Raffaella Gandolfi

Steroid hydroxylations with Botryodiplodia malorum and Colletotrichum lini

An improved procedure for the microbial hydroxylations of dehydroepiandrosterone (DHEA, 1) and 15 beta,16 beta-methylene-dehydroepiandrosterone (2) was studied using whole cells of Botryodiplodia malorum and Colletotrichum lini. C. lini catalyzed 7 alpha- and 15 alpha-hydroxylation of 1 and 7 alpha-hydroxylation of 2, while B. malorum gave 7 beta-hydroxylation of both the substrates. The stability of the enzymatic activity was higher in the presence of co-substrates (i.e., glucose or mannitol) allowing for repeated batches of the biotransformations. The yields of 7 alpha,15 alpha-dihydroxy-1 production were improved obtaining 5.8 gl(-1) (recovered product) from 7.0 gl(-1) of substrate. The structures of the hydroxylated products were assigned by a combination of two-dimensional NMR proton-proton and proton-carbon correlation techniques.

An easy and efficient method for the production of carboxylic acids and aldehydes by microbial oxidation of primary alcohols

Abstract An easy and efficient methodology for obtaining aldehydes or carboxylic acids by oxidation of the corresponding primary alcohols with acetic acid bacteria is reported. When the biotransformation is performed in water the acids are obtained; aldehydes can be accumulated by using a water/isooctane two-liquid phase system.

Acetic acid bacteria as enantioselective biocatalysts

Acetic acid bacteria (five strains of Acetobacter and five strains of Gluconobacter) were used for the biotransformation of different primary alcohols (2-chloropropanol and 2-phenylpropanol) and diols (1,3-butandiol, 1,4-nonandiol and 2,3-butandiol). Most of the tested strains efficiently oxidized the substrates. 2-Chloropropanol and 1,3-butandiol were oxidized with good rates and low enantioselectivity (enantiomeric excess = 18-46% of the S-acid), while microbial oxidation of 2-phenylpropanol furnished (S)-2-phenyl-1-propionic acid with enantiomeric excess (e.e.) >90% with 10 strains. The dehydrogenation of 2,3-butandiol was strongly dependent on the stereochemistry of the substrate; the meso form gave S-acetoin with all the tested strains, the only exception being a Gluconobacter strain. The formation of diacetyl was observed only by using R,R-2,3-butandiol with Acetobacter strains. Oxidation of 1,4-nonandiol gave γ-nonanoic lactone in one step, although with moderate enantioselectivity. 2002 Elsevier Science B.V. All rights reserved.

Microbial bioreductions of γ- and δ-ketoacids and their esters

Abstract A series of yeasts were used in the bioreductions of aliphatic and aromatic γ- and δ-ketoacids and esters to investigate the preparation of enantiomerically pure γ- and δ-lactones through the intermediacy of their corresponding γ- and δ-hydroxyacids and esters. Bioreduction of ethyl 4-oxononanoate 2a with Pichia etchellsii afforded the γ-nonanolide (+)- 5a with 99% e.e., while Pichia minuta proved to be the best choice for the bioreduction of ethyl 2-oxocyclohexylacetate 2e , which afforded cis -(−)- 5e and trans -(−)- 5e with 98 and 99% e.e., respectively. Reduction of 3-(2-oxocyclohexyl)propionic acid 3e with Pichia glucozyma gave predominantly the corresponding δ-lactone trans -(−)- 6e with 94% e.e., whose absolute configuration was determined by means of CD spectroscopy.

Chemoselective oxidation of primary alcohols to aldehydes with Gluconobacter oxydans

Abstract The production of aliphatic and aromatic aldehydes by oxidation of primary alcohols was achieved with Gluconobacter oxydans DSM 2343. The biotransformation was optimised studying the oxidation of 2-phenyl-1-ethanol to 2-phenylacetaldehyde. A high molar conversion (95% chromatographic conversion, 83% of isolated yield) was obtained using cells grown on glycerol as the main carbon source and directly used in the cultural medium after 24 h at 28°C, pH 4.5 and 5 g L −1 substrate concentration. The conversion of structurally different primary alcohols was performed under these conditions allowing the chemoselective production of aldehydes, sometimes with very good yields.

Biotransformations in two-liquid-phase systems: Production of phenylacetaldehyde by oxidation of 2-phenylethanol with acetic acid bacteria

Abstract Phenylacetaldehyde can be obtained by oxidation of 2-phenylethanol with acetic acid bacteria in two-liquid-phase systems where the aldehyde is removed into the organic phase before its further conversion to acid. Two Acetobacter strains (ALEF and ALEG) were able to accumulate aldehyde when aliphatic hydrocarbons were used. A two-liquid-phase system, composed of water and isooctane (v/v, 1/1), was particularly suited for a significant accumulation of the aldehyde: Acetobacter sp. ALEG furnished 9 g/l of phenylacetaldehyde within 4 h starting from 10 g/l of alcohol and still 8 g/l were recovered after 24 h in the organic phase, whereas strain ALEF gave 3.5 g/l of aldehyde from 5.0 g/l of substrate. Acetobacter sp. ALEG also showed satisfactory long-term stability, being able to perform the transformation with 80% of the original activity after 3 days of contact with the solvent.

Mycelium-bound carboxylesterase from Aspergillus oryzae: an efficient catalyst for acetylation in organic solvent

Dry mycelium of a strain of Aspergillus oryzae efficiently catalyzed the esterification between free acetic acid and primary alcohols (geraniol and ethanol) in organic solvent. The growth conditions to obtain high activity of mycelium-bound enzymes were firstly evaluated. A medium containing Tween 80 as carbon source furnished mycelium with the highest activity in the hydrolysis of alpha-naphthyl esters (alpha-N-acetate, butyrate, caprylate). Dry mycelium was employed to select suited conditions for an efficient acetylation of ethanol and geraniol in heptane. Maximum productions were obtained using 30 g l(-)(1) of lyophilized cells: 12.4 g l(-)(1) of geranyl acetate were produced at 80 degrees C starting from 75 mM geraniol and acetic acid (84% molar conversion) and 4.1 g l(-)(1) of ethyl acetate at 50 degrees C from 50 mM ethanol and acetic acid (94% molar conversion) after 24 h. The stability of the mycelium-bound carboxylesterases are notable since only 10-30% loss of activity was observed after 14 days at temperatures between 30 and 50 degrees C.

Enantioselective oxidation of prochiral 2-methyl-1,3-propandiol by Acetobacter pasteurianus

The microbial oxidation of prochiral 2-methyl-1,3-propandiol into (R)-3-hydroxy-2-methyl propionic acid with Acetobacter pasteurianus DSM 8937 is reported. The biotransformation was optimised furnishing (R)-3-hydroxy-2-methyl propionic acid with 97% enantiomeric excess and 100% molar conversion of 5 g/L within 2 h. A simple fed-batch procedure allowed for the obtainment of 25 g/L of the enantiomerically enriched acid. (R)-3-Hydroxy-2-methyl propionic acid is an important building block for the synthesis of Captopril, a widely used antihypertensive drug.

Lyophilised yeasts: easy-to-handle biocatalysts for stereoselective reduction of ketones

Abstract The use of lyophilised yeasts as biocatalysts for the reduction of carbonyl compounds has been studied. First, a comparison of the performances of fresh and lyophilised cells of seven yeasts was performed using ethyl acetoacetate and acetophenone as typical substrates. Lyophilised cells gave from low to high conversions but, with a few exceptions, always good enantioselectivity, and they were then employed for the reduction of structurally different carbonyl compounds. Highly enantioselective reduction of carbonyls was often achieved, even with aromatic ketones. Both enantiomers of the alcohols were, in most cases, obtained with high enantiomeric excess by simply choosing a suitable yeast.

Cell-bound and extracellular carboxylesterases from Streptomyces: hydrolytic and synthetic activities

Aim: The distribution of cell‐bound and extracellular carboxylesterases was investigated among the genus Streptomyces using 420 strains.