Dominique Böttcher

Strategies for the discovery and engineering of enzymes for biocatalysis

Protein engineering is the most important method to overcome the limitations of natural enzymes as biocatalysts. The past few years have seen a tremendous increase in novel concepts to facilitate the design of mutant libraries for focused directed evolution mostly guided by advanced bioinformatic tools. In addition, advanced high-throughput methods were developed using, for example, FACS analysis or microfluidic systems. These achievements significantly facilitate the tailor-made design of enzymes to make them suitable for industrial applications.

The alpha/beta-hydrolase fold 3DM database (ABHDB) as a tool for protein engineering.

Aligning the haystack to expose the needle: The 3DM method was used to generate a comprehensive database of the a/s-hydrolase fold enzyme superfamily. This database facilitates the analysis of structure–function relationships and enables novel insights into this superfamily to be made. In addition high-quality libraries for protein engineering can be easily designed.

Protein engineering of microbial enzymes

Protein engineering has emerged as an important tool to overcome the limitations of natural enzymes as biocatalysts. Recent advances have mainly focused on applying directed evolution to enzymes, especially important for organic synthesis, such as monooxygenases, ketoreductases, lipases or aldolases in order to improve their activity, enantioselectivity, and stability. The combination of directed evolution and rational protein design using computational tools is becoming increasingly important in order to explore enzyme sequence-space and to create improved or novel enzymes. These developments should allow to further expand the application of microbial enzymes in industry.

Fully automatized high‐throughput enzyme library screening using a robotic platform

A fully automatized robotic platform has been established to facilitate high‐throughput screening for protein engineering purposes. This platform enables proper monitoring and control of growth conditions in the microtiter plate format to ensure precise enzyme production for the interrogation of enzyme mutant libraries, protein stability tests and multiple assay screenings. The performance of this system has been exemplified for four enzyme classes important for biocatalysis such as Baeyer–Villiger monooxygenase, transaminase, dehalogenase and acylase in the high‐throughput screening of various mutant libraries. This allowed the identification of novel enzyme variants in a sophisticated and highly reliable manner. Furthermore, the detailed optimization protocols should enable other researchers to adapt and improve their methods. Biotechnol. Bioeng. 2016;113: 1421–1432.

Protein Engineering of Carboxyl Esterases by Rational Design and Directed Evolution

In the past few years a considerable number of mutagenesis methods and high-throughput screening (HTS) systems have been developed and improved. In parallel, computer programs or software packages for molecular modeling have been further investigated. Thus, the number of examples for successful directed evolution and rational design is increasing constantly. In this review the essential mutagenesis methods and HTS systems, especially for esterases, are described and various examples for the application of these protein engineering tools are provided.

Enantioselective kinetic resolution of phenylalkyl carboxylic acids using metagenome-derived esterases.

Enantiomerically pure β‐arylalkyl carboxylic acids are important synthetic intermediates for the preparation of a wide range of compounds with biological and pharmacological activities. A library of 83 enzymes isolated from the metagenome was searched for activity in the hydrolysis of ethyl esters of three racemic phenylalkyl carboxylic acids by a microtiter plate‐based screening using a pH‐indicator assay. Out of these, 20 enzymes were found to be active and were subjected to analytical scale biocatalysis in order to determine their enantioselectivity. The most enantioselective and also enantiocomplementary biocatalysts were then used for preparative scale reactions. Thus, both enantiomers of each of the three phenylalkyl carboxylic acids studied could be obtained in excellent optical purity and high yields.

Identification of pig liver esterase variants by tandem mass spectroscopy analysis and their characterization

Pig liver esterase (PLE) is probably the most important carboxyl esterase in organic synthesis and is commercially obtained by extraction of the animal tissue. However, problems occur in its application due to the presence of several isoenzymes (α-, β- and γ-PLE). The functional expression of the γ-isoenzyme was already shown and differences in the enantioselectivity compared to the commercial preparations were confirmed. The amino acid and nucleotide sequences of the α- and β-PLE are still unknown. In this work, putative sequences of the α-isoenzyme were identified from a commercial PLE preparation by 2D gel electrophoresis, digestion with proteases and analysis using Matrix-assisted laser desorption/ionization–time of flight (TOF) and electrospray ionisation quadrupole–TOF mass spectrometry. Based on these results, three amino acid exchanges were introduced into the gene encoding γ-rPLE by site-directed mutagenesis, and the proteins were expressed in E. coli Origami (DE3). The produced PLE mutants were characterised with respect to their substrate specificity and enantioselectivity. No significant differences in the activity towards methyl butyrate were found, but several variants showed substantially enhanced enantioselectivity in the resolution of (R,S)-1-phenyl-2-butyl acetate with E = 100 for the best mutant V236P/A237G.

Plasma-Modified Polypropylene as Carrier for the Immobilization of Candida antarctica Lipase B and Pyrobaculum calidifontis Esterase

In this work, the immobilization of two hydrolases on plasma‐modified polypropylene carriers was investigated. Treating Accurel MP1001 with an oxygen plasma was found most suitable to increase the hydrophilicity and to allow for efficient immobilization. Thus, for lipase B from Candida antarctica and for an esterase from Pyrobaculum calidifontis esterase (PestE) a 5‐fold and 14‐fold increase, respectively, in immobilization yield resulted compared to untreated carrier. In contrast to the oxygen‐modified support, modification of the polypropylene carrier with ammonia plasma showed no positive effect. Furthermore, it could be shown that immobilized PestE catalyzed enantioselective transesterification of α‐phenylethanol in vinyl acetate, whereas the free enzyme showed no activity. Both hydrolases could be recycled five times without significant loss of activity.

High-throughput screening of activity and enantioselectivity of esterases

A procedure for the high-throughput screening of esterases is described. This includes enzyme expression in microtiter plates and the measurement of activity and enantioselectivity (E) of the esterase variants using acetates of secondary alcohols as model substrates. Acetic acid released is converted in an enzyme cascade leading to the stoichiometric formation of NADH, which is quantified in a spectrophotometer. The method allows screening of several thousand mutants per day and has already been successfully applied to identify an esterase mutant with an E>100 toward an important building block for organic synthesis. This protocol can also be used for lipases and possibly other hydrolases that are expressed in soluble form in conventional Escherichia coli strains. This protocol can be completed in 3–4 days.

Insights into the physiological role of pig liver esterase: isoenzymes show differences in the demethylation of prenylated proteins.

The possible physiological role of PLE (E.C. 3.1.1.1) located in the endoplasmic reticulum (ER) of pig liver cells in the conversion of endogenous compounds was investigated as it was reported, that PLE acts as prenylated methylated protein methyl esterase (PMPMEase) hydrolysing methylesters of prenylated proteins. Using the specific PMPMEase substrate benzoyl-glycyl-farnesyl-cysteine methyl ester (BzGFCM), six different PLE isoenzymes expressed recombinantly in the yeast Pichia pastoris were found active. Activities ranged from 1.6-15.6mU per mg protein and it is suggested that Pro285 has a major influence on high activity. In addition, the role of the C-terminal HAEL retention signal for translocation of pig liver esterase (PLE) in the endoplasmic reticulum (ER) of eukaryotic cells was studied using the gamma-isoenzyme of PLE expressed in Pichia pastoris. Using truncated versions (HAE, HA, H and without retention signal) of the enzyme it was found that in contrast to earlier reports no influence of the signal peptide on the expression rate of PLE was found. However, higher enzyme activities were obtained in the periplasmatic fraction compared to the supernatant irrespective of the presence or absence of HAEL and the trimeric formation seems to occur in the supernatant of P. pastoris X33 enabling an easier transition of monomeric forms through cell membranes.