Uwe T. Bornscheuer

Engineering the third wave of biocatalysis

Over the past ten years, scientific and technological advances have established biocatalysis as a practical and environmentally friendly alternative to traditional metallo- and organocatalysis in chemical synthesis, both in the laboratory and on an industrial scale. Key advances in DNA sequencing and gene synthesis are at the base of tremendous progress in tailoring biocatalysts by protein engineering and design, and the ability to reorganize enzymes into new biosynthetic pathways. To highlight these achievements, here we discuss applications of protein-engineered biocatalysts ranging from commodity chemicals to advanced pharmaceutical intermediates that use enzyme catalysis as a key step.

Oils and Fats as Renewable Raw Materials in Chemistry

Oils and fats of vegetable and animal origin have been the most important renewable feedstock of the chemical industry in the past and in the present. A tremendous geographical and feedstock shift of oleochemical production has taken place from North America and Europe to southeast Asia and from tallow to palm oil. It will be important to introduce and to cultivate more and new oil plants containing fatty acids with interesting and desired properties for chemical utilization while simultaneously increasing the agricultural biodiversity. The problem of the industrial utilization of food plant oils has become more urgent with the development of the global biodiesel production. The remarkable advances made during the last decade in organic synthesis, catalysis, and biotechnology using plant oils and the basic oleochemicals derived from them will be reported, including, for example, ω-functionalization of fatty acids containing internal double bonds, application of the olefin metathesis reaction, and de novo synthesis of fatty acids from abundantly available renewable carbon sources.

Hydrolases in Organic Synthesis: Regio- and Stereoselective Biotransformations

1 Introduction. 2 Designing Enantioselective Reactions. 2.1 Kinetic Resolutions. 2.2 Asymmetric Syntheses. 3 Choosing Reaction Media: Water and Organic Solvents. 3.1 Hydrolysis in Water. 3.2 Transesterifications and Condensations in Organic Solvents. 3.3 Other Reaction Media. 3.4 Immobilization. 4 Protein Sources and Optimization of Biocatalyst Performance. 4.1 Accessing Biodiversity. 4.2 Creating Improved Biocatalysts. 4.3 Catalytic Promiscuity in Hydrolases. 5 Lipases and Esterases. 5.1 Availability, Structures and Properties. 5.2 Survey of Enantioselective Lipase-Catalyzed Reactions. 5.3 Chemo-and Regioselective Lipase-Catalyzed Reactions. 5.4 Reactions Catalyzed by Esterases. 6 Proteases and Amidases. 6.1 Occurrence and Availability of Proteases and Amidases. 6.2 General Features of Subtilisin, Chymotrypsin, and Other Proteases and Amidases. 6.3 Structures of Proteases and Amidases. 6.4 Survey of Enantioselective Protease-and Amidase-Catalyzed Reactions. 7 Phospholipases. 7.1 Phospholipase A1. 7.2 Phospholipase A2. 7.3 Phospholipase C. 7.4 Phospholipase D. 8 Epoxide Hydrolases. 8.1 Introduction. 8.2 Mammalian Epoxide Hydrolases. 8.3 Microbial Epoxide Hydrolases. 9 Hydrolysis of Nitriles. 9.1 Introduction. 9.2 Mild Conditions. 9.3 Regioselective Reactions of Dinitriles. 9.4 Enantioselective Reactions. 10 Other Hydrolases. 10.1 Glycosidases. 10.2 Haloalcohol Dehalogenases. 10.3 Phosphotriesterases. Abbreviations. References. Index.

Improved biocatalysts by directed evolution and rational protein design

The efficient application of biocatalysts requires the availability of suitable enzymes with high activity and stability under process conditions, desired substrate selectivity and high enantioselectivity. However, wild-type enzymes often need to be optimized to fulfill these requirements. Two rather contradictory tools can be used on a molecular level to create tailor-made biocatalysts: directed evolution and rational protein design.

Lipids as renewable resources: current state of chemical and biotechnological conversion and diversification

Oils and fats are the most important renewable raw materials of the chemical industry. They make available fatty acids in such purity that they may be used for chemical conversions and for the synthesis of chemically pure compounds. Oleic acid (1) from “new sunflower,” linoleic acid (2) from soybean, linolenic acid (3) from linseed, erucic acid (4) from rape seed, and ricinoleic acid (5) from castor oil are most important for chemical transformations offering in addition to the carboxy group one or more C-C-double bonds. New plant oils containing fatty acids with new and interesting functionalities such as petroselinic acid (6) from Coriandrum sativum, calendic acid (7) from Calendula officinalis, α-eleostearic acid (8) from tung oil, santalbic acid (9) from Santalum album (Linn.), and vernolic acid (10) from Vernonia galamensis are becoming industrially available. The basic oleochemicals are free fatty acids, methyl esters, fatty alcohols, and fatty amines as well as glycerol as a by-product. Their interesting new industrial applications are the usage as environmentally friendly industrial fluids and lubricants, insulating fluid for electric utilities such as transformers and additive to asphalt. Modern methods of synthetic organic chemistry including enzymatic and microbial transformations were applied extensively to fatty compounds for the selective functionalization of the alkyl chain. Syntheses of long-chain diacids, ω-hydroxy fatty acids, and ω-unsaturated fatty acids as base chemicals derived from vegetable oils were developed. Interesting applications were opened by the epoxidation of C-C-double bonds giving the possibility of photochemically initiated cationic curing and access to polyetherpolyols. Enantiomerically pure fatty acids as part of the chiral pool of nature can be used for the synthesis of nonracemic building blocks.

Biocatalytic Routes to Optically Active Amines

Optically active amines and amino acids play an important role in the pharmaceutical, agrochemical, and chemical industries. They are frequently used as synthons for the preparation of various pharmaceutically active substances and agrochemicals, but also as resolving agents to obtain chiral carboxylic acids. Consequently, there is a need for efficient methods to obtain the desired enantiomer of a given target structure in optically pure form. Beside a range of chemical methods using for example, asymmetric synthesis with transition metal catalysts, enzymes represent a useful alternative to access this important class of compounds. This review covers biocatalytic approaches using hydrolases (i.e. lipases, amidases), monoamine oxidase and other enzymes. Special focus is given on the application of ω‐transaminases with emphasis on concepts to allow efficient asymmetric synthesis starting from prostereogenic ketones.

Optimizing lipases and related enzymes for efficient application.

Although numerous reactions have been performed using lipases and related enzymes (e.g. esterases and phospholipases), it is still a challenge to identify the most suitable biocatalyst and best reaction conditions for an efficient application. Frequently used methods such as immobilization and optimization of the reaction medium cannot be transferred from one reaction system or substrate to another. However, in the past few years, rational protein design and directed evolution have emerged as efficient alternative methods to optimize biocatalytic reactions.

Rational assignment of key motifs for function guides in silico enzyme identification

Biocatalysis has emerged as a powerful alternative to traditional chemistry, especially for asymmetric synthesis. One key requirement during process development is the discovery of a biocatalyst with an appropriate enantiopreference and enantioselectivity, which can be achieved, for instance, by protein engineering or screening of metagenome libraries. We have developed an in silico strategy for a sequence-based prediction of substrate specificity and enantiopreference. First, we used rational protein design to predict key amino acid substitutions that indicate the desired activity. Then, we searched protein databases for proteins already carrying these mutations instead of constructing the corresponding mutants in the laboratory. This methodology exploits the fact that naturally evolved proteins have undergone selection over millions of years, which has resulted in highly optimized catalysts. Using this in silico approach, we have discovered 17 (R)-selective amine transaminases, which catalyzed the synthesis of several (R)-amines with excellent optical purity up to >99% enantiomeric excess.

Lipase-catalyzed syntheses of monoacylglycerols

The application of lipases for the production of monoacylglycerols is reviewed. Glycerolysis or selective hydrolysis of fats and oils, esterification of fatty acids or esters with glycerol, and reactions employing protected glycerols are presented. From the most recent literature, reaction systems ranging from organic solvents to reverse micelles and solid-phase systems, for example, are covered, including examples for the continuous production of partial glycerides.

Efficient Asymmetric Synthesis of Chiral Amines by Combining Transaminase and Pyruvate Decarboxylase

Chiral amines and amino acids play an important role in the pharmaceutical, agrochemical and chemical industry. They are frequently used as synthons for the preparation of various pharmaceutically active substances and agrochemicals, or as resolving agents for chiral acids. Consequently, there is a need for efficient methods to obtain the desired R or S enantiomer in an optically pure form. The most frequently used enzymatic method for the production of optically active amines is the kinetic resolution of racemic starting material by enantioselective hydrolysis of, for ACHTUNGTRENNUNGexample, N-acyl amides by peptidases, amidases or lipases. lternatively, transaminases can be used in kinetic resolution (Scheme 1A). The maximum yield in all of these processes is limited to 50% unless a racemization step is included to