Process considerations for the scale-up and implementation of biocatalysis

With increasing emphasis on renewable feed-stocks and green chemistry, biocatalytic processes will have an important role in the next generation of industrial processes for chemical production. However, in comparison with conventional industrial chemistry, the use of bioprocesses in general and biocatalysis in particular is a rather young technology. Although significant progress has been made in the implementation of new processes (especially in the pharmaceutical industry) no fixed methods for process design have been established to date. In this paper we present some of the considerations required to scale-up a biocatalytic process and some of the recently developed engineering tools available to assist in this procedure. The tools will have a decisive role in helping to identify bottlenecks in the biocatalytic development process and to justify where to put effort and resources.     

Enzymatic reactions in dense gases

The developments on applications of supercritical fluids as alternative solvents for biocatalytic processes that have taken place over the past two decades have been reviewed. An overview of process parameters influencing enzyme activity and stability, the influence of process parameters on reaction rates and productivity are presented. Applications of various types of reactors for enzymatic reaction in dense fluids, limitations of using enzymes as biocatalyst in supercritical fluids as well as future trends are presented. Main advantages of using dense gases as solvents for biocatalyzed reactions are the tunability of solvent properties and simple down stream processing features that can be readily combined with other unit operations. Although many enzymes are stable in supercritical fluids (SCFs) one should pay considerable attention to finding the correct reaction conditions for each substrate/enzyme/SCF system. One of the persistent problems is the instability and deactivation of enzymes under pressure and temperature. At present the most stable enzymes are hydrolases (lipases and esterases) for which pressure effect is lower than temperature deactivation.     

Tandem catalysis: Direct catalytic synthesis of imines from alcohols using manganese octahedral molecular sieves

Tandem processes involving catalysts can offer unique and powerful strategies for converting simple starting materials into more complex products in a single reaction vessel. Imines were synthesized directly from alcohols via a tandem catalytic process using manganese octahedral molecular sieves (OMS-2) as catalyst. The synthesis proceeds through two steps: an oxidation of the alcohols to carbonyls followed by the nucleophilic attack by an amine on the carbonyl to form the imine. OMS-2 acts as a bifunctional catalyst and catalyzes two mechanistically distinct processes in a single reaction vessel under the same conditions. Conversions up to 100% were obtained for benzylic alcohols with this efficient, environmentally friendly catalytic reaction. The advantages of this process are that the intermediates need not be isolated and the catalysts can be reused upon simple filtration without loss of activity.     

Efficient Biocatalytic Cleavage and Recovery of Organic Substrates Supported on Soluble Polymers

The applicability of novel solution‐phase supports in combination with enzymes for biocatalytic transformations is reported. Ex novo designed styrene‐based copolymers, bearing a phenylacetic residue in variable loadings and linked as a pendant group to the macromolecular backbone, through a spacer of variable length, have been synthesized and characterized. These derivatives are compatible and can be used as soluble supports in combination with immobilized penicillin G acylase (PGA – EC 3.5.1.11) for the biocatalytic cleavage of the covalently anchored organic substrate in quantitative yields, in water or water/dimethylformamide solvent mixtures, with recovery of the immobilized enzyme with negligible losses in activity.     

Rx-COSMO-CAMPD: Enhancing Reactions by Integrated Computer-Aided Design of Solvents and Processes based on Quantum Chemistry

Solvents strongly affect reaction-based chemical processes. Process design, therefore, needs to integrate solvent design. For this purpose, the integrated computer-aided molecular and process design (CAMPD) method Rx-COSMO-CAMPD is proposed. It employs a hybrid optimization scheme combining a genetic algorithm to explore the molecular design space with gradient-based optimization of the process. To overcome limitations of molecular design based on group-contribution methods, reaction kinetics and thermodynamic properties are predicted using advanced quantum-chemical methods. Rx-COSMO-CAMPD is demonstrated in a case study of a carbamate-cleavage process where promising solvents are designed efficiently. The results show that the integrated solvent and process design with Rx-COSMO-CAMPD outperforms computer-aided molecular design without process optimization in the identification of solvents that enable optimal process performance.     

Computational Mechanistic Investigations of Biocatalytic Nitrenoid C−H Functionalizations via Engineered Heme Proteins

Engineered heme proteins were developed to possess numerous excellent biocatalytic nitrenoid C−H functionalizations. Computational approaches such as density functional theory (DFT), hybrid quantum mechanics/molecular mechanics (QM/MM), and molecular dynamics (MD) calculations were employed to help understand some important mechanistic aspects of these heme nitrene transfer reactions. This review summarizes advances of computational reaction pathway results of these biocatalytic intramolecular and intermolecular C−H aminations/amidations, focusing on mechanistic origins of reactivity, regioselectivity, enantioselectivity, diastereoselectivity as well as effects of substrate substituent, axial ligand, metal center, and protein environment. Some important common and distinctive mechanistic features of these reactions were also described with brief outlook of future development.     

Process limitations in a whole-cell catalysed oxidation: Sensitivity analysis

Biocatalytic oxidation processes have to date presented major problems for scale-up, in part due to the complexity of the number of process variables. In this paper we have analysed the key limitations in such processes using the Baeyer-Villiger monooxygenase catalysed synthesis of optically pure lactones as an illustrative example. Limitations in product concentration, catalyst longevity and reaction rate were quantified and their effect on previously defined process metrics identified. Of particular interest is the way these metrics change with catalyst concentration. Using this assessment, the sensitivity of the metrics to potential changes to process and catalyst were analysed. We believe such an analysis is of general use to guide development efforts for a given biocatalytic reaction.     

基于分子矩阵的炼油过程的全厂模拟

针对复杂的炼油生产过程及其物流成分,采用分子矩阵表征相关的物流分子组成,对催化重整、加氢脱硫、汽油稳定塔和油品调和四个过程建立了基于分子矩阵的过程模型. 通过物性-分子矩阵转换关系可以方便地从已知物流物性计算分子矩阵以及由分子矩阵得到物流物性. 基于分子矩阵的模型计算不仅可以提供比传统虚拟组分模型更为详尽的分子信息,而且可以统一传统建模中不同过程虚拟组分和集总参数之间的差异,将多个过程模型集成实现全厂的模拟.     

Efficient production of cyclic adenosine monophosphate from adenosine triphosphate by the N-terminal half of adenylate cyclase from Escherichia coli

In this study, we aimed at developing an efficient biocatalytic process for bio-production of cyclic adenosine monophosphate (cAMP) from adenosine triphosphate (ATP). First, adenylate cyclase from Escherichia coli MG1655 (EAC) and Bordetella Pertussis (BAC) were expressed in E. coli BL21 (DE3) and comparatively analyzed for their activities. As a result, EAC from E. coli MG1655 exhibited a higher activity. However, amount of EAC were obtained in an insoluble form. Therefore, we expressed the first 446 amino acids of EAC (EAC446) to avoid the inclusion body. The effects of induction temperature, incubation time, and incubation pH were further evaluated to improve the expression of EAC446. Subsequently, the reaction process for the production of cAMP with ATP as a starting material was investigated. As none of cAMP was detected in the whole-cell based biocatalytic process, the reaction catalyzed by the crude enzyme was determined for cAMP production. What's more, the reaction temperature, reaction pH, metal ion additives and substrate concentration was optimized, and the maximum cAMP production of 18.45 g·L^-1 was achieved with a yield of 95.4% after bioconversion of 6 h.     

Rieske Oxygenases and Other Ferredoxin-Dependent Enzymes: Electron Transfer Principles and Catalytic Capabilities

Enzymes that depend on sophisticated electron transfer via ferredoxins (Fds) exhibit outstanding catalytic capabilities, but despite decades of research, many of them are still not well understood or exploited for synthetic applications. This review aims to provide a general overview of the most important Fd-dependent enzymes and the electron transfer processes involved. While several examples are discussed, we focus in particular on the family of Rieske non-heme iron-dependent oxygenases (ROs). In addition to illustrating their electron transfer principles and catalytic potential, the current state of knowledge on structure–function relationships and the mode of interaction between the redox partner proteins is reviewed. Moreover, we highlight several key catalyzed transformations, but also take a deeper dive into their engineerability for biocatalytic applications. The overall findings from these case studies highlight the catalytic capabilities of these biocatalysts and could stimulate future interest in developing additional Fd-dependent enzyme classes for synthetic applications.     

A combined process of biocatalysis and cell activity regeneration for the asymmetric reduction of 3‐oxo ester with immobilized baker's yeast

BACKGROUND: A process combining biocatalytic reaction and cell activity regeneration was designed for the asymmetric reduction of 3‐oxo ester. By immobilizing resting baker's yeast (Saccharomyces cerevisiae) in calcium alginate beads, the high yield and long catalyst life were achieved in the aqueous phase in this process with methyl acetoacetate (MAA) as the model substrate. RESULTS: Two combined fixed‐bed reactors were able to work steadily for at least 16 days. The activity of immobilized baker's yeast could be retained by re‐culture with culture medium regularly. The re‐culture time for bead reactivation was optimized to be 30 h. High yield (about 80%) and high enantiomeric excess (>95%) were maintained after 12 batches of asymmetric reduction. The immobilized beads retained their original shapes even after a long reaction time in the fixed‐bed reactor, while the beads broke after reaction of five batches in a flask. CONCLUSION: The combined process of biocatalysis and cell activity regeneration was successfully achieved in the asymmetric reduction and decreased the breakage of beads as well as increased the efficiency of catalyst. Copyright © 2008 Society of Chemical Industry     

Biocatalytic Metal-Organic Frameworks: Promising Materials for Biosensing

The highly efficient and specific catalysis of enzymes allows them to recognize a myriad of substrates, which enables biosensing. However, the fragility of natural enzymes severely restricts their practical applications. Metal-organic frameworks (MOFs) with porous networks and attractive functions have been intelligently employed as supports to encase enzymes and protect them against harsh environments. More importantly, customizable construction and composition affords the intrinsic enzyme-like activity of some MOFs (known as nanozymes), which provides an alternative route for the construction of robust enzyme mimics. This review will introduce the concept of these biocatalytic MOFs, with special emphasis on how biocatalytic processes that operate in these materials can reverse the plight of native enzyme-based biosensing. In addition, the present challenges and future outlooks in this research field are briefly discussed.     

Efficient production of cyclic adenosine monophosphate from adenosine triphosphate by the N-terminal half of adenylate cyclase from Escherichia coli

In this study, we aimed at developing an efficient biocatalytic process for bio-production of cyclic adenosine monophosphate(c AMP) from adenosine triphosphate(ATP). First, adenylate cyclase from Escherichia coli MG1655(EAC) and Bordetella Pertussis(BAC) were expressed in E. coli BL21(DE3) and comparatively analyzed for their activities. As a result, EAC from E. coli MG1655 exhibited a higher activity. However, amount of EAC were obtained in an insoluble form. Therefore, we expressed the first 446 amino acids of EAC(EAC446) to avoid the inclusion body. The effects of induction temperature, incubation time, and incubation p H were further evaluated to improve the expression of EAC446. Subsequently, the reaction process for the production of c AMP with ATP as a starting material was investigated. As none of c AMP was detected in the whole-cell based biocatalytic process, the reaction catalyzed by the crude enzyme was determined for c AMP production. What's more,the reaction temperature, reaction p H, metal ion additives and substrate concentration was optimized, and the maximum c AMP production of 18.45 g·L~(-1) was achieved with a yield of 95.4% after bioconversion of 6 h.     

酵母耦合原位分离技术不对称合成(R)-扁桃酸甲酯

以酿酒酵母(Saccharomyces cerevisiae AS2.1392)全细胞为催化剂不对称还原苯甲酰甲酸甲酯合成(R)-扁桃酸甲酯,该催化剂催化速度快、操作稳定性好。研究了底物和产物浓度对反应初速度的影响,建立了底物和产物抑制模型,并采取分批加入底物和添加树脂的方式解除底物和产物抑制。通过考察不同树脂对底物和产物的吸附量以及对生物还原反应的影响,筛选出了一种较适合的大孔吸附树脂NKA-Ⅱ。在优化的树脂加入量和加入模式下,当底物浓度为180mmol/L时,产物产率由35.0%提高到71.2%,对映体过量值(ee)保持在95%左右。     

Metadynamics as a tool for exploring free energy landscapes of chemical reactions

The metadynamics or hills method is a relatively new molecular dynamics technique aimed to enhance the sampling of separated regions in phase space and map out the underlying free energy landscape as a function of a small number of order parameters or collective variables. The high efficiency allows for the application of metadynamics in combination with first principles dynamics methods, in particular with Car-Parrinello molecular dynamics, to study processes in which changes in the electronic structure play a dominant role, such as chemical reactions. The option to choose several independent collective variables is important to tackle complex and concerted transformations that lack an obvious a priori choice for a single reaction coordinate. In this Account, we discuss the role of metadynamics in the search of transition states, local minima, reaction paths, free energy profiles, and reaction coordinates among a growing list of alternative methods.     

Non-innocent ligands in copper complexes catalyzed oxidation of thiols

Copper(II) chloride complexes with nitrogen-containing ligands (amines, amides, heterocycles) were studied as catalysts of selective oxidation of thiols to disulfides. This process has an important biochemical analogue – the formation of disulfide bridge in proteins [Wang C, Wesener SR, Zhang H, Cheng Y-Q. An FAD-dependent pyridine nucleotide-disulfide oxidoreductase is involved in disulfide bond formation in FK228 anticancer depsipeptide. Chem and Biol. 2009;16:585–593]; which affects their activity [Zhang L, Chou CP, Moo-Young M. Disulfide bond formation and its impact on the biological activity and stability of recombinant therapeutic proteins produced by Escherichia coli expression system. Biotech Adv. 2011;29:923–929]. Also, thiol oxidation is an important process of oil sweetening. The search for effective and stable catalysts for this reaction is still topical [Ganguly SK, Das G, Kumar S, Sain B, Garg MO. Mechanistic kinetics of catalytic oxidation of 1-butanethiol in light oil sweetening. Catal Tod. 2012;198:246–251]. This article describes a mechanistic study of thiol oxidation and non-innocent ligands transformations during this reaction. The studied ligands are capable of oxidizing thiols, the reaction being followed by reoxidation of the reduced forms of the ligands with air oxygen. The oxidative activity of the ligand correlates with catalyst activity for thiol oxidation.     

Chemistry of pitch carbonization

The conversion of pitch to carbon is a complex process encompassing a multitude of physical and chemical transformations among the many pitch components. Studies on both individual aromatic compounds and pitches have shown that polymerization through loss of side chains and hydrogen is the main chemical reaction. Molecular rearrangements are also prevalent. A continual increase in molecular weight through polymerization and loss of low molecular weight volatiles results in the transformation of pitch to mesophase, coke and ultimately carbon. Stable free-radicals are formed during both the polymerization and rearrangement processes. These various aspects are reviewed to develop a general mechanistic sequence for pitch carbonization.     

A Promising approach to the kinetics of crystallization processes: The sample controlled thermal analysis

Constant Rate Thermal Analysis (CRTA) method implies controlling the temperature in such a way that the reaction rate is maintained constant all over the process. This method allows determining simultaneously both the kinetic parameters and the kinetic model from a single experiment as the shape of the CRTA α‐T curves strongly depends on the kinetic model. CRTA method has been developed in the market only for thermogravimetric and thermodilatometric systems and, therefore, its use has been limited until now to the kinetic study of processes involving changes in mass or size of the samples, respectively. To overcome this obstacle, a method has been developed in this work for using the DSC signal for controlling the process rate in such a way that CRTA would be applied to the kinetic analysis of either phase transformations or crystallizations. The advantages of CRTA for performing the kinetics of crystallization processes have been here successfully demonstrated for the first time after selecting the crystallization of zirconia gel as test reaction.     

Designing a biocatalytic process involving deep eutectic solvents

During the last decade, deep eutectic solvents (DESs) have emerged as a promising alternative to traditional organic solvents, from both environmental and technological perspectives. The number of structural combinations encompassed by DESs is tremendous; thus, it is possible to design an optimal DES for each specific enzymatic reaction system. In (bio)catalytic processes, a DES can serve as solvent/co‐solvent, as an extractive reagent for an enzymatic product, and as a pretreatment solvent of enzymatic biomass. To date, hydrolases are the most studied enzymes in DESs, which is not surprising given that lipases are the most important industrial enzymes. At the same time, there are a limited number of papers dealing with synthetic reactions in DESs involving other hydrolytic enzymes (epoxide hydrolases, phospholipase, proteases and haloalkane dehalogenases), lyases, and dehydrogenases (as a part of the whole Saccharomyces cerevisae and Escherichia coli cell biocatalysis). When designing efficient biocatalytic processes involving DESs, independent of the reaction type and enzyme used, the following steps should be included: (i) preparation and characterisation of the DES, (ii) screening of the DES for optimal enzyme performance, (iii) selection and optimisation of the biocatalytic protocol, and (iv) recovery of the product/DES and DES recycling with possible scale‐up. In this paper, we will present some practical aspects that we experienced while working with these solvents, together with some major observations that are available in the literature. © 2020 Society of Chemical Industry     

Light-Driven ATP Regeneration in Diblock/Grafted Hybrid Vesicles

Light-driven ATP regeneration systems combining ATP synthase and bacteriorhodopsin have been proposed as an energy supply in the field of synthetic biology. Energy is required to power biochemical reactions within artificially created reaction compartments like protocells, which are typically based on either lipid or polymer membranes. The insertion of membrane proteins into different hybrid membranes is delicate, and studies comparing these systems with liposomes are needed. Here we present a detailed study of membrane protein functionality in different hybrid compartments made of graft polymer PDMS-g-PEO and diblock copolymer PBd-PEO. Activity of more than 90 % in lipid/polymer-based hybrid vesicles could prove an excellent biocompatibility. A significant enhancement of long-term stability (80 % remaining activity after 42 days) could be demonstrated in polymer/polymer-based hybrids.