Roland Wohlgemuth

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.

Towards large-scale synthetic applications of Baeyer-Villiger monooxygenases

Biocatalysis is coming of age, with an increasing number of reactions being scaled-up and developed. The diversity of reactions is also increasing and oxidation reactions have recently been considered for scale-up to commercial processes. One important chemical conversion, which is difficult to achieve enantio- or enantiotopo- selectively, is the Baeyer-Villiger (BV) oxidation of ketones. Using cyclohexanone monooxygenase to catalyse the reaction produces optically pure esters and lactones with exquisite enantiomeric excess values. Recently, these enzymes and their many applications in synthetic chemistry have been explored. The scale-up of these conversions has been examined with the idea of implementing the first commercial Baeyer-Villiger monooxygenase-based process. Here, we review the state-of-the-art situation for the scale-up and exploitation of these enzymes.

High-Yield Biocatalytic Amination Reactions in Organic Synthesis

The amino functionality gives important biological activity in pharmaceutical com-pounds. The formation of chiral amines and amino acids can be accomplished by seve-ral chemical routes but enzymatic formation of amines offers many advantages in pre-paring chiral amino compounds or amination of fragile compounds compared to stoi-chiometric or catalytic chemical transformations. Biocata- lytic routes to amines primarily use enzymes of the transaminase class (also known as aminotransferases) which transfer the amino func- tion from a donor organic compound to a ketone or aldehyde acceptor. Although known since 1937, the transamination reaction experi- ences re-newed interest due to the advances in biochemistry and molecular biology and the excellent selectivity of biocatalysts. Other en- zymes that have been used to synthesize chiral amines are from the phenylalanine ammonia lyase class that use ammonia as the amino source. The use of recombinant enzymes for the biocatalytic preparation of amines is expanding at a great rate and the range of enzymes revealed in DNA se-quence databases is of the order of tens of thousands. Since a large number of ketone substrates like ketones, hy- droxyketones and ketoacids can be made by chemical syn-thesis, the growing toolbox of � - , � -, � -, � -, � - and � -transaminases enable the synthe-sis of various new chemical entities by biocatalytic amination reactions. In order to simplify the isolation and purification of the product, it is useful to drive the amination reaction to completion. The biocatalytic processes that have been developed show different strategies of overcoming the kinetic limitations of the transaminase reactions and show how some enzymes have been used in processes to make large quantities of chiral compounds with amino functionalities.

Guidelines for reporting of biocatalytic reactions.

Enzymes and whole cells are being increasingly applied in research and industry, but the adoption of biocatalysis relies strongly on useful scientific literature. Unfortunately, too many published papers lack essential information needed to reproduce and understand the results. Here, members of the scientific committee of the European Federation of Biotechnology Section on Applied Biocatalysis (ESAB) provide practical guidelines for reporting experiments. The document embraces the recommendations of the STRENDA initiative (Standards for Reporting Enzymology Data) in the context of pure enzymology and provides further guidelines and explanations on topics of crucial relevance for biocatalysis. In particular, guidelines are given on issues such as the selectivity, specificity, productivity and stability of biocatalysts, as well as on methodological problems related to reactions in multiphase systems. We believe that adoption and use of these guidelines could greatly increase the value and impact of published work in biocatalysis, and hence promote the further growth of applications.

Microscale technology and biocatalytic processes: opportunities and challenges for synthesis

Despite the expanding presence of microscale technology in chemical synthesis and energy production as well as in biomedical devices and analytical and diagnostic tools, its potential in biocatalytic processes for pharmaceutical and fine chemicals, as well as related industries, has not yet been fully exploited. The aim of this review is to shed light on the strategic advantages of this promising technology for the development and realization of biocatalytic processes and subsequent product recovery steps, demonstrated with examples from the literature. Constraints, opportunities, and the future outlook for the implementation of these key green engineering methods and the role of supporting tools such as mathematical models to establish sustainable production processes are discussed.

The use of enzymes in organic synthesis and the life sciences: perspectives from the Swiss Industrial Biocatalysis Consortium (SIBC)

The potential of biotechnology by means of biocatalysis or biosynthesis in organic synthesis is far from being fully exploited. For this reason a group of life science companies active in pharmaceuticals, flavour and fragrance, vitamin and fine chemicals businesses describe some examples of the use of enzymes in industrial organic synthesis and discuss why enzymes are still the exception rather than the rule in organic synthesis.

Asymmetric biocatalysis with microbial enzymes and cells.

Microbial enzymes and cells continue to be important tools and natures privileged chiral catalysts for performing asymmetric biocatalysis from the analytical small scale to the preparative and large scale in synthesis and degradation. The application of biocatalysts for preparing molecular asymmetry has achieved high efficiency, enantioselectivity and yield and is experiencing today a worldwide renaissance. Recent developments in the discovery, development and production of stable biocatalysts, in the design of new biocatalytic processes and in the product recovery and purification processes have made biocatalytic approaches using microbial cells and enzymes attractive choices for the synthesis of chiral compounds. The methodologies of kinetic resolution and kinetic asymmetric transformation, dynamic kinetic resolution and deracemization, desymmetrization, asymmetric synthesis with or without diastereo control and multi-step asymmetric biocatalysis are finding increasing applications in research. The ever-increasing use of hydrolytic enzymes has been accompanied by new applications of oxidoreductases, transferases and lyases. Isomerases, already used in large-scale processes, and ligases, are emerging as interesting biocatalysts for new synthetic applications. The production of a wide variety of industrial products by asymmetric biocatalysis has even become the preferred method of production.

Preparative scale Baeyer-Villiger biooxidation at high concentration using recombinant Escherichia coli and in situ substrate feeding and product removal process

An efficient biocatalytic process based on the use of adsorbent resin (in situ substrate feeding and product removal) makes experiments at high substrate concentration possible by overcoming limitations due to substrate and product inhibition. This process was successfully applied to the preparative scale Baeyer–Villiger biooxidation of (−)-(1S,5R)-bicyclo[3.2.0]hept-2-en-6-one (25 g). Whole cells of recombinant E. coli (1 liter) overexpressing cyclohexanone monooxygenase were used as a biocatalyst and the substrate was preloaded onto the adsorbent resin. The corresponding lactone was obtained in 75–80% yield. Time for cell growth and biotransformation is about 24 h each and oxygen supply can be improved by using a tailor-made bubble column.

The locks and keys to industrial biotechnology

The sustainable use of resources by Nature to synthesize the required products at the right place, when they are needed, continues to be the role model for total synthesis and production in general. The combination of molecular and engineering science and technology in the biotechnological approach needs no protecting groups at all and has therefore been established for numerous large-scale routes to both natural and synthetic products in industry. The use of biobased raw materials for chemical synthesis, and the economy of molecular transformations like atom economy and step economy are of growing importance. As safety, health and environmental issues are key drivers for process improvements in the chemical industry, the development of biocatalytic reactions or pathways replacing hazardous reagents is a major focus. The integration of the biocatalytic reaction and downstream processing with product isolation has led to a variety of in situ product recovery techniques and has found numerous successful applications. With the growing collection of biocatalytic reactions, the retrosynthetic thinking can be applied to biocatalysis as well. The introduction of biocatalytic reactions is uniquely suited to cost reductions and higher quality products, as well as to more sustainable processes. The transfer of Natures simple and robust sensing and control principles as well as its reaction and separation organization into useful technical systems can be applied to different fermentations, biotransformations and downstream processes. Biocatalyst and pathway discovery and development is the key towards new synthetic transformations in industrial biotechnology.

Characterisation of a recombinant NADP-dependent glycerol dehydrogenase from Gluconobacter oxydans and its application in the production of L-glyceraldehyde.

The acetic acid bacterium Gluconobacter oxydans has a high potential for oxidoreductases with a variety of different catalytic abilities. One putative oxidoreductase gene codes for an enzyme with a high similarity to the NADP+‐dependent glycerol dehydrogenase (GlyDH) from Hypocrea jecorina. Due to this homology, the GlyDH (Gox1615) has been cloned, over‐expressed in Escherichia coli, purified and characterised. Gox1615 shows an apparent native molecular mass of 39 kDa, which corresponds well to the mass of 37.213 kDa calculated from the primary structure. From HPLC measurements, a monomeric structure can be deduced. Kinetic parameters and the dependence of the activity on temperature and pH were determined. The enzyme shows a broad substrate spectrum in the reduction of different aliphatic, branched and aromatic aldehydes. Additionally, the enzyme has been shown to oxidize a variety of different alcohols. The highest activities were observed for the conversion of D‐glyceraldehyde in the reductive and L‐arabitol in the oxidative direction. Since high enantioselectivities were observed for the reduction of glyceraldehyde, the kinetic resolution of glyceraldehyde was investigated and found to yield enantiopure L‐glyceraldehyde on preparative scale.