Biotransformation of benzaldehyde to L-phenylacetylcarbinol (L-PAC) by Torulaspora delbrueckii free cells in the presence of beta-cyclodextrin (2023)

Studies were conducted to investigate the possibility of reducing the toxic and inhibitory effects of the benzaldehyde substrate during its biotransformation into L-PAC by Torulaspora delbrueckii free cells using beta-cyclodextrin (beta-CD). The use of different concentrations of benzaldehyde and acetaldehyde in the presence of 2% beta-CD showed that the body can tolerate higher concentrations of benzaldehyde and acetaldehyde in the presence of beta-CD. Semi-continuous feeding of benzaldehyde and acetaldehyde was found to increase L-PAC yield compared to single feeding.

benzaldehyde; Biotransformation; cyclodextrin; L-Phenylacetycarbinol; torulaspora delbrueckii

Biotransformation of benzaldehyde to L-phenylacetylcarbinol (L-PAC) by cells free ofTorulaspora delbrueckiiin the presence of beta-cyclodextrin

Vilas. B. Shukla e Pushpa R. Kulkarni ^* *Corresponding author

Division of Food Technology and Fermentation; Abt. Chemical Technology; University of Mumbai; (UCCT); Nathalal Parekh Marg; Matunga; Mumbai - 400 019; India

ABSTRACT

Studies were conducted to investigate the possibility of reducing the toxic and inhibitory effects of the substrate benzaldehyde during its biotransformation into L-PAC by free cells of Torulaspora delbrueckii usingB-Ciclodextrina(B-CD). Using different levels of benzaldehyde and acetaldehyde in the presence of 2%B-CD showed that in the presence ofB-CD, the body could tolerate higher concentrations of benzaldehyde and acetaldehyde. Semi-continuous feeding of benzaldehyde and acetaldehyde was found to increase L-PAC yield compared to single feeding.

Key words:Benzaldehyde, Biotransformation, Cyclodextrin, L-Phenylacetycarbinol,Torulaspora delbrueckii

INTRODUCTION

Most of the literature on the synthesis of L-phenylacetylcarbinol (L-PAC) and benzyl alcohol by yeast fermentation deals with optimization of yield per free cells (Agrawal et al., 1987; Cardillo et al., 1991; Zeeman et al. ., 1992). Studies on the formation of L-PAC from benzaldehyde showed that under normal fermentation conditions using yeast, quantitative conversion of benzaldehyde to L-PAC was never achieved because by-products such as benzyl alcohol, PAC-diol were formed (Smith and Hendlin, 1953; Gupta et al., 1979; Netraval and Vojtisek, 1982; Agrawal and Basu, 1989; Tripathi et al., 1988, Long et al., 1989). Yeast cannot be used for multiple batches because of the toxic and inhibitory effects of the substrate and products (Long et al., 1989; Coughlin et al., 1991). The use of cyclodextrin to reduce benzaldehyde toxicity for bioconversion using immobilized cells has been reported (Coughlin et al., 1991; Mahmoud et al., 1990). With these facts in mind, an attempt was made to study the effect of adding b-cyclodextrin on the biotransformation of benzaldehyde to L-PAC by the cells in FIG.Torulaspora delbrueckiiand the results are reported here.

MATERIALS AND METHODS

Materials -Microbial media components (Hi-Media Ltd. Mumbai), AR grade solvents and chemicals (S.D. Fine Chemicals Ltd. Mumbai and Merck India Ltd., Mumbai) and b-cyclodextrin (a sample gift from S.A. Chemicals Ltd., Mumbai) used. A molasses yeast isolate identified as Torulaspora delbrueckii was used. The composition of the maintenance medium used [Long et al., 1989] was: 2% glucose, 1% peptone, 1% yeast extract, 1% agar and pH 5.5. The growth medium [Long et al., 1989] contained 2% glucose, 2% peptone, 1% yeast extract and had a pH of 5.5. The biotransformation medium [Nikolova and Ward, 1991] with the composition glucose 5%, peptone 0.6% and pH 4.5 was used.

culture growth -1 ml of Zellsuspension IsolatesTorulaspora delbrueckiicontains 10⁶Cells were inoculated into 9 ml of growth medium and incubated on a rotary shaker at 30±2^ÖC at 240 rpm for 24 hours. The culture thus obtained was inoculated into 100 ml of the same medium and allowed to grow for 24 hours. under the same conditions and the cells were harvested by centrifugation at 10,000 rpm for 15 min at 15^ÖThe obtained biomass was washed with water, centrifuged and used for biotransformation studies.

Biotransformation of Benzaldehyde into L-PAC -The biotransformation medium (100 ml) was inoculated with 3.0 g (wet weight) of the cell mass obtained above. The vial was incubated on a shaker at 30±2^ÖC and 240 rpm for 1 h to adapt the cells to the medium. Benzaldehyde and acetaldehyde were added at concentrations ranging from 700 to 1200 mg/100 ml and 700 to 1200 ml/100 ml, respectively, and the flasks were again incubated in a shaker at 30 ± 2 for biotransformation^ÖC e 240 rpm.

Effect of b-cyclodextrin addition on benzaldehyde biotransformation -Previous studies had shown thatTorulaspora delbrueckiiCells can tolerate benzaldehyde up to 600 mg/100 ml of biotransformation medium and 600 ml of a 30-35% aqueous acetaldehyde solution. Therefore, the effect of 2% b-cyclodextrin (b-CD) was studied at benzaldehyde and acetaldehyde concentrations ranging from 700 mg to 1200 mg/100 ml and 700 ml to 1200 ml/100 ml, respectively. The reaction was allowed to proceed at 30 ± 2 for 3 h.^ÖC and 240 rpm. From these studies, 900 mg of benzaldehyde and 1000 ml of acetaldehyde were considered optimal. To study the effect of b-CD levels, b-CD concentration was varied in the range of 0.5 to 2.5%. Semi-continuous feeding of different amounts of benzaldehyde and acetaldehyde was also performed 4 times at intervals of 30 minutes in the presence of 2% b-CD.

Analysis of biotransformation products -After biotransformation, the medium was centrifuged at 10,000 rpm for 15 minutes. The supernatant was extracted three times with equal volumes of diethyl ether. The combined extract was dried over anhydrous sodium sulfate and concentrated in a temperature-controlled water bath. The residue obtained was dissolved in methanol and subjected to GC analysis. The conditions used for the GC were the following: The GC model used was the Chemito-8510 with the Oracle-1 computational integrator. A 4 meter column with 5% OV-17 was used. The injector temperature was 250^ÖC and the detector temperature (FID) was 250^ÖC. The column schedule was as follows, 75^ÖC for 3 min, then 10^ÖC/min up to 250^ÖC and hold for 5 min. The retention times of benzaldehyde, benzyl alcohol, L-PAC, PAC-diol were 11 min, 13 min, 17 min and 18.5 min, respectively. The concentrations of these compounds were determined using the peak area method (Shukla and Kulkarni, 1999 ). Certainly. Each experiment was repeated three times and found to be reproducible within ± 5 percent.

RESULTS AND DISCUSSION

Biotransformation of benzaldehyde by immobilized cell mass ofSaccharomyces cerevisiaeATCC 834 at higher levels of benzaldehyde has been reported using b-cyclodextrin (Mahmoud et al., 1990; Coughlin et al., 1991).

Cyclodextrins are known to provide a protective effect by forming inclusion complexes (Szetji, 1998; Uekama et al., 1998).

In the present work, the investigation of the effect of benzaldehyde levels in the presence of 2% b-CD showed that free cells of Torulaspora delbrueckii could easily tolerate up to 900 mg of benzaldehyde (Abb.1) confirms the protective effect of b-CD.

The same organism has been reported to tolerate only 600 mg of benzaldehyde in the absence of b-CD (Shukla, 1999). It is also known that the addition of acetaldehyde increases the production of L-PAC (Groeger et al., 1966; Subramanian et al., 1987). Previous reports from our laboratory have shown that the addition of acetaldehyde up to 600 mL has been found to be beneficial (Shukla, 1999). In the present work, the effect of adding acetaldehyde in the presence of 2% b-CD at a concentration of 600 mg of benzaldehyde was investigated. as shown inAbb. 2with the addition of acetaldehyde up to 1000 mL, the production of L-PAC increased and then remained constant up to 1200 mL. A concomitant decrease in benzyl alcohol was observed as the acetaldehyde concentration increased from 700 to 1200 mL.

To study the effect of b-CD level on the biotransformation product profile, different levels of b-CD were used. as shown inAbb. 3at lower levels, i. H. 0.5 to 1.5% b-CD, less L-PAC was formed, giving more residual benzaldehyde. When the b-CD level was increased to 2.5 g, L-PAC production increased while benzyl alcohol production remained almost constant at all levels from 0.5 to 2.0%. This suggests that higher levels of b-CD probably prevent the oxidation of the benzaldehyde molecule and redirect it towards the production of L-PAC and benzyl alcohol.

To confirm the beneficial effect of semi-continuous addition of benzaldehyde and acetaldehyde on L-PAC production, as well as to explore the organism's tolerance to high concentrations of benzaldehyde and acetaldehyde in the presence of b-CD, semi-continuous feeding of both were performed at different levels in the presence examined by b-CD. Semi-continuous feeding four times with 30 minutes intervals resulted in a product profile as inAbb.4showing. With increasing total cumulative dose of benzaldehyde and acetaldehyde, the yield of L-PAC steadily increased. The body can produce a maximum of 527 mg/100 ml of L-PAC with a total content of 1200 mg of benzaldehyde and 1200 ml of acetaldehyde. Netraval and Vojtisek (1982) also declared a yield of 6.3 g/L L-PACS. carlsbergensisBudvar with semi-continuous feeding of benzaldehyde, acetaldehyde and sucrose, while Cardillo et al. (1991) reported a 60% conversion of benzaldehyde to L-PAC whenS. delbrückiiwas used.

In the presence of 2% b-CD,Torulaspora delbrueckiiCells can tolerate 900 mg of benzaldehyde and 1000 ml of acetaldehyde as opposed to 600 mg of benzaldehyde and 600 ml of acetaldehyde in the absence and presence of 2% b-CD,Torulaspora delbrueckiiCells could produce 527 mg/100 ml of L-PAC when benzaldehyde and acetaldehyde were fed semi-continuously four times at 300 mg and 300 ml, respectively.

Received April 25, 2000;

Revised: April 16, 2001;

Assumed on November 5, 2001.

* Corresponding author