory and experiments in the 21st century. This essay reviews some recent empirical and theoretical studies which are beginning to shed light on the complexity of these feedbacks and makes suggestions for future directions and the training of the next generation of students.     

Methods to assess the impact of extraction of non-timber tropical forest products on plant populations

Thousands of plant and animal species in tropical regions provide a variety of non-timber products that are used by billions of people all over the world. Conservation and long term utilization of these species require that they be harvested on a sustainable basis. However, the extent to which non-timber forest products are exploited without adverse effects on natural populations is not known. There is in fact considerable evidence for non-sustainable harvest of non-timber products. We outline methods that may be used to assess the impact of harvest on population processes of the species that are being harvested. We present sampling protocols for rapid assessment as well as long term monitoring of populations. We briefly consider the limitations of these methods and suggest that the monitoring protocols we outline should be part of an overall management plan designed to extract and utilize non-timber tropical forest products on a long term basis.     

Transfusion medicine in the era of proteomics

Blood components (BCs) are highly complex mixtures of plasma proteins and cells. At present, BC and blood derivatives (BDs) quality control is mainly focused on standardized quantitative assessment, providing relatively limited information about products. Unfortunately, during the production, inactivation, and storage processes there is the risk of changes in their integrity, especially at the protein level, which could cause negative effects on transfusion. It is therefore a major challenge to identify significant alterations of these products, and, in this context, proteomics can play a potentially relevant role in transfusion medicine (TM) to assess the protein composition of blood-derived therapeutics, particularly for identifying modified proteins. It can provide comprehensive information about changes occurring during processing and storage of BCs and BDs and can be applied to assess or improve them, therefore potentially enabling a global assessment of processing, inactivation and storage methods, as well as of possible contaminants and neoantigens that may influence the immunogenic capacity of blood-derived therapeutics. Thus, proteomics could become a relevant part of quality-control process to verify the identity, purity, safety, and potency of various blood therapeutics. A more detailed understanding of the proteins found in blood and blood products, and the identification of their interactions, may also yield important information for the design of new small molecule therapeutics and also for future improvements in TM.

Proteomics, together with genomics in the near future, will presumably have an impact on disease diagnosis and prognosis as well as on further advances in the production, pathogen inactivation and storage processes of blood-based therapeutics.     

Profiling protease activities by dynamic proteomics workflows

Proteases play prominent roles in many physiological processes and the pathogenesis of various diseases, which makes them interesting drug targets. To fully understand the functional role of proteases in these processes, it is necessary to characterize the target specificity of the enzymes, identify endogenous substrates and cleavage products as well as protease activators and inhibitors. The complexity of these proteolytic networks presents a considerable analytic challenge. To comprehensively characterize these systems, quantitative methods that capture the spatial and temporal distributions of the network members are needed. Recently, activity-based workflows have come to the forefront to tackle the dynamic aspects of proteolytic processing networks in vitro, ex vivo and in vivo. In this review, we will discuss how mass spectrometry-based approaches can be used to gain new insights into protease biology by determining substrate specificities, profiling the activity-states of proteases, monitoring proteolysis in vivo, measuring reaction kinetics and defining in vitro and in vivo proteolytic events. In addition, examples of future aspects of protease research that go beyond mass spectrometry-based applications are given.     

Routes to Potential Bioproducts from Lignocellulosic Biomass Lignin and Hemicelluloses

An essential feature of proposed fermentation-based lignocellulose to biofuel conversion processes will be the co-production of higher value chemicals from lignin and hemicellulose components. Over the years, many routes for chemical conversion of lignin and hemicelluloses have been developed by the pulp and paper industry and we propose that some of these can be applied for bioproducts manufacturing. For lignin products, thermochemical, chemical pulping, and bleaching methods for production of polymeric and monomeric chemicals are reviewed. We conclude that peroxyacid chemistry for phenol and ring-opened products looks most interesting. For hemicellulose products, preextraction of hemicelluloses from woody biomass is important and influences the mixture of solubilized material obtained. Furfural, xylitol, acetic acid, and lactic acid are possible targets for commercialization, and the latter can be further converted to acrylic acid. Pre-extraction of hemicelluloses can be integrated into most biomass-to-biofuel conversion processes.     

Residual dipolar couplings: Synergy between NMR and structural genomics

Structural genomics is on a quest for the structure and function of a significant fraction of gene products. Current efforts are focusing on structure determination of single-domain proteins, which can readily be targeted by X-ray crystallography, NMR spectroscopy and computational homology modeling. However, comprehensive association of gene products with functions also requires systematic determination of more complex protein structures and other biomolecules participating in cellular processes such as nucleic acids, and characterization of biomolecular interactions and dynamics relevant to function. Such NMR investigations are becoming more feasible, not only due to recent advances in NMR methodology, but also because structural genomics is providing valuable structural information and new experimental and computational tools. The measurement of residual dipolar couplings in partially oriented systems and other new NMR methods will play an important role in this synergistic relationship between NMR and structural genomics. Both an expansion in the domain of NMR application, and important contributions to future structural genomics efforts can be anticipated.     

Nutrient stress is a target for new antibiotics

Novel approaches are required to address the looming threat of pan-resistant Gram-negative pathogens and forestall the rise of untreatable infections. Unconventional targets that are uniquely important during infection and tractable to high-throughput drug discovery methods hold high potential for innovation in antibiotic discovery programs. In this context, inhibitors of bacterial nutrient stress are particularly exciting candidates for future antibiotic development. Amino acid, nucleotide, and vitamin biosynthesis pathways are critical for bacterial growth in nutrient-limiting conditions in the laboratory and the host. Although historically dismissed as dispensable for pathogens, a wealth of transposon mutagenesis and single-mutant studies have emerged which demonstrate that several such pathways are critical for infection. Indeed, high-throughput screens of diverse synthetic compounds and natural products have uncovered inhibitors of nutrient biosynthesis. Herein, we review bacterial nutrient biosynthesis and its role during host infection. Further, we explore screening platforms developed to search for inhibitors of these targets and highlight successes among these. Finally, we feature important and sometimes surprising connections between bacterial nutrient biosynthesis, antibiotic activity, and antibiotic resistance.     

分离纯化中蛋白质的不稳定性及其对策

分离纯化出高纯度有生物活性的蛋白质一直是一项艰巨的工作。在基因工程药物蛋白质的生产中,收率低和纯度低是亟待解决的关键问题。这里除了生物组份复杂、难以分离外,许多目标蛋白质自身活性的丧失也是一个重要原因。由于药物蛋白质昂贵的市场价格,回收率稍有提高就有可能带来巨大的经济效益,所以,有关蛋白质在分离纯化中的失活及其对策在近年来受到了极大的关注。1997年第十届欧洲生物技术大会上,蛋白质的稳定被单独列为一个专题。1999年在加拿大召开的第九届生物产品回收技术会议也对蛋白质的稳定性进行讨论。目前重点探索的是蛋白质失活的机制,采取相应的对策以保护活性,并在分离纯化中采用高效的分离纯化技术缩短操作周期,提高产物的活性回收率。     

The affinity concept in bioseparation: evolving paradigms and expanding range of applications

The meaning of the word affinity in the context of protein separation has undergone evolutionary changes over the years. The exploitation of molecular recognition phenomenon is no longer limited to affinity chromatography modes. Affinity based separations today include precipitation, membrane based purification and two-phase/three-phase extractions. Apart from the affinity ligands, which have biological relationship (in vivo) with the target protein, a variety of other ligands are now used in the affinity based separations. These include dyes, chelated metal ions, peptides obtained by phage display technology, combinatorial synthesis, ribosome display methods and by systematic evolution of ligands by exponential enrichment (SELEX). Molecular modeling techniques have also facilitated the designing of biomimetic ligands. Fusion proteins obtained by recombinatorial methods have emerged as a powerful approach in bioseparation. Overexpression in E. coli often result in inactive and insoluble inclusion bodies. A number of interesting approaches are used for simultaneous refolding and purification in such cases. Proteomics also needs affinity chromatography to reduce the complexity of the system before analysis by electrophoresis and mass spectrometry are made. At industrial level, validation, biosafety and process hygiene are also important aspects. This overview looks at these evolving paradigms and various strategies which utilize affinity phenomenon for protein separations.     

Protein loading,elution, and resolution behavior in a novel device that integrates ultrafiltration and chromatographic separation

Hollow fiber membranes and chromatographic resin beads are commonly employed in a variety of bioseparation processes. A new class of integrated separation devices is being studied in which the shell side of a hollow fiber device is filled with adsorbents/chromatographic resin beads. Such devices and the corresponding separation methods integrate feed broth clarification by the microfiltration/ultrafiltration membrane with bioproduct purification by the shell-side resin beads either as an adsorbent or as beads in elution chromatography. A mathematical model has been developed for the prediction of the chromatographic behavior of such an integrated device. Simulations have been done to study the effects of axial dispersion, feed flow rate, water permeation rate, fiber packing density, and void fraction. Numerical solutions were obtained by solving the governing equations. This model can reasonably describe the concentration profiles as well as the breakthrough and elution behaviors in the integrated device.     

Anything but Conventional Chromatography Approaches in Bioseparation

While packed bed chromatography, known as conventional chromatography, has been serving the biopharmaceutical industry for decades as the bioseparation method of choice, alternative approaches are likely to take an increasing leading role in the next few years. The high number of new biological drugs under development, and the need to make biopharmaceuticals widely accessible, has been driving the academia and industry in the quest of anything but conventional chromatography approaches. In this perspective paper, these alternative approaches are discussed in view of current and future challenges in the downstream processing field.     

Application of methods for viral clearance in stem cell production

Regenerative medicine therapies will allow in the future the transplant of cells of human origin in some diseases that until now have been incurable. The assurance of the safety and quality, especially from a microbiological point of view, is very important for these therapeutic products. Depending on the starting material, there are several sources of pathogen presence, mainly human viruses. Also, the use of feeders of animal origin as layers in which the stem cells can grow may permit the transmission of animal pathogens to these cells. However, cell sources are limited due to the low availability of spare in vitro fecundation human embryos and the low rate of success in the derivation of human stem cell lines. Thus, in several cases, it will be necessary to evaluate the possibility of removing or inactivating these microorganisms. In this paper, we summarize the main methods of viral clearance and we have provided an overview of the main features taking into account in the viral clearance techniques.     

By-products resulting from lignocellulose pretreatment and their inhibitory effect on fermentations for (bio)chemicals and fuels

Lignocellulose might become an important feedstock for the future development of the biobased economy. Although up to 75 % of the lignocellulose dry weight consists of sugar, it is present in a polymerized state and cannot be used directly in most fermentation processes for the production of chemicals and fuels. Several methods have been developed to depolymerize the sugars present in lignocellulose, making the sugars available for fermentation. In this review, we describe five different pretreatment methods and their effect on the sugar and non-sugar fraction of lignocellulose. For several pretreatment methods and different types of lignocellulosic biomass, an overview is given of by-products formed. Most unwanted by-products present after pretreatment are dehydrated sugar monomers (furans), degraded lignin polymers (phenols) and small organic acids. Qualitative and quantitative effects of these by-products on fermentation processes have been studied. We conclude this review by giving an overview of techniques and methods to decrease inhibitory effects of unwanted by-products.     

Isolation and purification of recombinant proteins, antibodies and plasmid DNA with hydroxyapatite chromatography

Hydroxyapatite and related stationary phases increasingly play a role in the downstream processing of high-value biological materials, such as recombinant proteins, therapeutic antibodies and pharmaceutical-grade plasmid DNA. Chromatographic hydroxyapatite is an inorganic, ceramic material identical in composition, if not in structure, to calcium phosphate found in human bones and teeth. The interaction of hydroxyapatite with biomacromolecules is complex and highly dynamic, which can make predicting performance difficult, but also allows the design of very selective isolation processes. This review discusses the currently commercially available chromatographic materials, different retention mechanisms supported by these materials and differential exploitation for the design of highly specific isolation procedures. The state of the art of antibody purification by hydroxy- and fluoroapatite is reviewed together with tested routines for method development and implementation. Finally, the isolation of plasmid DNA is discussed, since the purification of DNA therapeutics at a sufficiently large scale is an emerging need in bioprocess development and perhaps the area in bioseparation where apatite chromatography can make its most important contribution to date.     

重组蛋白质纯化技术

90年代以来 ,基因重组技术得到很大的发展 ,基因工程产品的分离纯化的成本约占其全部成本的 6 0 %～ 80 %,因此重组蛋白的分离纯化技术越来越重要。着重介绍了扩张柱床吸附层析技术 ,径向膜层析技术 ,灌注层析技术 ,液液萃取技术 ,置换层析技术和金属螯合亲和层析技术近年来进展情况以及它们的优缺点和应用范围。     

生物炼制过程中木质素高值转化研究进展

木质素高值转化对于提升生物炼制经济性,促进社会经济绿色发展具有重要意义。然而,木质素结构复杂且不均一,其高值化利用仍存在技术壁垒,使得木质素应用尚未形成规模。文中首先综述了当前生物炼制过程中木质素高值转化面临的主要挑战。然后通过比较不同预处理技术对木质素分离、性质及其利用的主要影响,详细阐述了基于生物炼制理念发展的新型组合预处理技术。其次,针对木质素本征结构特性导致其利用效率低等问题,进一步详述了溶剂分级、膜分级、梯度沉淀分级等分级利用策略对克服木质素不均一性,改善其可加工性能的重要影响。再次,针对木质素利用策略,系统比较了木质素热化学转化和生物转化,结合生物质预处理及木质素分级,阐述了以生物炼制理念进行木质素高值转化的新策略。最后,总结了木质素利用过程中存在的挑战性问题,展望了木质素高效分离、分级及转化过程发展的新策略和新趋势。