Intelligent accelerators
Biocatalysts in the chemical industry – new processes present opportunities and challenges

Experts have long been aware of the advantages offered by biocatalysts in chemical processes. However, practical application has often been a problem. An interdisciplinary team at Degussa’s biotechnology project engineering center is providing the basis for more timely use of these biological helpers.

Biocatalysts (enzymes) are present in microorganisms and animal cells in large numbers. These enzymes, which catalyze a wide variety of metabolic processes (e.g. energy production, cell development, etc.), have been used for many years in material transformation applications. The range of applications includes the production of sugar by breaking down starch into individual sugar molecules or conversion of glucose into fructose. Compared to traditional chemical methods, processes that use biological catalysts tend to be long-term solutions. Biological catalysts are highly selective, function at ambient temperature and pressure and are used primarily in aqueous systems. Despite recent initial work on gene research and screening technologies which have been used to detect a variety of catalysts, the number of biological catalytic process used in practical application remains significantly below that of traditional chemical synthesis. The reasons why this has been the case in the past are the relatively long and complex development processes and to some extent low product concentrations. In addition, process stability was often inadequate to meet the needs of day to day production.
Successful commercialization of the processes requires an up-front analysis of the entire process. Issues that require clarification include substrate accessibility, efficiency, cost of the biological catalyst, separation of the product from the reaction medium and the infrastructure that will be needed. The time it takes to design new processes also plays a crucial role. To minimize development time, it is important to establish technology platforms that are as universal and standardized as possible. This opens the door to short development cycles and routine use of the technology.
Four-stage development
Process development is typically broken down into four stages: screening for suitable basic activity, optimizing the catalyst for the engineering application, development of a suitable production process for the biocatalyst and finally the actual biological transformation. It is important to keep in mind that the biological transformation process is often merely one reaction step in a multi-stage synthesis. More recent approaches tend to combine several reaction steps (e.g. two or more enzymes) in one reactor.
Currently, screening is either activity-based (e.g. in strain collections) or it is performed at the genetic level (metagenome, in-silico). The latest high-throughput screening techniques can be used to quickly detect suitable base activity which is then optimized using evolutionary or rational processes. The most relevant aspects in this context are stability of the catalyst in the presence of oxidants and high temperatures, enhancement of specific activity, reduction of product inhibiton and modification of the substrate or enantioselectivity. Since the volume of enzyme produced with wild-type strains in not sufficient for efficient production, catalysts are produced by recombinant methods using production strains (bacteria, yeast, fungus). When good expression rates can be achieved, the target protein can make up as much as 50% of the total protein in the cell. With the platform approach in mind, fermentation processes have been developed which can be used for as many enzyme systems as possible and which can be reliably scaled up from the laboratory to the production environment. The latest fermentation techniques can be used to achieve high volumetric activity in the fermenters and minimize the fermentation volumes that are needed. This keeps the lid on the cost of producing biocatalysts.

High selectivity
Biocatalysts can be used either as whole cell catalysts or as isolated enzymes. The whole cell approach has the advantage that no costs are incurred to isolate the enzymes, but it can only be used if no interference is caused by other cell activity. There is a tendency to exploit whole cell systems for reaction steps that contain more than one enzyme. Free enzymes are either immobilized on suitable substrates and subsequently used in fixed bed or suspension reactors, or they are used in an enzyme membrane reactor. A suitable membrane retains the enzyme in the reactor, and only the product and solvent pass through the membrane, forming the basis for a repetitive fed batch or continuous process flow. Biocatalysts are highly selective, and it is often relatively easy to isolate the product. Crystallization or precipitation from the reaction solution can often be used to isolate the product in purified form. New approaches attempt to combine the production of biocatalysts and biological transformation in one step to reduce the number of processing steps and the amount of equipment used. If the right technology platforms for screening, enzyme optimization, enzyme production and reaction design are developed, new processes can currently be taken from initial concept to full-scale production in less than twelve months. Various examples are described below which show how biocatalysts have helped achieve substantial product or process improvement.
Production of chiral compounds
The demand for single-enantiomer compounds will increase dramatically in the near future in pharmaceutical building block applications. Fifty percent of all new products are already based on single-enantiomer substances. Due to their high enantioselectivity, biocatalysts offer distinct advantages compared to chemical catalysts or chemical resolution. To minimize elaborate recycling steps or large amounts of waste, methods have been developed which make it possible to ideally produce 100 percent of the desired enantiomer in one reaction step. Examples include reductive amination including integrated co-factor regeneration of achiral substrates or dynamic kinetic enzymatic resolution. The product can, for example, be isolated directly from the reaction solution by continuous crystallization.
Cosmetic ester
A large number of esters (e.g. dodecyloleate) are currently used in the cosmetic industry as emulsifying agents. In the past, these esters were produced from alcohol and carboxylic acid at high temperatures using acid as catalysts. High temperature produces unwanted byproducts and product discoloration. This resulted in the need for more elaborate and expensive process steps to achieve the desired level of product purity. When lipases are used as catalysts, the reaction temperature can be reduced significantly, and there is no discoloration. Appropriate reaction management can produce a nearly complete conversion, eliminating the need for additional processing steps.
Acrylamide
Acrylamide is used in applications such as production of flocculent auxiliary agents. Annual consumption is in the lower five-figure range. Chemical saponification based on acrylonitrile requires copper ions as a catalyst which must be separated out of the reaction mixtures at significant cost. The use of nitrile hydratase eliminates the need for toxic copper, and the reaction solution can be used in subsequent process steps directly following separation from the biocatalyst.
Outlook
The examples described in this article show how processes based on biocatalysts have replaced chemical processes in recent years. A large number of new enzymes emanating from large-scale genome and screen projects will continue to increase opportunities to use biocatalysts. However, successful commercialization requires a timely analysis of the entire process to evaluate optimization potential, which the biological catalyst can deliver, and special process considerations related to the use of these catalysts. Isolated development of enzyme systems that does not take into account of the overall process and the existing/required infrastructure is seldom likely to produce a successful outcome. It is essential that interdisciplinary teams be formed at an early stage which include chemists, biologists and process engineers to design the process. The creation of technology platforms will continue to result in a further significant reduction in the development time and the cost of new
processes.