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Expanding the reaction scope of natural metalloenzymes can provide new opportunities for biocatalysis. Mononuclear non-heme iron-dependent enzymes represent a large class of biological catalysts involved in the biosynthesis of natural products and catabolism of xenobiotics, among other processes. Here, we report that several members of this enzyme family, including Rieske dioxygenases as well as α-ketoglutarate-dependent dioxygenases and halogenases, are able to catalyze the intramolecular C-H amination of a sulfonyl azide substrate, thereby exhibiting a promiscuous nitrene transfer reactivity. One of these enzymes, naphthalene dioxygenase (NDO), was further engineered resulting in several active site variants that function as C-H aminases. Furthermore, this enzyme could be applied to execute this non-native transformation on a gram scale in a bioreactor, thus demonstrating its potential for synthetic applications. These studies highlight the functional versatility of non-heme iron-dependent enzymes and pave the way to their further investigation and development as promising biocatalysts for non-native metal-catalyzed transformations.

Carboxylesterases (EC 3.1.1.1) and triacylglycerol lipases (EC 3.1.1.3) constitute a ubiquitous superfamily of enzymes that hydrolyze carboxylic ester bonds. They are characterized by their α/β-barrel structure and their enzymatic activity is defined by a catalytic triad that usually consists of a nucleophilic serine in a GXSXG pentapeptide motif and an acidic residue (aspartic acid or glutamic acid) that is hydrogen bonded to a histidine residue. Collectively, lipolytic enzymes can catalyze hydrolytic, esterification, transesterification, and amidation reactions over a broad range of substrates. Many of these enzymes have been isolated and characterized from extremophilic microorganisms, like those isolated from the genus Pyrococcus or Bacillus, comprising enzymes from bacteria and archaea domains. Additionally, many extremophilic enzymes have been characterized from mesophilic fungi and yeast, with some of them being commercially available such as the lipases from Candida antarctica or Rhizomucor miehei. These enzymes have been extensively used in industry, encompassing application in the manufacturing of drugs, foodstuffs, detergents, paper, and leather, clearly demonstrating their versatility for biotechnological applications.

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.

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.

Janibacter sp. strain R02 (BNM 560) was isolated in our laboratory from an Antarctic soil sample. A remarkable trait of the strain was its high lipolytic activity, detected in Rhodamine-olive oil supplemented plates. Supernatants of Janibacter sp. R02 displayed superb activity on transesterification of acyl glycerols, thus being a good candidate for lipase prospection. Considering the lack of information concerning lipases of the genus Janibacter, we focused on the identification, cloning, expression and characterization of the extracellular lipases of this strain. By means of sequence alignment and clustering of consensus nucleotide sequences, a DNA fragment of 1272bp was amplified, cloned and expressed in E. coli. The resulting recombinant enzyme, named LipJ2, showed preference for short to medium chain-length substrates, and displayed maximum activity at 80°C and pH 8-9, being strongly activated by a mixture of Na+ and K+. The enzyme presented an outstanding stability regarding both pH and temperature. Bioinformatics analysis of the amino acid sequence of LipJ2 revealed the presence of a consensus catalytic triad and a canonical pentapeptide. However, two additional rare motifs were found in LipJ2: an SXXL β-lactamase motif and two putative Y-type oxyanion holes (YAP). Although some of the previous features could allow assigning LipJ2 to the bacterial lipase families VIII or X, the phylogenetic analysis showed that LipJ2 clusters apart from other members of known lipase families, indicating that the newly isolated Janibacter esterase LipJ2 would be the first characterized member of a new family of bacterial lipases.

One of the major challenges in biocatalysis is the discovery or development of novel biocatalysts suitable for different applications. Within the last decades, directed evolution has emerged as a powerful tool for generating diversity and has allowed the development of biocatalysts with the desired properties. An alternative way to increase diversity among biocatalysts is to further explore nature on the search for novel activities or broader substrate specificity. Recent estimations indicate that we have been able to culture in the lab only 1% of the existing microorganisms, thus the strategy of searching for enzyme activities among existing microorganisms could still provide a manifold of useful biocatalysts. The endophytic community is a particularly interesting group of microorganisms since they have adapted themselves to live in an environment rich in biologically active compounds. Furthermore, several endophytes are able to produce secondary metabolites mimicking their host plant. It has been proposed that the endophytes have acquired these capabilities through years of co-evolution with their host plant. These characteristics indicate that endophytic microorganisms can be a rich source of novel enzymes; despite that, they have been scarcely explored in biocatalysis. This review attempts to cover all the precedents in the area of biotransformation and biocatalysis mediated by endophytic microorganisms.

The biotransformation of (4R)-(−)-carvone by Mentha pulegium (pennyroyal) leaves and its endophytic bacteria was performed in order to search for novel biocatalysts with enoate reductase activity. The obtained results clearly indicated that endophytes play an important role in the biotransformation of (4R)-(−)-carvone with pennyroyal plant tissues. The best activity was associated to the endophytic bacteria Pseudomonas proteolytica FM18Mci1 and Bacillus sp. FM18civ1. Enoate reductase activity for the reduction of (4R)-(−)-carvone and (4S)-(+)-carvone as model substrates was evaluated for each strain. Finally, both isolated strains were evaluated for the kinetic resolution of racemic carvone. The two bacteria gave (1R, 4R) or (1R, 4S)-dihydrocarvone as major products. P. proteolytica FM18Mci1 had preference for the 4S-(−)-carvone, reaching a conversion 95% in 24 h. In contrast, Bacillus sp. FM18civ1 had preference for (4R)-(−)-carvone. The results obtained in the kinetic resolution of carvone indicated that the Bacillus strain could be useful for resolving a racemic mixture of carvone.

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.