Alternative pig liver esterase (APLE) - cloning, identification and functional expression in Pichia pastoris of a versatile new biocatalyst.

Isolated from pig liver as a crude, inhomogeneous enzyme fraction, pig liver esterase (PLE) was found to metabolize a wide range of substrates; often in a highly stereoselective manner. This crude esterase preparation, however, contains several iso-enzymes at proportions varying from batch to batch. Racemic methyl-(4E)-5-chloro-2-isopropyl-4-pentenoate is cleaved enantioselectively by crude PLE, but not by recombinantly expressed gamma-isoform of PLE. Concluding that another PLE iso-enzyme must carry the relevant activity, we cloned and sequenced cDNAs of several PLE isoforms and functionally expressed them in Pichia pastoris. One novel isoform termed alternative pig liver esterase (APLE) was found to hydrolyze methyl-(2R,4E)-5-chloro-2-isopropyl-4-pentenoate in a highly stereoselective manner (E>200). When heterologously expressed and directed for secretion in P. pastoris, APLE was found to be localized in the periplasm. The presence or absence of a putative C-terminal ER retention signal did neither influence functional expression nor cellular localization. The recombinant enzyme, purified by ion exchange chromatography, had a specific activity of 36U (mg protein)(-1) towards racemic methyl-(4E)-5-chloro-2-isopropyl-4-pentenoate.

Potential and capabilities of hydroxynitrile lyases as biocatalysts in the chemical industry.

The application of hydroxynitrile lyases (HNLs) as catalysts for the stereoselective condensation of HCN with carbonyl compounds has been reported as early as 1908. This enzymatic C-C bond coupling reaction furnishes enantiopure cyanohydrins which serve as versatile bifunctional building blocks for chemical synthesis. Screening of natural sources led to the discovery of both (R)- and (S)-selective HNLs, and several distinctly different classes of these enzymes with substantial differences concerning sequence, structure, and mechanism have been found. Especially during the last two centuries, HNLs have been developed into valuable biocatalysts, which can be produced in recombinant form by overexpression in microbial hosts, resulting in the implementation of industrial processes utilizing these enzymes. Recently, protein engineering in combination with in silico methods gave rise to the development of a tailor-made HNL for large-scale manufacturing of a specific target cyanohydrin.

Counteracting expression deficiencies by anticipating posttranslational modification of PaHNL5-L1Q-A111G by genetic engineering.

(R)-2-chloromandelic acid represents a key pharmaceutical intermediate. Its production on large scale was hampered by low turnover rates and moderate enantiomeric excess (ee) using enzyme as well as metal catalysts. The cloning and heterologous overexpression of an (R)-hydroxynitrile lyase from Prunus amygdalus opened a way to large-scale production of this compound. Especially the rationally designed mutation of alanine to glycine at amino acid position 111 of the mature protein tremendously raised the yield for enantioselective conversion of 2-chlorobenzaldehyde to (R)-2-chloromandelonitrile, which can be hydrolysed to the corresponding alpha hydroxy acid. However, expression of this mutein was less efficient than for the unmodified enzyme. Subsequent LC/MS/MS-analysis of the protein sequence revealed that mutation A111G triggered the posttranslational deamidation of the neighbouring residue asparagine (N110) to aspartic acid. This finding on the one hand could explain the decreased secretion efficiency of the mutant as compared to the wildtype enzyme, but on the other hand raised the question which of the two residues was truly accountable for the enhanced conversion. The muteins N110D, A111G and N110DA111G were constructed and compared in terms of protein productivity and performance in chemical syntheses. The expression level of the double mutein was augmented significantly and the enantioselectivity remained high. Reduced protein expression of mutein PaHNL5-L1Q-A111G was remedied by mutational anticipation of posttranslational deamidation.

Stereoselective biocatalytic synthesis of (S)-2-hydroxy-2-methylbutyric acid via substrate engineering by using "thio-disguised" precursors and oxynitrilase catalysis.

3-Tetrahydrothiophenone (4) and 4-phenylthiobutan-2-one (7) were used as masked 2-butanone equivalents to give the corresponding cyanohydrins 5 (79 % yield, 91 % ee) and 8 (95 % yield, 96 % ee) in an enzymatic cyanohydrin reaction applying the hydroxynitrile lyase (HNL) from Hevea brasiliensis. After hydrolysis and desulphurisation the desired intermediate (S)-2-hydroxy-2-methylbutyric acid (10) was obtained with 99 % ee. Interestingly, when applying (R)-selective HNL from Prunus amygdalus again the (S)-cyanohydrin 5 was formed (62 % ee). The absolute configuration of 5 was verified by crystal structure determination of the corresponding hydrolysis derived carboxylate. The fact that both enzymes yield the same enantiomer was analysed and interpreted by molecular modelling calculations.

Structural studies of peri-interactions and bond formation between electron-rich atomic centres and N-phenylcarboxamides or nitroalkenyl groups.

Structural studies of peri-interactions with dimethylamino groups in naphthalene systems indicate that the N-phenylcarboxamide group has a through-space electron attracting power closer to that of a carboxylic ester than a N,N-dialkylcarboxamide, while 2-nitroalkenyl groups have a lower through-space electron attracting power. However, addition of a benzoyl group to the 2-position of the nitroethenyl group leads to cyclisation to give a zwitterion, in which the carbanion is stabilised by full conjugation with the nitro group and partial conjugation with the carbonyl group. An interesting case where a steric interaction overrides an electrophile/nucleophile attraction is also described. The limitations to the interpretation of short contact distances from crystallographic measurements are discussed.

Reliable high-throughput screening with Pichia pastoris by limiting yeast cell death phenomena.

Comparative screening of gene expression libraries employing the potent industrial host Pichia pastoris for improving recombinant eukaryotic enzymes by protein engineering was an unsolved task. We simplified the protocol for protein expression by P. pastoris and scaled it down to 0.5-ml cultures. Optimising standard growth conditions and procedures, programmed cell death and necrosis of P. pastoris in microscale cultures were diminished. Uniform cell growth in 96-deep-well plates now allows for high-throughput protein expression and screening for improved enzyme variants. Furthermore, the change from one host for protein engineering to another host for enzyme production becomes dispensable, and this accelerates the protein breeding cycles and makes predictions for large-scale production more accurate.

Innovative oxidation methods in fine chemicals synthesis.

Oxidation reactions are key steps of the synthetic pathway of chemicals, from the source, crude oil, to the ever more complicated products of the fine chemicals industry. Catalytic processes appear to be best suited to accommodate the new conditions of fine chemicals synthesis, i.e., short development time, increasing concerns about waste streams and economic pressure to use raw materials. Among various developments in oxidation chemistry, fine-tuning the reactivity of common oxidants, expanding the application of 'new' oxidants, new transformations by combination of known reagents, innovative application of oxidation reactions in multi-step synthesis, the use of new reaction media and new developments in bio-oxidations constitute the main research areas. This review could be defined as a 'spotlight' approach, pointing to significant findings in these different areas rather than giving an overview, and focusing on findings that show where progress could be made.