Fabrizio Aragozzini

Isolation of a novel carboxylesterase from Bacillus coagulans with high enantioselectivity toward racemic esters of 1,2-O-isopropylideneglycerol

An intracellular carboxylesterase from Bacillus coagulans NCIMB 9365 with high stereospecific hydrolytic activity toward racemic esters of 1,2-O-isopropylideneglycerol was purified and characterized. The microorganism contained two intracellular carboxylesterases designated as carboxylesterase A and B. Purification was achieved in three steps: precipitation with ammonium sulfate from the crude cellular lysate, ion exchange, and gel filtration chromatography. Carboxylesterase A is stereoselective, has a molecular weight of 70–73 kDa with an isoelectric point of 4.7, and a maximum activity (assayed on α-naphthylcaprylate) at pH 7.0 and 65°C. The enzyme showed good affinity toward (r)-benzoyl-1,2-isopropylideneglycerol (Km = 1.05 mm) and its activity on this substrate was competitively inhibited by (s)-benzoyl-1,2-isopropylideneglycerol. The purified enzyme yielded (s)-1,2-O-isopropylideneglideneglycerol with high enantiomeric excesses (e.e.) by resolution of various racemic esters (97% e.e starting from benzoate ester).

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.

Continuous production of isovaleraldehyde through extractive bioconversion in a hollow-fiber membrane bioreactor

A membrane bioreactor was developed to perform an extractive bioconversion aimed at the production of isovaleraldehyde by isoamyl alcohol oxidation with whole cells of Gluconobacter oxydans. A liquid/liquid extractive system using isooctane as extractant and assisted by a hollow-fiber hydrophobic membrane was chosen to recover the product. The aqueous bioconversion phase and the organic phase were maintained apart with the aid of the membrane. The extraction of alcohol and aldehyde was evaluated by performing equilibrium and mass transfer kinetic studies. The bioprocess was then performed in a continuous mode with addition of the substrate to the aqueous phase. Fresh solvent was added to the organic phase and exhausted solvent was removed at the same flow rate. The extractive system enabled a fast and selective in situ removal of the aldehyde from the water to the organic phase. High conversions (72–90%) and overall productivity (2.0–3.0 g l−1 h−1) were obtained in continuous experiments performed with different rates of alcohol addition (1.5–3.5 g l−1 h−1). Cell deactivation was observed after 10–12 h of operation.

Aldehyde production by alcohol oxidation with Gluconobacter oxydans

The microbial oxidation of various primary alcohols to the corresponding aldehydes has been investigated. A focused screening performed amongst some acetic acid bacteria showed that a newly isolated strain of Gluconobacter oxydans oxodizes various short-chain aliphatic alcohols to the corresponding aldehydes with negligible acid production. 3-Methyl-1-butanol (isoamyl alcohol) proved to be the better substrate with high yields (more than 90%) without by-product formation. This biotransformation also occurs with continuous or semicontinuous addition of substrate since the volatile product is removed from the medium under vigorous aeration conditions. Product recovery is attained either by the use of cold traps or by reversible complex formation.

Production of geranyl acetate and other acetates by direct esterification catalyzed by mycelium of Rhizopus delemar in organic solvent

Dry mycelium of Rhizopus delemar MIM catalyzed the formation of geranyl acetate using 110 mM geraniol and acetic acid at 55°C in heptane to give 11.9 g/l (55% molar conversion). Geranyl acetate was produced at 72.5-75 g/l after 10 days by semi-continuous addition of the substrates. Rhizopus delemar also catalyzed the direct acetylation of different primary alcohols with molar conversions ranging from 65 to 98%.

Enantioselective oxidation of (RS)-2-phenyl-1-propanol to (S)-2-phenylpropanoic acid with Gluconobacter oxydans: simplex optimization of the biotransformation

Abstract The microbial oxidation of racemic 2-phenyl-1-propanol by Gluconobacter oxydans DSM 50049 was investigated. Whole bacterial cells were used to produce ( S )-(+)-2-phenylpropanoic acid with high enantiomeric excess (E>200). A simplex sequential method was employed as an experimental design to guide the optimization process. Temperature of 26–28°C, pH 6.0–6.2, substrate concentration of 20–25 mM and agitation of 150 rpm have been found the best conditions to achieve the highest reaction rates and enantioselectivities.

Multigram-scale production of aliphatic carboxylic acids by oxidation of alcohols with Acetobacter pasteurianus NCIMB 11664

Acetobacter pasteurianus NCIMB 11664 was selected for multigram-scale production of different aliphatic carboxylic acids through oxidation of the corresponding alcohols after screening different acetic acid bacteria. Continuous production was carried out using an air-lift reactor, with overall yields of 1-propionic, 1-butyric, 2-methyl-1-butyric and 3-methyl-1-butyric acids ranging from 45 to 61 g dm−3. ©1997 SCI

Stereoselective reduction of non-cyclic carbonyl compounds by some eumycetes

SummaryTwelve strains of Eumycetes were able to perform the reduction of 2-pentanone, acetophenone and ethyl acetoacetate, sometimes in a yield suitable for preparative work. For each substrate, preferential reduction to the R-configurated alcohol was observed with one or more strains.

Microbial biotransformations in water/organic solvent system. Enantioselective reduction of aromatic β- and γ-nitroketones

Abstract The production of single enantiomers of γ- and β-nitroalcohols by microbial bioreduction has been studied. A restricted screening among 14 yeasts was performed using 1-phenyl-4-nitro-1-butanone 1 as substrate. Pichia minuta (CBS 1708) and Pichia etchellsii (CBS 2011) gave the highest enantiopreference for (S)-alcohol 3 formation (e.e. 85% and 80%, respectively), while Kluyveromyces marxianus (CBS 397) was the only strain able to preferentially furnish the (R) enantiomer (e.e. 70%). These three microorganisms, along with bakers yeast, were then employed in reactions performed in water/organic solvent systems using different solvents (hexane, benzene and dibutyl ether) affording alcohol 3 in high enantiomeric excesses (>95–97%). These strains were also employed to reduce 1-phenyl-3-nitro-1-propanone 2, maintaning the same stereobias observed with γ-nitroketone 1 and showing high enantioselectivity in both simple aqueous (>85–97%) and biphasic media (>97%).

Production of flavour esters by Rhizopus oryzae

Microbial production of different alipathic esters with flavour characteristic has been studied. Lyophilized whole cells of Rhizopus oryzae CBS 112-07 were found to be particularly suitable to catalyse the synthesis of different flavour esters (hexyl acetate, propionate, butyrate, caprylate; geranyl acetate, propionate, butyrate and 2- and 3-methylbutyl acetate, butyrate) in n-heptane. This strain was therefore utilized for the semipreparative production of geranyl butyrate by semicontinous and continous addition of the substrates with satisfactory yields (144 g l−1 in 264 h and 142 g l−1 in 48 h respectively).