Bacteria produce and secrete lipases, which can catalyze both the hydrolysis and the synthesis of long-chain acylglycerols. These reactions usually proceed with high regioselectivity and enantioselectivity, and, therefore, lipases have become very important stereoselective biocatalysts used in organic chemistry. High-level production of these biocatalysts requires the understanding of the mechanisms underlying gene expression, folding, and secretion. Transcription of lipase genes may be regulated by quorum sensing and two-component systems; secretion can proceed either via the Sec-dependent general secretory pathway or via ABC transporters. In addition, some lipases need folding catalysts such as the lipase-specific foldases and disulfide-bond-forming proteins to achieve a secretion-competent conformation. Three-dimensional structures of bacterial lipases were solved to understand the catalytic mechanism of lipase reactions. Structural characteristics include an alpha/beta hydrolase fold, a catalytic triad consisting of a nucleophilic serine located in a highly conserved Gly-X-Ser-X-Gly pentapeptide, and an aspartate or glutamate residue that is hydrogen bonded to a histidine. Four substrate binding pockets were identified for triglycerides: an oxyanion hole and three pockets accommodating the fatty acids bound at position sn-1, sn-2, and sn-3. The differences in size and the hydrophilicity/hydrophobicity of these pockets determine the enantiopreference of a lipase. The understanding of structure-function relationships will enable researchers to tailor new lipases for biotechnological applications. At the same time, directed evolution in combination with appropriate screening systems will be used extensively as a novel approach to develop lipases with high stability and enantioselectivity.

The term B-factor, sometimes called the Debye–Waller factor, temperature factor, or atomic displacement parameter, is used in protein crystallography to describe the attenuation of X-ray or neutron scattering caused by thermal motion. This review begins with analyses of early protein studies which suggested that B-factors, available from the Protein Data Bank, can be used to identify the flexibility of atoms, side chains, or even whole regions. This requires a technique for obtaining normalized B-factors. Since then the exploitation of B-factors has been extensively elaborated and applied in a variety of studies with quite different goals, all having in common the identification and interpretation of rigidity, flexibility, and/or internal motion which are crucial in enzymes and in proteins in general. Importantly, this review includes a discussion of limitations and possible pitfalls when using B-factors. A second research area, which likewise exploits B-factors, is also reviewed, namely, the development of the so-called B-FIT-directed evolution method for increasing the thermostability of enzymes as catalysts in organic chemistry and biotechnology. In both research areas, a maximum of structural and mechanistic insights is gained when B-factor analyses are combined with other experimental and computational techniques.

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Combinatorial methods in the development of enantioselective homogeneous catalysts constitute a new branch of catalysis research. The goal is to prepare libraries of potential asymmetric catalysts, rather than choosing the traditional one-catalyst-at-a-time approach. Several conceptional advancements have been reported in the parallel preparation of chiral ligands. Currently the most meaningful systems constitute modularly constructed ligands on solid supports, which allow high degrees of structural diversity and thus the maximum probability of finding enantioselective catalysts or even new types of ligands for asymmetric catalysis. Search strategies have been developed which amongst other things, lead to catalysts not likely to have been discovered by traditional methods. Genuine application of such strategies involve thousands of catalysts and require high-throughput screening systems capable of assaying enantioselectivity. The first high-throughput ee-screening systems were in fact developed for use in the directed evolution of enantioselective enzymes, a process based on "evolution in the test tube" in which the appropriate methods of random mutagenesis, gene expression, and ee assays are combined. Since no screening system is likely to be universal, different approaches are necessary. Thus far these include assays based on UV/Vis, fluorescence, circular dichroism, mass spectrometry, and even modified gas chromatography as well as special forms of capillary electrophoresis. One of the most efficient systems involves the concept of the mass-spectrometric detection of deuterium-labeled pseudo-enantiomers and pseudo-prochiral compounds with which about 1000 exact ee determinations can be achieved per day, although the assay is restricted to kinetic resolution and/or reactions of prochiral compounds bearing enantiotopic groups. Super-high-throughput screening for enantioselectivity is possible in many cases by making use of chirally modified capillary array electrophoresis in a parallel step. Accordingly, 7000 to 30 000 ee determinations can be carried out per day. These and other analytical developments are expected to stimulate further research in the combinatorial search for asymmetric homogeneous catalysts and in the directed evolution of enantioselective enzymes for use in organic chemistry.