Synthesis of Chiral Acyclic Pyrimidine Nucleoside Analogues from DHAP-Dependent Aldolases.

Dihydroxyacetone phosphate (DHAP)-dependent aldolases catalyze the aldol addition of DHAP to a variety of aldehydes and generate compounds with two stereocenters. This reaction is useful to synthesize chiral acyclic nucleosides, which constitute a well-known class of antiviral drugs currently used. In such compounds, the chirality of the aliphatic chain, which mimics the open pentose residue, is crucial for activity. In this work, three DHAP-dependent aldolases: fructose-1,6-biphosphate aldolase from rabbit muscle, rhanmulose-1-phosphate aldolase from Thermotoga maritima, and fuculose-1-phosphate aldolase from Escherichia coli, were used as biocatalysts. Aldehyde derivatives of thymine and cytosine were used as acceptor substrates, generating new acyclic nucleoside analogues containing two new stereocenters with conversion yields between 70% and 90%. Moreover, structural analyses by molecular docking were carried out to gain insights into the diasteromeric excess observed.

Novel fungal organophosphorus hydrolases in acidic media: an application to apples decontamination.

Organophosphorus pesticides bring significant improvements in agriculture, but their toxicity causes environmental and health negative impacts. The aim of this work was the development of robust biocatalysts to be applied in bioremediation. Four fungi were evaluated as hydrolase sources capable of degrading organophosphorus pesticides: Aspergillus niger, Fusarium sp., Penicillium chrysogenum, and Penicillium nalgiovense. The hydrolysis rates of methyl paraoxon obtained under acidic conditions were in the range of 10 to 21 mg L-1 d-1, which is remarkable since most similar biocatalysts are active under alkaline conditions. Penicillium chrysogenum activity was outstanding, and it was selected to prepare, characterize, and study the applications of its enzymatic extract. It was used to evaluate the bioremediation of apple surfaces at pH 2 in the presence of SDS, achieving complete methyl paraoxon degradation under proposed conditions. These results indicate that this biocatalyst could complement industrialized fruit washing processes for the elimination of organophosphorus pesticides.

Synthetic Activity of Recombinant Whole Cell Biocatalysts Containing 2-Deoxy-D-ribose-5-phosphate Aldolase from Pectobacterium atrosepticum.

In nature 2-deoxy-D-ribose-5-phosphate aldolase (DERA) catalyses the reversible formation of 2-deoxyribose 5-phosphate from D-glyceraldehyde 3-phosphate and acetaldehyde. In addition, this enzyme can use acetaldehyde as the sole substrate, resulting in a tandem aldol reaction, yielding 2,4,6-trideoxy-D-erythro-hexapyranose, which spontaneously cyclizes. This reaction is very useful for the synthesis of the side chain of statin-type drugs used to decrease cholesterol levels in blood. One of the main challenges in the use of DERA in industrial processes, where high substrate loads are needed to achieve the desired productivity, is its inactivation by high acetaldehyde concentration. In this work, the utility of different variants of Pectobacterium atrosepticum DERA (PaDERA) as whole cell biocatalysts to synthesize 2-deoxyribose 5-phosphate and 2,4,6-trideoxy-D-erythro-hexapyranose was analysed. Under optimized conditions, E. coli BL21 (PaDERA C-His AA C49M) whole cells yields 99 % of both products. Furthermore, this enzyme is able to tolerate 500 mM acetaldehyde in a whole-cell experiment which makes it suitable for industrial applications.

Citrobacter koseri immobilized on agarose beads for nucleoside synthesis: a potential biocatalyst for preparative applications.

The biocatalyzed synthesis of purine nucleosides and their analogs is a case widely studied due to the high pharmaceutical interest of these compounds, providing the whole-cell biocatalysts, a useful tool for this purpose. Vidarabine and fludarabine are commercial examples of expensive bioactive nucleosides that can be prepared using a microbial transglycosylation approach. Citrobacter koseri whole-cells immobilized on agarose beads proved to be an interesting option to transform this biotransformation in a preparative process. The entrapment matrix provided a useful and resistant multipurpose biocatalyst regarding its stability, mechanical strength, microbial viability and reuse. Immobilized biocatalyst retained the initial activity for up to 1 year storage and after 10 years, the biocatalyst did not show cell leaking and still exhibited residual activity. In addition, the biocatalyst could be reused in batch 68 times keeping up to 50% of the initial biocatalytic activity and for at least 124 h in a continuous process.

Alternative low-cost process for large-scale production of Bacillus thuringiensis in a simple and novel culture system.

An industrial-scale, profitable method for production of the most widely used bioinsecticide, Bacillus thuringiensis (Bt), is challenging because of its widespread application. The aim of this study is to present a strategy to develop a low-cost, large-scale bioprocess to produce Bt H14. This study was first focused on the design of a culture medium composed of economical and available components, such as glycerol and lysed Saccharomyces cerevisiae. The production goal of 1200 ITU was achieved using a medium composed of 20:20 g L-1of glycerol:lysed yeast in batch cultures. Efforts were subsequently focused on the design of an appropriate culture system, and an original two-stage culture system was proposed. First, yeast (the primary component of the culture medium) are cultivated using a minimal mineral medium and lysed, and in the second stage, Bt is cultivated in the same bioreactor using the lysed yeasts as culture medium (supplemented with a feeding pulse of 10 g L-1 glycerol). This system was called fed batch one pot (FOP). A new inoculation strategy is also presented in this study, since these Bt cultures were inoculated directly with heat pre-treated spores instead of vegetative bacteria to facilitate the bioprocess. This study was developed from the laboratory to production-scale bioreactors (measuring from 500 mL to 2500 L), and the efficiency of the proposed strategy was evident in LD50 tests results, achieving 1796 ITU in large-scale processes. Both the use of non-conventional sources and the process development for biomass production are important for cost-effective production of Bt-based insecticides in mosquito control projects.

New and highly active microbial phosphotriesterase sources.

Many toxic insecticides used worldwide as well as some chemical warfare agents are phosphotriester derivatives. Therefore, detoxification of organophosphorus compounds has become the subject of many studies and in particular bioremediation, based on the phosphotriesterase catalysed hydrolysis of these compounds, has shown to be an effective and ecological methodology. In order to identify new bacterial phosphotriesterases, a simple and sensitive fluorimetric screening method on solid media was employed that allowed the selection of six strains with phosphotriesterase activity. Since pH and temperature are important parameters for bioremediation of contaminated soils and waters, the influence of these variables on the rate of the enzymatic hydrolysis was assessed. This study afforded notable results, being the most remarkable one the increased activity exhibited by Nocardia asteroides and Streptomyces setonii strains at 50°C, 7 and 30 times higher than at 30°C, respectively. Compared with the results obtained with Brevundimonas diminuta, whose activity is usually considered as reference, an increase of 26 and 75 times is observed, respectively.

Self-sufficient redox biotransformation of lignin-related benzoic acids with Aspergillus flavus.

Aromatic carboxylic acids are readily obtained from lignin in biomass processing facilities. However, efficient technologies for lignin valorization are missing. In this work, a microbial screening was conducted to find versatile biocatalysts capable of transforming several benzoic acids structurally related to lignin, employing vanillic acid as model substrate. The wild-type Aspergillus flavus growing cells exhibited exquisite selectivity towards the oxidative decarboxylation product, 2-methoxybenzene-1,4-diol. Interestingly, when assaying a set of structurally related substrates, the biocatalyst displayed the oxidative removal of the carboxyl moiety or its reduction to the primary alcohol whether electron withdrawing or donating groups were present in the aromatic ring, respectively. Additionally, A. flavus proved to be highly tolerant to vanillic acid increasing concentrations (up to 8 g/L), demonstrating its potential application in chemical synthesis. A. flavus growing cells were found to be efficient biotechnological tools to perform self-sufficient, structure-dependent redox reactions. To the best of our knowledge, this is the first report of a biocatalyst exhibiting opposite redox transformations of the carboxylic acid moiety in benzoic acid derivatives, namely oxidative decarboxylation and carboxyl reduction, in a structure-dependent fashion.

Biocatalytic approaches applied to the synthesis of nucleoside prodrugs.

Nucleosides are valuable bioactive molecules, which display antiviral and antitumour activities. Diverse types of prodrugs are designed to enhance their therapeutic efficacy, however this strategy faces the troublesome selectivity issues of nucleoside chemistry. In this context, the aim of this review is to give an overview of the opportunities provided by biocatalytic procedures in the preparation of nucleoside prodrugs. The potential of biocatalysis in this research area will be presented through examples covering the different types of nucleoside prodrugs: nucleoside analogues as prodrugs, nucleoside lipophilic prodrugs and nucleoside hydrophilic prodrugs.

A comparative study on phosphotransferase activity of acid phosphatases from Raoultella planticola and Enterobacter aerogenes on nucleosides, sugars, and related compounds.

Natural and modified nucleoside-5'-monophosphates and their precursors are valuable compounds widely used in biochemical studies. Bacterial nonspecific acid phosphatases (NSAPs) are a group of enzymes involved in the hydrolysis of phosphoester bonds, and some of them exhibit phosphotransferase activity. NSAP containing Enterobacter aerogenes and Raoultella planticola whole cells were evaluated in the phosphorylation of a wide range of nucleosides and nucleoside precursors using pyrophosphate as phosphate donor. To increase the productivity of the process, we developed two genetically modified strains of Escherichia coli which overexpressed NSAPs of E. aerogenes and R. planticola. These new recombinant microorganisms (E. coli BL21 pET22b-phoEa and E. coli BL21 pET22b-phoRp) showed higher activity than the corresponding wild-type strains. Reductions in the reaction times from 21 h to 60 min, from 4 h to 15 min, and from 24 h to 40 min in cases of dihydroxyacetone, inosine, and fludarabine, respectively, were obtained.

Selection of a new whole cell biocatalyst for the synthesis of 2-deoxyribose 5-phosphate.

2-deoxyribose 5-phosphate (DR5P) is a key intermediate in the biocatalyzed preparation of deoxyribonucleosides. Therefore, DR5P production by means of simpler, cleaner, and economic pathways becomes highly interesting. One strategy involves the use of bacterial whole cells containing DR5P aldolase as biocatalyst for the aldol addition between acetaldehyde and D: -glyceraldehyde 3-phosphate or glycolytic intermediates that in situ generate the acceptor substrate. In this work, diverse microorganisms capable of synthesizing DR5P were selected by screening several bacteria genera. In particular, Erwinia carotovora ATCC 33260 was identified as a new biocatalyst that afforded 14.1-mM DR5P starting from a cheap raw material like glucose.

Biocatalysed halogenation of nucleobase analogues.

The synthesis of halogenated nucleosides and nucleobases is of interest due to their chemical and pharmacological applications. Herein, the enzymatic halogenation of nucleobases and analogues catalysed by microorganisms and by chloroperoxidase from Caldariomyces fumago has been studied. This latter enzyme catalysed the chlorination and bromination of indoline and uracil. Pseudomonas, Citrobacter, Aeromonas, Streptomyces, Xanthomonas, and Bacillus genera catalysed the chlorination and/or bromination of indole and indoline. Different products were obtained depending on the substrate, the biocatalyst and the halide used. In particular, 85% conversion from indole to 5-bromoindole was achieved using Streptomyces cetonii.

Synthesis of 9-beta-d-arabinofuranosylguanine by combined use of two whole cell biocatalysts.

Unlike the preparation of other purine nucleosides, transglycosylation from a pyrimidine nucleoside and guanine is difficult because of the low solubility of this base. Thus, another strategy, based on the coupled action of two whole cell biocatalyzed reactions, transglycosylation and deamination, was used. Enterobacter gergoviae and Arthrobacter oxydans were employed to synthesize 9-beta-d-arabinofuranosylguanine (AraG), an efficient anti leukemic drug.

Arthrobacter oxydans as a biocatalyst for purine deamination.

Deaminases are enzymes that catalyze the hydrolysis of amino groups of nucleosides or their bases. Because these enzymes play important roles in nucleotide metabolism, they are relevant targets in anticancer and antibacterial therapies. Mammalian deaminases are commercially available but the use of bacterial whole cells, especially as biocatalysts, is continuously growing because of their economical benefits. Moreover, deaminases are useful for the preparative chemoenzymatic transformation of nucleoside and base analogues into a variety of derivatives. The purine deaminase activities of Arthrobacter oxydans, a gram-positive bacterium utilized widely in bioremediation, were studied. The presence of adenosine, adenine and guanine deaminases was demonstrated and some purine bases and nucleosides were analyzed as substrates. Using A. oxydans whole cells as the biocatalyst, different purine compounds such as the anti-HIV, 2',3'-dideoxyinosine (73%, 2 h) were obtained.

Coupled biocatalysts applied to the synthesis of nucleosides.

Biocatalytic procedures offer a good alternative to the chemical synthesis of nucleosides since biocatalyzed reactions are regio- and stereoselective and afford reduced by-products contents. Among them, enzymatic transglycosylation between a pyrimidine nucleoside and a purine base catalyzed by nucleoside phosphorylases or microorganisms that contain them, has attracted considerable attention. In addition, the combination to other enzymatic steps has been explored. In this work we investigate the coupled action of nucleoside phosphorylases with other enzymatic activities: deaminase and phosphopentomutase. Unlike the preparation of other purine nucleosides, transglycosylation from a pyrimidine nucleoside and guanine is difficult because of the low solubility of this base. Therefore, another strategy, based on microbial transglycosylation followed by deamination, is here explored. The direct use of furanose 1-phosphate, the intermediate in the transglycosylation reaction, is an attractive alternative when pyrimidine nucleosides are not available. Its preparation from the more stable furanose 5-phosphate and phosphopentomutase is here applied to different sugars and bases.

Microbial hydrolysis of acetylated nucleosides.

Enzymatic hydrolysis of acetylated nucleosides using microbial whole cells has been carried out for the first time. Unlike Candida antarctica B lipase-catalysed alcoholysis, none of the tested microorganisms displayed a common deacetylation profile. Depending on the substrate and the biocatalyst used, 5'-selective deprotection or mixtures of mono O-acetylated products were obtained.

Immobilized Escherichia coli BL21 as a catalyst for the synthesis of adenine and hypoxanthine nucleosides.

Different supports, such as alginate, agar, agarose, and polyacrylamide, were used to immobilize Escherichia coli BL 21 by entrapment techniques. The transglycosylation reaction involved in the synthesis of adenosine from uridine and adenine was chosen as a model system to study the characteristics of these biocatalysts. Whole cells immobilized on agarose proved to be optimal and could be used up to 30 times without significant loss of activity. This biocatalyst was further employed to test its ability in the synthesis of other adenine and hypoxanthine nucleosides. Ribo-, 2'-deoxyribo-, and arabinonucleosides could be prepared in high yields starting from the corresponding pyrimidine nucleosides and purine bases. Similar product yields were obtained with both free and immobilized cells, though, in the latter case, a longer reaction time was necessary.