Biocatalysis--key to sustainable industrial chemistry

The ongoing trends to process improvements, cost reductions and increasing quality, safety, health and environment requirements of industrial chemical transformations have strengthened the translation of global biocatalysis research work into industrial applications. One focus has been on biocatalytic single-step reactions with one or two substrates, the identification of bottlenecks and molecular as well as engineering approaches to overcome these bottlenecks. Robust industrial procedures have been established along classes of biocatalytic single-step reactions. Multi-step reactions and multi-component reactions (MCRs) enable a bottom-up approach with biocatalytic reactions working together in one compartment and recations hindering each other within different compartments or steps. The understanding of the catalytic functions of known and new enzymes is key for the development of new sustainable chemical transformations.     

Environmental metagenomics: An innovative resource for industrial biocatalysis

The current existing enzymes have been identified from cultivable micro-organisms, most frequently from bacteria. These bacterial biocatalytic capabilities have been widely used for biotransformations, resulting in the development of profitable industrial bioprocesses in the fields of feed and food processing, textiles, agro-chemistry, cosmetics, pharmaceuticals and fine chemistry. However, the originality of this bioresource is progressively drying up, while requests from industry for novel biocatalytic activities are increasing in the face of economic and environmental pressure. Metagenomics, through access to the huge reservoir of uncultivated bacteria which represents the majority of the present biodiversity, opens the door to new industrial sources of enzymes. Surmounting hurdles encountered with this technology (e.g. DNA extraction to obtain high quality DNA libraries with proper statistical representativity, setting up of relevant high throughput screenings assays, combining functional and genome-based identifications), gives unique opportunities to access novel biocatalysts that better fit with the required industrial specifications, thus providing new biocatalysis tool boxes.     

Environmental metagenomics: An innovative resource for industrial biocatalysis

The current existing enzymes have been identified from cultivable micro-organisms, most frequently from bacteria. These bacterial biocatalytic capabilities have been widely used for biotransformations, resulting in the development of profitable industrial bioprocesses in the fields of feed and food processing, textiles, agro-chemistry, cosmetics, pharmaceuticals and fine chemistry. However, the originality of this bioresource is progressively drying up, while requests from industry for novel biocatalytic activities are increasing in the face of economic and environmental pressure. Metagenomics, through access to the huge reservoir of uncultivated bacteria which represents the majority of the present biodiversity, opens the door to new industrial sources of enzymes. Surmounting hurdles encountered with this technology (e.g. DNA extraction to obtain high quality DNA libraries with proper statistical representativity, setting up of relevant high throughput screenings assays, combining functional and genome-based identifications), gives unique opportunities to access novel biocatalysts that better fit with the required industrial specifications, thus providing new biocatalysis tool boxes.     

Combinatorial biocatalysis: taking the lead from nature

Combinatorial biocatalysis is an emerging technology in the field of drug discovery. The biocatalytic approach to combinatorial chemistry uses enzymatic, chemoenzymatic, and microbial transformations to generate libraries from lead compounds. Important recent advances in combinatorial biocatalysis include iterative derivatization of small molecules and complex natural products, regioselectively controlled libraries, novel one-pot library syntheses, process automation, and biocatalyst enhancements.     

Assessment of intraparticle biocatalytic distributions as a tool in rational formulation

Research has shown that the intraparticle biocatalytic distribution has extensive effects on the properties of various (industrial) biocatalytic particles and their performance in (bio-) chemical reactions. In recent years, advances in molecular chemistry have led to the development of many different specific (immuno-) labeling and light-microscopic detection techniques. Furthermore, high-quality image-digitizing devices and enhanced computing power have made image analysis readily accessible. These technologies may lead to the assessment and improvement of the internal biocatalyst profile as an integral part of biocatalytic particle optimization.     

Systematic Approach to Solvent Selection for Biphasic Systems with a Combination of COSMO‐RS and a Dynamic Modeling Tool

Biphasic aqueous‐organic systems are important reaction systems for catalytic processes. This is especially true for biocatalysis where the range of accessible products can be significantly extended. In such systems, the aqueous phase is the reactive phase in which the biocatalyst is dissolved and the organic phase is nonreactive and acts as substrate reservoir and as in situ product extraction solvent. Here, the choice of the nonreactive phase is highly important for the overall performance of the system. In this contribution, a systematic approach to solvent selection for biphasic aqueous‐organic systems is presented with respect to partition coefficients. The model reaction is the stereoselective carbon‐carbon coupling of two 3,5‐dimethoxy‐benzaldehyde molecules to (R)‐3,3',5,5'‐tetramethoxy‐benzoin catalyzed by benzaldehyde lyase (EC 4.1.2.38) from Pseudomonas fluorescens. A systematic approach to solvent selection consisting of two steps is proposed: Firstly, the conductor‐like screening model for real solvents (COSMO‐RS) is used to facilitate a fast solvent screening. Since this is an ab initio approach it allows a pre‐screening without laborious experimental input. The proposed ranking of solvents, based on the ratio of partition coefficients at infinite dilution, is a sound basis for the successive steps. Secondly, a dynamic model is fitted to experimental data in order to obtain detailed and reliable results for mass transfer and partition coefficients. Therefore, the method makes efficient use of the experimental data and substantiates quantitative results with guided experiments.     

Directed evolution for enzyme development in biocatalysis

As an important sector of the chemical industry, biocatalysis requires the continuous development of enzymes with tailor-made activity, selectivity, stability, or tolerance to unnatural environments. This is now routinely achieved by directed evolution based on iterative cycles of genetic diversification and activity screening. Here, we highlight its recent developments. First, the design of “smarter” libraries by focused mutagenesis may be a crucial start-up for a fast and successful outcome. Then library assembly and expression are also key steps that benefits from modern molecular biology progresses. Finally, various strategies may be considered for library screening depending on the final objective: while low-throughput direct assays have been very successful in generating enzymes for important biocatalytic processes, even in bringing completely new chemistries to the enzyme world, ultrahigh-throughput screening methods are emerging as powerful approaches for engineering the next generation of industrial enzymes.     

Novel screening methods--the key to cloning commercially successful biocatalysts

Providing sufficient biocatalyst to support the demands of multi tonne product supply can be problematical. Here we describe how screening for and cloning a gamma-lactamase overcame biocatalyst supply issues, and greatly improved the actual biocatalytic process. The isolation of an expressing gamma-lactamase clone from a gene library necessitated a combination of classical molecular biology techniques together with innovative screening methods to identify a functional clone. Once isolated the enzyme was characterised with regard to its process performance and proved to be active at 500 g L(-1) substrate. Further development of the recombinant fermentation and downstream processing has resulted in the ability to produce sufficient biocatalyst from one 5001 fermentation to resolve 5 metric tonnes of (+/-)-lactam, whilst simplifying the process chemistry greatly.     

生物信息学在工业生物催化研究中的应用

随着石油等不可再生资源的日益减少以及环境污染问题的日益严重,应用工业生物催化技术改造或取代传统化工工艺已经成为新世纪化学工业可持续发展的研究热点。工业生物催化技术的研究对象是生物催化剂及其催化过程。近来,利用生物信息学技术进行工业生物催化研究已经越来越受到人们的重视。随着工业生物催化的发展,生物信息学将直接指导并加快新型高效生物催化剂的发现及功能改造进程。     

Biocatalytic combinatorial synthesis

Combinatorial biocatalysis, based on a principle of the combinatorial use of biosynthetic steps rather than the combinatorial use of reagents, offers a complementary approach to combinatorial chemistry, which, used individually or in connection with synthetic organic transformations, provides access to analogues not readily accessible by chemical synthetic means alone. The issues and strategies particular to this approach are discussed. Examples are given demonstrating these principles as well as the unique advantages of achieving chemo-, regio- and stereoselectivity under mild reaction conditions that biocatalytic methods offer.     

Whole cell biocatalysis in nonconventional media

Summary In this paper biocatalytic reactions carried out by whole cells in nonconventional media are reviewed. Similar relationships are observed between solvent hydrophobicity and catalytic activity in reactions carried out by isolated enzymes and whole cells. In addition to the effect of organic solvent on biocatalyst stability, microbial cells are susceptible to damaging effects caused by the organic phase. In general, more hydrophobic solvents manifest lower toxicity towards the cells. Whole cell biocatalysts require more water than isolated enzymes and two-phase systems have been most widely used to study whole cell biocatalysis. Immobilization makes cell biocatalysts more resistant to organic solvents and helps achieve homogeneous biocatalyst dispersion. Cell entrapment methods have been widely used with organic solvent systems and mixtures of natural and/or synthetic polymers allow adjustment of the hydrophobicity-hydrophilicity balance of the support matrix. Some examples of stereoselective catalysis using microbial cells in organic solvent media are presented.     

Biotransformations in microstructured reactors: more than flowing with the stream?

The state of the art in the application of microstructured flow reactors for biocatalytic process research is reviewed. A microstructured reactor that is fully automated and analytically equipped presents a powerful screening tool with which to perform biocatalyst selection and optimization of process conditions at intermediary stages of process development. Enhanced mass transfer provided by the microstructured reactor can be exploited for process intensification, particularly during multiphase biocatalytic processing where mass transfer across phase boundaries is often limiting. Reversible immobilization of enzymes in microchannels remains a challenge for flexible realization of biotransformations in microstructured reactors. Compartmentalization in microstructured reactors could be useful in performing multistep chemoenzymatic conversions.     

Chemical biotechnology for the specific oxyfunctionalization of hydrocarbons on a technical scale

Oxygenases catalyze, among other interesting reactions, highly selective hydrocarbon oxyfunctionalizations, which are important in industrial organic synthesis but difficult to achieve by chemical means. Many enzymatic oxygenations have been described, but few of these have been scaled up to industrial scales, due to the complexity of oxygenase based biocatalysts and demanding process implementation. We have combined recombinant whole-cell catalysis in a two-liquid phase system with fed-batch cultivation in an optimized medium and developed an industrially feasible process for the kinetically controlled and complex multistep oxidation of pseudocumene to 3,4-dimethylbenzaldehyde using the xylene monooxygenase of Pseudomonas putida mt-2 in Escherichia coli. Successful scale up to 30 L working volume using downscaled industrial equipment allowed a productivity of 31 g L(-1) d(-1) and a product concentration of 37 g L(-1). These performance characteristics meet present industry requirements. Product purification resulted in the recovery of 469 g of 3,4-dimethyl- benzaldehyde at a purity of 97% and an overall yield of 65%. This process illustrates the general feasibility of industrial biocatalytic oxyfunctionalization.     

Biocatalysis for pharmaceutical intermediates: the future is now

Biocatalysis is continuing to gain momentum and is now becoming a key component in the toolbox of the process chemist, with a place alongside chemocatalysis and chromatographic separations. The pharmaceutical industry demands a speed of development that must be on a parallel with conventional chemistry and high optical purity for complex compounds with multiple chiral centres. This review describes how these demands are being addressed to make biocatalysis successful, particularly by the use of micro-scale technology for high-speed catalyst screening and process development alongside discipline integration of biology and engineering with chemistry. Developments in recombinant technology will further expand the repertoire of biocatalysis in the coming years to new chemistries and enable catalyst design to fit the process. Further development of biocatalysis for green chemistry and high productivity processes can also be expected.     

On oxygen limitation in a whole cell biocatalytic Baeyer-Villiger oxidation process

In this article, a recombinant cyclohexanone monooxygenase (CHMO), overexpressed in Escherichia coli has been used to study the oxidation of bicyclo[3.2.0]hept-2-en-6-one to its two corresponding lactones at very high enantiomeric excess. The reaction is a useful model for the study of biocatalytic oxidations to create optically pure molecules. The major limitations to a highly productive biocatalytic oxidation in this case are oxygen supply, product inhibition, and biocatalyst stability. In this article, we investigate the effects of whole cell biocatalyst concentration on the rate of reaction at a range of scales from shake flasks to 75 L bioreactors. At low cell concentrations (<2 g(dcw)/L) the maximum specific rate (0.65 g/g(dcw).h) is observed. However, at higher cell concentrations (> 2 g(dcw)/L), the reaction becomes oxygen limited and both the specific rate and absolute rate decrease with further increases in cell concentration. The role of oxygen limitation in reducing the rate of reaction with scale was investigated by increasing the maximum oxygen transfer rate in the reactor at a high cell concentration and observing the increase in product formation rate. We propose a qualitative model demonstrating the relationship between oxygen limitation, biocatalyst concentration, and the rate of reaction. This conceptual model will be a useful guide in the industrial scale-up of whole cell mediated Baeyer-Villiger biocatalysis.     

Maximizing the stability of metabolic engineering‐derived whole‐cell biocatalysts

A systematic and powerful knowledge‐based framework exists for improving the activity and stability of chemical catalysts and for empowering the commercialization of respective processes. In contrast, corresponding biotechnological processes are still scarce and characterized by case‐by‐case development strategies. A systematic understanding of parameters affecting biocatalyst efficiency, that is, biocatalyst activity and stability, is essential for a rational generation of improved biocatalysts. Today, systematic approaches only exist for increasing the activity of whole‐cell biocatalysts. They are still largely missing for whole‐cell biocatalyst stability. In this review, we structure factors affecting biocatalyst stability and summarize existing, yet not completely exploited strategies to overcome respective limitations. The factors and mechanisms related to biocatalyst destabilization are discussed and demonstrated inter alia based on two case studies. The factors are similar for processes with different objectives regarding target molecule or metabolic pathway complexity and process scale, but are in turn highly interdependent. This review provides a systematic for the stabilization of whole‐cell biocatalysts. In combination with our knowledge on strategies to improve biocatalyst activity, this paves the way for the rational design of superior recombinant whole‐cell biocatalysts, which can then be employed in economically and ecologically competitive and sustainable bioprocesses.     

Integrated product formation and recovery.

As continues to be demonstrated, the in situ recovery of selected products froma bioreactor can have a significant positive impact on production. The strategies that are focused on here are: aqueous two-phase biocatalysis; non-aqueous biocatalysis; and membrane-enhanced biocatalysis. Additional fundamental understanding of molecular partitioning and biocatalytic activity in these environments will facilitate the rational selection of the components involved in these processing strategies.     

Tools for Selective Enzyme Reaction Steps in the Synthesis of Laboratory Chemicals

The need for more selective reactions steps and the compatibility between process steps which follow on from each other has been a major driving force for organic synthesis. The synthesis of chiral compounds, metabolites, new chemical entities and natural products by a combination of chemical and enzyme reaction steps has become well established, due the existence of stable enzymes as selective catalysts which are inherently chiral by nature. Auxiliary tools such as suitable transfer reagents for reaching complete conversion, easy and robust reaction control as well as tools for straightforward workup and purification of the final product have been developed. Selective enzyme reaction steps in the area of hydrolyses, oxidation steps including hydroxylation and the Baeyer‐Villiger oxidation, carbon‐carbon bond formation and glycosylation reactions have compared favorably with existing methods of classical organic synthesis. The tools developed during optimization and scale‐up of these enzyme reaction steps have the potential to shorten development time. The introduction of selective enzyme reactions into an entire synthetic process has resulted in harmonization of improvements in economic efficiency with resultant solutions to health, safety and environment problems. This will become even more important in industrial synthetic chemistry in the future, for convenient solutions to certain intractable synthetic problems and for expanding the repertoire of chemistry by modular biocatalysts. Efficient isolation procedures for the final product are essential to take full advantage of the biocatalytic conversion to obtain high product yields.