Combination of Oxyanion Gln114 Mutation and Medium Engineering to Influence the Enantioselectivity of Thermophilic Lipase from Geobacillus zalihae

The substitution of the oxyanion Q114 with Met and Leu was carried out to investigate the role of Q114 in imparting enantioselectivity on T1 lipase. The mutation improved enantioselectivity in Q114M over the wild-type, while enantioselectivity in Q114L was reduced. The enantioselectivity of the thermophilic lipases, T1, Q114L and Q114M correlated better with log p as compared to the dielectric constant and dipole moment of the solvents. Enzyme activity was good in solvents with log p < 3.5, with the exception of hexane which deviated substantially. Isooctane was found to be the best solvent for the esterification of (R,S)-ibuprofen with oleyl alcohol for lipases Q114M and Q114L, to afford E values of 53.7 and 12.2, respectively. Selectivity of T1 was highest in tetradecane with E value 49.2. Solvents with low log p reduced overall lipase activity and dimethyl sulfoxide (DMSO) completely inhibited the lipases. Ester conversions, however, were still low. Molecular sieves employed as desiccant were found to adversely affect catalysis in the lipase variants, particularly in Q114M. The higher desiccant loading also increased viscosity in the reaction and further reduced the efficiency of the lipase-catalyzed esterifications.     

Hidden Specificities in Enzyme Catalysis: Structural Basis of Substrate Structure-Selectivity Relationship of a Ketoreductase

Enzymes often convert both physiological and non-physiological substrates with high stereoselectivity; yet, for some enzymes, opposite product chirality is observed. A possible explanation is the existence of hidden specificities becoming apparent when non-physiological substrates confer different substrate–enzyme interactions than the physiological substrate. To test this hypothesis, a series of α-methylated β-keto esters were converted with Tyl-KR1, a ketoreductase from polyketide synthesis in Streptomyces fradiae. The conversions of six substrates with different physicochemical properties exhibited enantioselectivities ranging from 84 % ee for R,R to 84 % ee for S,S, yet high and uniform diastereoselectivity (anti, d.r.>9:1). The exchange of a single atom, namely an oxygen ester instead of a thioester, led to almost complete loss of enantioselectivity (<5 % ee). An additional S,S-selective binding mode as a hidden specificity in Tyl-KR1 has been identified through molecular modeling and site-directed mutagenesis.     

Characterization of a Self-Sufficient Cytochrome P450 Monooxygenase from Deinococcus apachensis for Enantioselective Benzylic Hydroxylation

A self-sufficient cytochrome P450 monooxygenase from Deinococcus apachensis (P450DA) was identified and successfully overexpressed in Escherichia coli BL21(DE3). P450DA would be a member of the CYP102D subfamily and assigned as CYP102D2 according to the phylogenetic tree and sequence alignment. Purification and characterization of the recombinant P450DA indicated both NADH and NADPH could be used by P450DA as a reducing cofactor. The recombinant E. coli (P450DA) strain was functionally active, showing excellent enantioselectivity for benzylic hydroxylation of methyl 2-phenylacetate. Further substrate scope studies revealed that P450DA is able to catalyze benzylic hydroxylation of a variety of compounds, affording the corresponding chiral benzylic alcohols in 86–99 % ee and 130–1020 total turnover numbers.     

Whole Cell Biotransformation of 5-(4-(2-(2-Pyridyl)methylamino)ethoxy)benzylidenethiazolidine-2,4-dione to its Benzyl Derivative Using a Yeast Reductase

Whole yeast cell reductive biotransformation of a benzylidene thiazolidinedione to its corresponding benzyl derivative has been scaled up from laboratory to small pilot plant scale. The fermentation conditions for the yeast Rhodotorula rubra were examined and cell titres increased by optimising the medium. Problems encountered in handling substrates of low water solubility are discussed. Various techniques are described for adapting the procedure for process scale operation using the general principles of process scale-up and integration. Liquid–liquid extraction using a dense solvent was performed on whole broth systems using a continuous flow liquid–liquid separator. Fatty acid impurities were removed using selective solvent extraction. © 1997 SCI.     

Determination of the Enantioselectivity for Kinetically Controlled Condensations Catalysed by Amidases and Peptidases

For kinetically controlled synthetic reactions catalysed by amidases and peptidases, the enantio‐ or stereoselectivity determined from initial rates with racemic mixtures (E_{syn, rac}) was found to differ from the enantioselectivity determined from measurements with isolated enantiomeric nucleophiles (E_syn). This was observed for kinetically controlled condensation of R‐phenyglycineamide with S‐, R‐ and racemic Phe and S‐, R‐ and racemic Leu catalysed by penicillin amidase from E. coli and for kinetically controlled condensation of N^α‐acetyl‐S‐tyrosine ethyl ester with S‐, R‐ and racemic AlaNH₂ catalysed by bovine α‐chymotrypsin. It is shown that only E_{syn, rac} determined with racemic nucleophiles is an intrinsic enzyme property which should be used to study the influence of the primary structure, physicochemical parameters and immobilisation on biocatalyst enantioselectivity in kinetically controlled synthetic reactions catalysed by these enzymes.     

Increased Enantioselectivity by Engineering Bottleneck Mutants in an Esterase from Pseudomonas fluorescens

Four hydrophobic and bulky amino acid residues (F126, F144, F159, and I225) were identified to form a bottleneck guarding the entrance to the active site of an esterase from Pseudomonas fluorescens (PFE I). Hence, a range of nonpolar amino acids were introduced into PFE I to broaden the substrate range and to increase enantioselectivity while preserving the hydrophobicity of the tunnel. First, single variants were created and then the most enantioselective ones were combined to find cooperative effects. This resulted in several mutants, which showed substantially enhanced enantioselectivity; for instance, in the kinetic resolution of 1‐phenyl‐1‐propyl acetate, with which the wild type only showed E=1.2, two mutants gave E>46. For 1‐phenyl‐1‐ethyl acetate enantioselectivity increased from ～50 to >100 for all mutants studied. Furthermore, higher conversions could be found at shorter reaction times; this indicates that the mutations not only enhanced selectivity, but that also the entrance into the active site was indeed facilitated by these mutations. The experimental results could be explained by computer modeling.     

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.     

Utilisation of C2–C4 gaseous hydrocarbons and isoprene by microorganisms

Microorganisms able to grow on low molecular weight aliphatic hydrocarbon gases, i.e. the n‐alkanes, ethane, propane and butane, and the terminal alkenes, ethylene, propylene and butylene, are not uncommon but mainly belong to certain taxonomic groups. These microbes are described in this review together with the pathways by which the hydrocarbons are assimilated. Microbial oxidation of the volatile alkadiene, isoprene, is also discussed. Avenues for possible commercial exploitation of these metabolic activities are also reviewed. Short‐chain n‐alkane‐utilising organisms have been investigated as tools in petroleum exploration and for production of single cell protein. More recently microbes grown on gaseous hydrocarbons other than methane have been evaluated for use in biotechnological production of epoxides, synthesis of chiral epoxyalkanes and as catalysts in bioremediation systems. Copyright © 2005 Society of Chemical Industry     

Chiral Phosphoric Acid‐Catalyzed Asymmetric Aza‐Friedel–Crafts Reaction of Indoles with Cyclic N‐Acylketimines: Enantioselective Synthesis of Trifluoromethyldihydroquinazolines

An enantioselective aza‐Friedel–Crafts (F‐C) reaction of indoles with cyclic N‐acylketimines has been developed. By using chiral phosphoric acid catalysts, a wide range of enantioenriched trifluoromethyldihydroquinazolines was obtained in excellent yields (up to 98%) with good to high enantioselectivities (up to 99% ee).