Alejandro A. Orden

Atypical regioselective biohydrolysis on steroidal oxiranes by Aspergillus niger whole cells : Some stereochemical features

5,6-Epoxycholestan-3beta-ol derivatives were hydrolyzed in a diastereoconvergent manner by growing and resting cells of several strains of Aspergillus niger, particularly A. niger ATCC 11394. These strains displayed opposite regioselectivity toward each isomer in an alpha and beta epoxide mixture, thus, the nucleophilic attack took place at the less substituted and the most substituted carbon atom on each diasteromer, respectively. These biocatalysts opened trisubstituted oxiranes but were unable to hydrolyze the disubstituted oxiranes in the tested sterol derivatives. These findings suggest that A. niger strains possess another hydrolytic ability different from the commercial A. niger epoxide hydrolase (EH) that did not accept this kind of steroidal oxiranes as substrates.

Deracemization of secondary alcohols by chemo-enzymatic sequence with plant cells.

A screening based on undifferentiated plant cells allowed identifying Gardenia jasminoides as the best biocatalyst to perform the kinetic resolution of 1-phenylethanol. This species was further tested for its ability to oxidize stereoselectively the (S)-isomers from racemic mixtures of secondary alcohols leaving their antipodes unaffected in Tris-HCl buffer. Those substrates which afforded the best results in the kinetic resolution were subjected to a chemo-enzymatic sequence of deracemization. G. jasminoides immobilized cells in calcium alginate were used for the oxidation of the (S)-enantiomers and, in a second step, NaBH(4) was added to the same vessel for the reduction of the corresponding ketone. The sequential repetition of these two steps allowed obtaining the R-alcohols in 82-90% yield in high optical purity (71-96% ee). Despite the viability of the cells is affected by the chemical reagent, their enzymes remain active due to the protective environment of the calcium alginate beads.

Effects of elicitor and copper sulfate on grindelic acid production in submerged cultures of Grindelia pulchella

Grindelia pulchella callus and cell suspension cultures were established from seedling leaves. When several phytoregulator supplementations were assayed in solid Murashige and Skoog medium containing 3% (w/v) of sucrose (MS medium), combinations of indole-3-butyric acid (IBA) and N 6 -benzylaminopurine (BA) resulted the most appropriate conditions to generate fast growing friable calli with detectable levels of grindelic acid. Moreover, the same basal media supplemented with 20.0 µM IBA/4.4 µM BA was found to be optimal for cell growth in submerged cultures (µ max = 0.26 days -1 ) while the addition of 20.0 µM IBA/18.0 µM BA resulted in a relative higher metabolite production (4.55 mg/gDW) when the inocula was 5% (v/v). Furthermore, three different stress factors and combinations of them were used to elicit cell suspensions. These experiments demonstrated that the combination of CuSO 4 and dimethylsulfoxide (DMSO) increase the grindelic acid production to 2.63 mg/gDW in the elicited essay versus 0.756 mg/gDW in the control, at expense of cell growth. In contrast, the addition of jasmonic acid (JA) alone and combined with DMSO neither affected cell growth nor grindelic acid accumulation.

Stereo- and regioselective hydroxylation of grindelic acid derivatives by Aspergillus niger

Stereo- and regioselective hydroxylation of grindelane derivatives on position 3β was catalyzed by cultures of Aspergillus niger. Grindelic acid (1), methyl grindelate (2), 15-hydroxy-7(8)-en-9α,13(S)-oxide-ent-labdane (3) and 7α,8α-epoxymethylgrindelate (4) were bioconverted into the corresponding 3β-hydroxy derivatives as the only biotransformation products. 13(S),15-dihydroxy-8(9)-en-ent-labdane (5) remained unreacted under the same conditions. The conformational and electronic studies of the substrates are discussed.

A study of Raphanus sativus and its endophytes as carbonyl group bioreducing agents

Abstract The reported anti-Prelog selectivity in Raphanus sativus hairy roots prompted us to search for similar activity in other R. sativus systems, such as in vitro germinated sprouts, edible roots, and its associated endophytic microorganisms. Two test substrates were used in these studies: acetophenone and rac-ethyl 2-ethyl-3-oxobutanoate. Endophytes were isolated under substrate selective pressure and individual strains were evaluated for their ability to reduce both substrates. Among the isolates, two bacteria and a fungus having the potential to be used as biocatalysts were found: Bacillus megaterium which reduces acetophenone with excellent anti-Prelog selectivity, and Pseudomonas sp. and Penicillium chrysogenum with enantioselectivity and stereoselectivity for the reduction of rac-ethyl 2-ethyl-3-oxobutanoate, respectively.

Treatment of endosulfan contaminated water with in vitro plant cell cultures

Endosulfan is a Persistent Organic Pollutant insecticide still used in many countries. It is commercially available as mixtures of two diastereomers, α- and β-endosulfan, known as technical grade endosulfan (TGE). A laboratory model based on the use of axenic plant cell cultures to study the removal and metabolization of both isomers from contaminated water matrixes was established. No differences were recorded in the removal of the two individual isomers with the two tested endemic plants, Grindelia pulchella and Tessaria absinthioides. Undifferentiated cultures of both plant species were very efficient to lower endosulfan concentration in spiked solutions. Metabolic fate of TGE was evaluated by analyzing the time course of endosulfan metabolites accumulation in both plant biomass and bioremediation media. While in G. pulchella we only detected endosulfan sulfate, in T. absinthioides the non-toxic endosulfan alcohol was the main metabolite at 48h, giving the possibility of designing phytoremediation approaches.

Plant tissue cultures as sources of new ene- and ketoreductase activities

While many redox enzymes are nowadays available for synthetic applications, the toolbox of ene-reductases is still limited. Consequently, the screening for these enzymes from diverse sources in the search of new biocatalyst suitable for green chemistry approaches is needed. Among 13 plant tissue cultures, Medicago sativa and Tessaria absinthioides calli, as well as Capsicum annuum hairy roots, were selected due to their ability to hydrogenate the CC double bond of the model substrate 2-cyclohexene-1-one. The three axenic plant cultures showed more preference toward highly activated molecules such as nitrostyrene and maleimide rather than the classical substrates of the well-known Old Yellow Enzymes, resembling the skills of the NAD(P)H-dependent flavin-independent enzymes. When the three biocatalytic systems were applied in the reduction of chalcones, T. absinthioides showed high chemoselectivity toward the CC double bond whereas the other two demonstrated abilities to biohydrogenate the CC double bounds and the carbonyl groups in a sequential fashion.