JOURNAL OF FERMENTATION AND BIOENGINEERING

Vol. 86, No. 1, 62-64. 1998

Resolution of 2-Alkanols by Microbially-Catalyzed Esterification FRANCESCO MOLINARI, LAURA MANTEGAZZA, RAFFAELLA VILLA, AND

FABRIZIO ARAGOZZINI*

Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Sezione Microbiologia Industriale, Universitci degli Studi di Milano, via Celoria 2, 20133 Milano, Italia

Received 16 May 1997/Accepted 30 April 1998

A method based on the use of whole lyophilized microbial cells has been developed for resolution of 2- alkanols. Firstly, the esterification of racemic 2-octanol with butyric acid was studied. Rhizopus oryzae CBS 112-07 grown on Tween 80 was selected as a suitable biocatalyst. Appropriate choice of solvent, temperature and reactant concentration allowed for highly selective esterification (>97% enantiomeric excess of R-ester, 41% molar conversion). Direct esterification of 2-octanol with butyric acid was compared with interesterifica- tion using different acylating reagents. The best results were obtained with butyric acid and tributyrin. Es- terification of other racemic 2-alkanols (2-butanol, 2-pentanol, 2-hexanol, 2-heptanol) showed that chain length played a crucial role in enantioselectivity, since lower enatiomeric excesses were observed using short chain alcohols.

[Key words: microbial biotransformation, 2-octanol, enantioselective esterification, 2-alkyl esters]

The resolution of racemic alcohols by esterification catalyzed by lipases or esterases in organic media with low water activity has been shown to be a valuable syn- thetic method. Lyophilized whole cells can also catalyze secondary alcohol esterification in organic solvent with butyric acid (1). This observation lead us to investigate the stereoselectivity of this biotransformation and to compare it with various interesterifications. Direct es- terification of 2-octanol has been observed with different lipases (porcine pancreatic, Penicillium expansum and Candida sp.) but they have not furnished very high enan- tioselectivity (2, 3). To improve the enantioselectivity, enzyme-catalyzed interesterifications with different esters have been studied, but optically pure 2-octyl esters are still difficult to obtain (4-8).

In this work we studied the microbially-catalyzed reso- lution of the racemic mixture of 2-octanol and of other secondary alcohols (2-butanol, 2-pentanol, 2-hexanol, 2- heptanol) by direct esterification in organic solvent.

MATERIALS AND METHODS

Materials Chemicals were all purchased from Fluka Chimica, Milan0 (Italy), except Tween 80 from Merck, Milan0 (Italy).

Microorganisms, media and culture conditions Micrococcus luteus ATCC 9341 (American Type Culture Collection, Parkville, USA) was routinely maintained on Difco nutrient broth (8 g.1 r, agar 15 g.1 I, pH 7), while Rhizopus delemar MIM (Microbiologia Industriale Milano, Italy) and Rhizopus oryzae CBS 112-07 (Centraalbureau Voor Schimmelcultures, Baarn, Holland) were maintained on malt extract (8 g.I r, agar 15 g.I~ r, pH 5.5).

Selection of the strain with the highest enantioselectivity was performed with microorganisms cultured on liquid medium (Difco yeast extract 1 g.I- r, (NH&SO4 1 g.1 I, K2HP04 1 g.l-~i, MgS04.7Hz0 0.1 g./-r, Tween 80 5 g. I r, pH 5.8; for M. futeus pH was 7.0). The cultures were inoculated with cell suspensions obtained from 24 h slants or with spores obtained from 72 h slants (R. dele-

* Corresponding author.

62

mar and R. oryzae). Submerged cultures were carried out in 2 I Erlenmeyer flasks containing 400 ml of medium and incubated for 24-48 h at 28°C on a reciprocal shaker (100 strokes minr).

R. oryzae was also cultured in the same basic synthetic medium containing different carbon sources (malt ex- tract, olive oil, soybean oil, oleic acid 5 g.1 ‘). Sub- merged cultures were inoculated with spores from 72 h slants or with pellets from 48 h submerged cultures. The latter type of inoculum furnished cells with the highest activity and enantioselectivity for 2-octanol esterifica- tion.

Biotransformations Biotransformations were per- formed with lyophilized cells. Ester synthesis was carried out in 10 ml screw capped test tubes by suspending lyophilized cells (20 g. 1-l) in n-heptane (5 ml) and then adding the alcohol and an equimolar amount of acid. The reaction mixtures were continuously stirred with a magnetic stirrer.

Analytical methods Alcohol and ester concentra- tions were determined together with the enantiomeric composition by gas-chromatographic (GC) analysis using a chiral capillary column (diameter 0.25 mm, length 25 m, thickness 0.25 11, DMePeBeta-CDX-PS086, MEGA, Legnano, Italy).

Samples (0.2ml) were taken at intervals and added to an equal volume of an internal standard solution in n- heptane. Primary alcohols were used as internal standard for corresponding secondary alcohol esterifications.

RESULTS

Selection of the microorganism and study of the direct esterification with butyric acid It has previously been observed that lyophilized cells of M. luteus ATCC 9341, R. delemar MIM and R. oryzae CBS 112-07 are able to catalyze the esterification of racemic 2-octanol with butyric acid in n-heptane (1). The enantioselectivity of these transformations was investigated in n-heptane with equimolar amounts of alcohol (1 g/l) and acid at 60°C (Table 1).

R. oryzae gave the highest enantiospecificity and was,

VOL. 86. 1998 MICROBIAL RESOLUTION OF 2-ALKANOLS 63

TABLE 1. Molar conversions and stereoselectivity of 2- octylbutyrate production catalyzed by lyophilized

cells (30 g. I-‘) in n-heptane at 60°C after 7 d

Microorganism Molar conversion (%)

e.e. ester (W

M. luteus ATCC 9341 47 50 (s) R. delemar MIM 35 69 (R) Rhizopus oryzae CBS 112-07 30 90 (R)

therefore, selected for further studies. Different growth times, carbon sources (malt extract, olive oil, soybean oil, Tween 80 and oleic acid) and types of inocula (pellets from submerged cultures or spores from solid media) were investigated. The best results were obtained with cells grown on Tween 80 in submerged cultures for 48 h and inoculated with pellets from submerged cul- tures. These cells were able to catalyze the esterification of racemic 2-octanol (1 g/l) at different temperatures as shown in Table 2.

In the range of temperatures between 30 and 60°C similar conversions were observed. After 5 d the reac- tions stopped and no changes in the enantiomeric compo- sition was observed over longer periods. At 75°C the transformation did not proceed after 48 h. The highest enantioselectivity was observed at 30°C with an enantio- merit ratio (E) > 150. The time course of the transforma- tion at 30°C is reported in Fig. 1.

Different organic solvents were evaluated. All the aliphatic hydrocarbons tested (pentane, hexane, heptane, isooctane and hexadecane) allowed for ester production (Table 3). No esterification was observed when benzene, toluene, dietyl ether and dibutyl ether were utilized.

With all the tested solvents, similar conversions were observed after 5 d. Heptane, being the best solvent in terms of enantioselectivity, was chosen as the solvent to be used for subsequent experiments.

Higher substrate concentrations were utilized (Table 4). Initial rates, calculated within the first 5 h, increased with increasing substrate concentration, although not in a linear fashion. Good molar conversions were observed starting from log/Z of 2-octanol with 4.8 g/l yield of almost pure (R)-2-octylbutyrate. The enantioselectivity remained very high in all the cases, although there was a slight decrease at higher substrate concentration.

Interesterifications and direct esterification using other substrates A comparison between direct esterification and interesterification was carried out. Biotransforma- tions were performed at 30°C with 5 g/f of racemic 2- octanol and an equimolar amount of different acylating agents. These were activated esters (isopropenyl and trichloroethyl butyrate) and other butyrate esters (ethyl butyrate and tributyrin). Results are shown in Table 5.

The tested interesterifications did not significantly

TABLE 2. Production of (R)-Zoctylbutyrate catalyzed by lyophilized cells (30 g. 1-i) of R. oryzae in n-heptane at

different temperatures

Temperature Conversion (“C) (%)

30 40 45 41 60 44 75 29

e.e. (R)-ester Initial rate (W (;~mol.mlllh~l)

>97 0.8 95 0.9 91 1.1 87 1.4

Molar conversion and e.e. after 5 d.

3 4

Time (cl)

FIG. 1. Time course of (R)-2-octylbutyrate production catalyzed by lyophilized cells (30 g.l-I) of R. oryzae in n-heptane at 30°C. Symbols: n , molar conversion; +, enantiomeric excess.

improve rates, molar conversions or enantioselectivities. Trichloroethyl- and isopropenylbutyrate, which are com- monly utilized to reach high degrees of interesterifica- tion, gave conversions lower than ethylbutyrate and tributyrin. Tributyrin furnished the highest initial rate, molar conversion and enantioselectivity. The influence of the chain length of the acid on the resolution of 2- octanol was evaluated at 30°C in n-heptane using acetic and octanoic acid. With octanoic acid, (R)-ester was produced with high enantioselectivity (>97%) and good molar conversions (42%) after 4 d. The acetylation afford- ed only 10% of molar conversion after 7 d, the (&ester was preferentially formed, although with low enantio- selectivity (e.e. 20%).

The study was then extended to the direct microbial esterification of other racemic secondary alcohols (0X7) with butyric acid. Results of esterification between these alcohols and butyric acid are reported in Table 6.

The chain length of the alcohol markedly affected enantioselectivity with a gradual lowering of the enan- tiopreference with shorter chain secondary alcohols for (R)-ester production, until the (s) ester was obtained as prevalent enantiomer with 2-butanol.

DISCUSSION

Intracellular microbial esterase and lipases can be directly exploited to catalyze acylation of different alco- hols by using lyophilized cells in organic solvents. It was previously found that lyophilized whole cells catalyzed direct esterification between the alcohol and the free carboxylic acid with good yields, without removing the formed water (9). This is a notable simplification com- pared with the need of purifying the enzymes involved.

High enantioselectivity is usually achieved in enzyme-

TABLE 3. Production of (R)-2-octylbutyrate catalyzed by lyophilized cells (30 g I- I) of R. oryzae in different

solvent at 30°C

Solvent Molar conversion (W

Pentane 41 Hexane 48 Heptane 41 Isooctane 40 Hexadecane 37

Molar conversion and e.e. after 5 d.

e.e. (R)-ester (36)

90 97

i97 97 87

64 MOLINARI ET AL. J. FERMENT. BIOENG.,

TABLE 4. Production of (R)-2-octylbutyrate catalyzed by TABLE 6. Production of different 2-alkylbutyrates by direct lyophilized cells (30 g. l-l) of R. oryzae in n-heptane esterification catalyzed by lyophilized cells (30 g. I- ‘) of

at 30°C with different substrate concentrations R. oryzae in n-heptane at 30°C

2-Octanol Conversion e.e. (R)-ester Initial rate (n.1 ‘) (W (00 (,~mol.ml-lh-l) Substrate

Time Molar conversion e.e. ester (d) (Pd) (PO’)

1 41 >91 0.8 2.5 39 91 1.5 5 38 91 3.1 1.5 35 91 3.5

10 32 95 3.8

Molar conversion and e.e. after 5 d.

TABLE 5. Production of (R)-2-octylbutyrate catalyzed by lyophilized cells (30 g. I-‘) of R. oryzae in n-heptane

at 30°C using different acylating reagents

Acylating reagent o Con,v+;ion e.e. (RI-ester Initial rate (061 (umol.ml-‘h-l)

Isopropenyl butyrate 19 81 1.5 Trichloroethyl butyrate 21 89 6.3 Tributyrin 40 91 11.3 Ethyl butvrate 30 95 6.9

Molar conversion and e.e. after 5 d.

catalyzed esterifications under conditions of rapid and irreversible interesterification using acylating agents such as enol esters or 2-haloethyl esters (10). It is thus quite interesting to also have biocatalysts able to give highly enantioselective acylation by using free acids. In this work it was found that lyophilized cells of R. oryzae CBS 112-07 furnished (R)-2-octyloctanoate, (R)-2-octyl-, (R)-2-heptyl- and (R)-2-hexylbutyrate with high optical purity by direct esterification of the racemic mixture of the alcohols and the acid. The direct esterification reached equilibrium at 40-45x molar conversion, without further changes in the stereochemical composi- tion. These are almost quantitative yields when dealing with resolution of a racemic mixture. Similar results were observed using tributyrin as acylating reagent. Interesterifications with trichloroethylbutyrate and isopropenylbutyrate did not improve selectivity and yields. The use of the free acid is an easy procedure and especially interesting when natural substrates have to be utilized.

The reasons why direct esterification can be so efficiently catalyzed by whole cells is puzzling. It may be due to favourable water partitioning. In cellular systems the released water may be trapped in microbial sites other than those involved in the acylation, thus avoiding a shift of the equilibrium toward hydrolytic reaction. It is also possible that the tolerance of the intracellular lipases/esterases towards the free acid is enhanced when they are “immobilized” in the whole cell structure.

The influence of temperature on the stereobias is im- portant. It is likely that more than one enzyme of the whole cell can catalyze the esterification of 2-octanol, but only the highly enantioselective one(s) are active at 30°C.

An interesting feature of this biotransformation is the use of a low concentration of biocatalyst. Although whole cells were employed, the specific activity was still satisfactory. Good yields were also observed at log/l of

2-Butanol 3 45 25 (s) 2-Pentanol 4 49 70 (R) 2-Hexanol 5 46 95 (R) 2-Heptanol 5 42 97 (R)

substrate concentrations, allowing for a productivity number of 6.8 (mmol of product/kg of cells. h of trans- formation) (11).

It can be concluded that lyophilized microbial cells of R. oryzae CBS 112-07 can be used as biocatalysts for the production of optically pure (R)-2-alkylbutyrates in or- ganic solvent. There is great interest for the identifica- tion of “new” microbial enzymes able to perform enan- tioselective transformations. The possibility to achieve high selectivity with whole cells without isolation of the enzymes involved is technically and economically interest- ing for preparative application.

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