OXIDATION OF SECONDARY ALCOHOLS TO METHYL KETONES BY IMMOBILIZED YEAST CELLS TUNG-LI HUANG, BING-SHIUN FANG, AND HUNG-YUAN FANG

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1 J. Gen. App!. Microbiol., 31, (1985) OXIDATION OF SECONDARY ALCOHOLS TO METHYL KETONES BY IMMOBILIZED YEAST CELLS TUNG-LI HUANG, BING-SHIUN FANG, AND HUNG-YUAN FANG Refining and Manufacturing Research Center, Chinese Petroleum Corporation, Chia Yee, Taiwan, Republic of China (Received November 14, 1984) As Hou et al. (1, 2) mentioned, cell-free extract from methanol-grown yeasts catalyzes the oxidation reaction of secondary alcohols to methyl ketones. Recently, we found that glucose-grown yeasts also produce secondary alcohol dehydrogenase that is constitutively formed. Among several methanol-grown yeasts, Hansenula polymorpha P-5 was the most suitable enzyme source because of its thermal stability and broad substrate specificity. In addition to the secondary alcohols, primary alcohols and 1,2-propanediol are also oxidized by the cell-free extract of H. polymorpha P-5. The oxidation of secondary alcohols to methyl ketones by immobilized yeast cells was examined and compared with the oxidation by the cell suspensions. Oxygen was essential for the conversion of 2- propanol to acetone. The secondary alcohol dehydrogenase activity of the immobilized cells was not affected by ph shock from 7.0 to 3.5, but with the cell suspension it was affected. The optimun ph for the cell suspension was about 8.5, but the immobilized yeast cells had a broad optimum ph range from 5.0 to 8.5. Only an 8 % decrease in the enzyme activity of the immobilized yeast cells occurred when the ph was below 4.0. For the immobilized yeast cells, the tolerance concentration of the secondary alcohol dehydrogenase to acetone was up to 500 (v/v). However, with cell suspensions, the enzyme was not tolerant to acetone. The optimal enzyme activity of cell suspensions lasted 2 days; that of immobilized yeast cells lasted 3 days. The enzyme activity of immobilized cells could be restored with glucose medium. During the last ten years, immobilized cells and enzymes have contributed importantly to enzyme technology. There are many examples of successful commercial application including organic synthesis, clinical analysis, chemical analysis, food processing and pharmaceutic industry. Using immobilized cells or enzymes has many advantages. Expensive en- 125

2 126 HUANG, FANG, and FANG VOL. 31 zymes may be reused in batch or continuous processes to minimize enzyme costs. Immobilization often improves the thermal and ph stability of the enzyme. The process can be carried out in continuous ways. Product refining costs are lowered because by-product formation is minimized. Recently, Hou et al. (1, 2) discovered that cell suspensions of methanolgrown yeasts catalyze the oxidation of various secondary alcohols to the corresponding methyl ketones. In our studies, we tried to develop this special kind of bioconversion system using immobilized cells. Compared to alcohol production by immobilized yeast cells, this system has these special characteristics: Oxygen is required during the whole conversion reaction period. The reactant is different from the growth substrate of H. polymorpha P-5, so the bioconversion process and regeneration operate separately. Coenzyme NAD+ is essential for the oxidation of 2-propanol to acetone, so another enzyme system in H. polymorpha P-5 is involved in recycling NAD. This bioconversion system is described in this report. Reaction conditions and stability are also reported. MATERIALS AND METHODS Yeast strains. H. pol ymorpha ATCC 26012, Candida boidinii ATCC 32195, Torulopsis methanolovescens ATCC 26176, Pichia pinus ATCC 34972, and H. polymorpha P-5 which was isolated from Taiwan local soil by enrichment culture with methanol as the sole carbon source. Cultivating and harvesting cells. Organisms were grown in 2-liter flasks containing 250 ml of mineral salt medium with 1.5 % (v/v) methanol. The mineral salt medium was composed of (NH4)2SO4, 0.2%; KH2PO4, 0.2%; NaH2PO4, 0.2 %; MgSO4.7H2O, 0.05%; FeSO4.7H2O, 20 ppm; CaC12.2H2O, 4 ppm; ZnSO4 7H2O, 4 ppm; CuSO4.5H2O, 1 ppm; MnSO4.5H2O, 1 ppm; thiamine, 1.5 ppm; biotin, 0.03 ppm; ph 4.7. Hansenula strains were cultured at 38 C and the other strains were cultured at 30 C. Cells were harvested at the exponential phase by centrifugation at 12,000 x g for 10 min. Preparation of immobilized cells. Two grams of Na-alginate was swelled in 100 ml distilled water, than 5 g of wet cells was added and mixed completely, and the mixture was dropped into 0.1 M CaC12 (ph 6.0) solution to form uniform spherical beads 2-3 mm in diameter. Ketone production. 1. By cell suspension: the harvested cells were suspended in 50 mm phosphate buffer (ph 7.0). A 20-ml cell suspension was put into a 125-m1 flask. Secondary alcohol (0.2 ml) was added, then incubated at room temperature on a rotary shaker at 200 rpm. 2. By immobilized cells : 10 grams of immobilized cell beads was put into 125-ml flasks. Secondary alcohol (10 ml, 1 %) was added and incubated at room temperature on a rotary shaker at 200 rpm. Assay of ketone product. The ketone product obtained from secondary

3 1985 Oxidation of Secondary Alcohols 127 Table 1 Oxidation of secondary alcohol to methyl ketone by cell suspension of yeasts in exponential and stationary phases. Fig. 1. Profile of the production of acetone by the oxidation of 2-propanol at 45 C by cell suspensions of yeasts. : H. polymorpha ATCC 26012, x ATCC 26176, 0: P. pinus ATCC H. polymorpha P-5, o: T. methanolovescens alcohol by cell suspension or by immobilized cells was estimated by flame ionization gas chromatography using a stainless steel column (12 feet by 1/8 inch) packed with 10% Carbowax 20 M on 80/100 chromosorb W column. The column temperature was maintained isothermally at 130 C, and the flow of carrier gas was 30 ml of nitrogen per min. Preparation of crude extract and enzyme assay. The methods were the same as Hou's (2). Assay of protein content. The protein content of cell suspensions and cellfree extracts was determined by the method of LowRY et al. (3). RESULTS 1. H. polymorpha P-5 is a suitable enzyme source of secondary alcohol dehydrogenase. Cell suspensions of several yeasts grown on methanol catalyzed the oxidation of 2-propanol to acetone. H. polymorpha P-5 had the highest conversion rate and

4 128 HUANG, FANG, and FANG VOL. 31 Fig. 2. Profile of NAD+ reduction by the cell-free extract of H. polymorpha P-5 with different alcohols as reaction substrate. The substrates for test were as follows: A, 2-propanol and 2-butanol; B,1-butanol; C,1,2-propanediol; D,1-propanol; E, glycerol; F, benzyl alcohol, after reaction for 3 min, ethanol was added; G, 1,2-ethanediol, after reaction for 5-min, 1,2-propanediol was added; H,1,3-propanediol. The reaction mixture, in a total volume of 3.0 ml, contained 50 mm phosphate buffer, ph 8.0, 5 iimol of NAD+, 0.5 ml crude extract, and substrate. The reactions were started by adding 100,yl of 0.1 M substrate, and the amount of NAD} reduction was measured spectrophotometrically at 340 nm. Fig. 3. Effect of aeration on the enzyme activity of immobilized cells. x : Aeration started at the beginning, : aeration started after standing for 28 hr. the exponential-phase cells possessed a much higher enzyme activity than the stationary-phase cells (Table 1). 2-Propanol was completely oxidized to acetone, and no further oxidation products of acetone were revealed by gas chromatographic analysis. Furthermore, the thermal stability of H. pol ymorpha was better than the others (Fig. 1). When the reaction temperature was raised to 45 C, the cell suspension of H. polymorpha ATCC and P-5 still kept a constant enzyme activity for 5 hr. However, the enzyme activity of T. methanolovescens ATCC and P. pinus ATCC lasted for only about 3 hr. In order to eliminate the permeability problem of different alcohols and coenzyme NAD+, the cell-free extract of H. polymorpha P-5, rather than cell suspensions, was used to examine the substrate specificity (Fig. 2). The results showed that in addition to secondary alcohols, primary alcohols and 1,2-propanediol were

5 1985 Oxidation of Secondary Alcohols 129 Fig. 4. Effect of reaction temperature on the enzyme activity of immobilized cells. x : room temperature (20-25 C), : 38 C. Fig. 5. Tolerance of secondary alcohol dehydrogenase activity for acetone, with immobilized cells. also oxidized. Glycerol, benzyl alcohol, 1,2-ethanediol and 1,3-propanediol were not oxidized. 2. Optimal conditions for the oxidation reaction of 2-propanol to acetone by immobilized H. polymorpha P-5 cells. The secondary alcohol dehydrogenase activity of H. polymorpha P-5 cell suspension was affected by culture age. The enzyme activity of exponential-phase cells was about 2.5 times as high as that of stationary-phase cells (Table 1), so the exponential-phase cells were used for immobilization. Oxygen was required for the conversion of 2-propanol to acetone by immobilized yeast cells. If the air supply stopped, no enzyme activity occurred (Fig. 3). The effect of temperature on the enzyme activity was also examined. Room temperature (20-25 C) seemed more suitable than 38 C (Fig. 4). However, a previous report (1) indicated that the optimun temperature for the production of methyl ketone by cell suspensions of H. pol ymorpha ATCC was about 40 C. Perhaps the acetone vaporized easily at the higher temperature, causing a loss of product which affected the difference in results. The recovery cost was highly affected by the final product concentration. Assuming that the enzyme activity was not inhibited by the product, the higher the product concentration was, the lower was the recovery cost. Figure 5 shows that while acetone accumulated up to 5 %, the enzyme activity was not affected. However, with cell suspensions, the same enzyme was inhibited by acetone (Fig. 6). When acetone was removed from the reaction mixture, the enzyme activity of the cell suspension was restored. This suggests that the secondary alcohol

6 130 HUANG, FANG, and FANG VOL. 31 Acetone concentration (v/v %) Fig. pension. 6. Inhibition of secondary alcohol dehydrogenase by acetone, with cell sus- Fig. 7. Effect of ph shock on the enzyme activity of immobilized cells and cell suspension. x : ph was shifted from 7.0 to 3.5 after 5-hr incubation; : ph was not shifted; --- : ph variation curve; x-x, - : immobilized cells; x--x, -- : cell suspensions. dehydrogenase of the cell suspensions was inhibited rather than deactivated by acetone. The acetone concentration in the reaction mixture may stimulate the permeability of 2-propanol and acetone in the Ca-alginate beads causing the acetone

7 1985 Oxidation of Secondary Alcohols 131 Fig. 8. Optimum ph profile of cell suspension and immobilized cells. -: immobilized cell beads in which cell concentration was 16 mg dry cell wt/g beads; : cell suspension. Fig. 9. The correlation between enzyme activity of immobilized yeast cells and cell concentration in the beads production rate in immobilized cells to increase when the accumulated acetone concentration was above 1.5 % (v/v) (Fig. 5). The oxidation of 2-propanol to acetone by immobilized cells was not affected by ph variations from 7.0 to 3.5, but in cell suspensions the enzyme was affected (Fig. 7). The optimum ph for cell suspensions was about 8.5. However, the immobilized yeast cells had a broad optimum ph range from 5.0 to 8.5 (Fig. 8). The enzyme activity of immobilized yeast cells decreased only 8 % even when the

8 132 HUANG, FANG, and FANG VOL. 31 Fig. 10. Enzyme reactivation of immobilized cells x : The immobilized cells were not regenerated; regenerated at the fourth day. by regeneration method. The immobilized cells were IWULII UFI II nfc `uuya) Fig. 11. Effects of aerobic or anaerobic regeneration on the immobilized cells. - : aerobic regeneration; x, --- : anaerobic regeneration. enzyme activity of ph was below 4.0. It was evident that the immobilization treatment improved the enzyme stability at ph values even as low as 2.5 to 4.0. The enzyme activity of immobilized yeast cells was only about 3500 of that of free cells when the cell concentration in the beads was 1.12 mg of dry cells/g of beads. The enzyme activity increased as the cell concentration in the beads increased. However, if the cell concentration was above 9.0 mg of dry cells/g of beads, enzyme activity leveled off (Fig. 9). This may result from an insufficiency of oxygen supply caused by the cells near the surface of the beads consuming all

9 1985 Oxidation of Secondary Accohols 133 of the oxygen allowing none to diffuse to the inner part beads at the higher cell concentrations. 3. The regeneration test of immobilized H. polymorpha P-5 cells. When H, polymorpha P-5 was grown in glucose medium, secondary alcohol dehydrogenase appeared in both cell-free extracts and cell suspensions. It is evident that this enzyme was constitutively formed. In order to restore the enzyme activity of immobilized cells, regeneration treatment with glucose medium (Table 2) was investigated. The results showed that if the immobilized cells was not regenerated, the initial enzyme activity lasted for only 3 days and was reduced to 65 % at the fourth day. However, with regeneration for 20 hr at the fourth day, the enzyme activity was restored completely (Fig. 10). Shaking may injure the uniform spherical beads of immobilized cells, so an anaerobic regeneration test was investigated. Regeneration with and without shaking gave slightly different results. The restoration of enzyme activity without shaking was delayed one day longer than it was with shaking (Fig. 11). DISCUSSION The kinetics of the alcohol-oxidizing enzyme of methanol-grown yeast is quite complex. Methanol is oxidized mainly by methanol oxidase which is inducibly formed and its prosthetic group has been identified as a flavin adenine dinucleotide (4-7). The oxidation of primary alcohols other than methanol is catalyzed by a constitutive NAD+-dependent alcohol dehydrogenase (8). Hou et al. first discovered and purified a NAD + -specific secondary alcohol dehydrogenase from methanol-grown yeast (2, 9). In our research, we also found that cell-free extract from H. polymorha P-5 catalyzes the oxidation of a series of alcohols including both primary and secondary alcohols. This is compatible with the previous reports (2, 8, 9). HoU et al. ever proved that 1,2-propanediol could not be oxidized by the purified secondary alcohol dehydrogenase. In our study, 1,2-propanediol was oxidized by the cell-free extract of H. polymorpha P-5. Apparently 1,2-propanediol may be oxidized by the primary alcohol dehydrogenase rather than the secondary alcohol dehydrogenase in the cell-free extract. Propanol was readily oxidized to acetone by immobilized H. polymorpha P-5. Because the enzyme activity of the immobilized cells was not affected by ph variations from 7.0 to 3.5, buffering for constant ph was not required during the whole reaction period. 2-Propanol in water was used directly as the reaction substrate. No side-reaction products and no additional impurities in the reaction solution make it easy to recover the product. The oxygen requirement for regeneration of methanol-grown cells is much greater than for glucose-grown cells. Violent vibration and aeration may destroy the uniform spherical beads of immobilized yeast cells, so glucose medium is used

10 134 HUANG, FANG, and FANG VOL. 31 to regenerate them. Decreased enzyme activity of immobilized cells may result from the loss of cells from the beads, so the enzyme activity of immobilized cells was restored completely after the regeneration treatment. In yeast cells grown anaerobically, no mitochondria appear in electron micrographs of thin sections. However, when anaerobically-grown cells are aerated, they acquire the ability to respire and at the same time mitochondria develop in the cells (10). Owing to the degeneration of mitochondria, the pool of oxidized NAD+ may be limited. Maybe this is the reason why the restoration of enzyme activity by regeneration treatment without shaking was delayed one day. After one day of aeration to oxidize 2-propanol to acetone, mitochondria are reproduced, and the secondary alcohol dehydrogenase activity can be restored completely. In order to avoid contamination by other metabolites, the oxidation reaction and regeneration treatment are done separately. Furthermore, if secondary alcohol is added to the growth medium, the growing cells may simply consume the growth substrate and not catalyze the oxidation of secondary alcohol to methyl ketone because the yeast cell cannot use secondary alcohol as its carbon source. That is why the immobilized resting cells rather than immobilized growing cells are used to catalyze the conversion of 2-propanol to acetone. However the half-life of the immobilized resting cells is much shorter than the immobilized Saccharomycete cells for ethanol production. So how to increase the half-life of immobilized H. polymorpha P-5 cells becomes the focus of future work. REFERENCES 1) C. T. Hou, R. PATEL, A. I. LASKIN, N. BARNABE, and I. MARCZAK, App!. Environ. Microbiol., 38,135 (1979). 2) R. N. PATEL, C. T. HOu, A. I. LASKIN, P. DERELANKO, and A. FELIX, App!. Environ. Microbiol., 38, 219 (1979). 3) 0. H. LOWRY, N. J. ROSEBROUGH, A. L. FARR, and R. J. RANDALL, J. Biol. Chem., 193, 265 (1951). 4) R. N. PATEL, C. T. HOU, A. I. LASKIN, and P. DERELANKO, Arch. Biochem. Biophys., 210, 481 (1981). 5) N. KATO, Y. OMORI, Y. TANI, and K. OGATA, Eur. J. Biochem., 64, 341 (1976). 6) Y. TANI, T. MIYA, and K. OGATA, Aggic. Biol. Chem., 36, 76 (1972). 7) N. KATO, Y. TANI, and K. OGATA, Agric. Biol. Chem., 38, 675 (1974). 8) H. SAHM and F. WAGNER, Eur. J. Biochem., 36, 250 (1973). 9) R. N. PATEL, C. T. HOU, A. I. LASKIN, P. DERELANKO, and A. FELIX, Eur. J. Biochem., 101, 401 (1979). 10) K. HUNTER and A. H. ROSE, Yeast Lipids and Membranes, In The Yeasts, Vol. 2, ed. by A. H. ROSE and J. S. HARRISON, Academic Press, London and New York (1971), p. 262.