No abstract

Aims: Identification, cloning, expression and characterization of a novel lipase -Lip I.3 -from strain Pseudomonas CR-611. Methods and Results: The corresponding gene was identified and isolated by PCR-amplification, cloned and expressed in Escherichia coli, and purified by refolding from inclusion bodies. Analysis of the deduced amino acid sequence revealed high homology with members of the bacterial lipase family I.3, showing 97% identity to a putative lipase from Pseudomonas fluorescens Pf0-1, and 93% identity to a crystallized extracellular lipase from Pseudomonas sp. MIS38. A typical C-terminal type I secretion signal and several putative Ca

Chiral amines are essential precursors in the production of biologically active compounds, including several important drugs. Among the biocatalytic strategies that have been developed for their synthesis, the use of ω-transaminases (ω-TA) appears as an attractive alternative allowing the stereoselective amination of prochiral ketones. However, the problems associated with narrow substrate specificity, unfavourable reaction equilibrium and expensive amine donors still hamper its industrial application. The search for novel enzymes from nature can contribute to expand the catalytic repertoire of ω-TA and help to circumvent some of these problems. A genome mining approach, based on the work described by Höhne et al., was applied for selection of potential R-ω-TA. Additional criteria were used to select an enzyme that differs from previously described ones. A candidate R-ω-TA from Capronia semiimmersa was selected, cloned and expressed in Escherichia coli. Interestingly, alignment of this enzyme with previously reported TA sequences revealed the presence of two additional amino acid residues in a loop close to the active site. The impact of this change was analysed with a structural model based on crystallized R-ω-TAs. Analysis of the substrate specificity of R-ω-TA from C. semiimmersa indicates that it accepts a diversity of ketones as substrates yielding the corresponding amine with good yields and excellent enantioselectivity. The expressed enzyme accepts isopropylamine as amine donor what makes it suitable for industrial processes.

Several Pseudomonas sp. CR611 Lip I.3 mutants with overall increased activity and a shift towards longer chain substrates were constructed. Substitution of residues Y29 and W310 by smaller amino acids provided increased activity on C18-substrates. Residues G152 and S154, modified to study their influence on interfacial activation, displayed a five and eleven fold increased activity.

No abstract

The development of biocatalytic tools for the synthesis of optically pure amines has been the focus of abundant research in recent years. Among other enzymes, imine reductases have attracted much attention associated with the possibility of attaining chiral secondary amines. Furthermore, the reductive aminase activity associated with some of these enzymes has facilitated the production of optically pure amines from a prochiral ketone, a transformation that opens doors to an incredible array of products. In this work, the genomes from native Streptomyces strains isolated in our lab have been explored on the search for novel imine reductases. Application of different structural criteria and sequence motif filters allowed the identification of two novel enzymes, Ss-IRED_S and Ss-IRED_R. While the former presented outstanding activity towards bulky cyclic imine substrates, the latter presented reductive aminase activity with the assayed ketones. A bioinformatic analysis based on modeling and docking studies was performed in order to explain the differences in enzyme activity, searching for additional criteria that could be used to analyze enzyme candidates in silico, providing additional tools for enzyme selection for a particular application. Our findings suggest that imine reductase activity could be predicted by this analysis, overall accounting for the number of docking positions that meet the catalytic requirements.

A recombinant Escherichia coli expressing an a-alkyl-b-ketoester reductase that yields the anti (2R ,3R ) a-alkyl-bhydroxyester as the major product was constructed. The enzyme was identified from genomic libraries of Paucimonas lemoignei, cloned and expressed in E. coli providing a whole cell biocatalyst with novel stereoselectivity. This constitutes the first report on a whole cell biocatalyst for the synthesis of (2R ,3R ) a-alkyl-b-hydroxy-esters.