Chapter 17
Microbial Oxidation of Alcohols by Candida boidinii: Selective Oxidation
Biotechnology for Improved Foods and Flavors Downloaded from pubs.acs.org by MACQUARIE UNIV on 02/17/19. For personal use only.
M . Nozaki, N. Suzuki, and Y. Washizu Central Research Laboratory, Takasago International Corporation, 1-4-11 Nishi-Yawata, Hiratsuka, Kanagawa 254, Japan
Chemistry of the microbial oxidation by Candida boidinii SA051 was investigated. Chemo-, regio- and stereo- selectivity and substrate specificity of this microbial oxidation will be discussed.
It is known that there are some types of microbial oxidation. The alcohol dehydrogenase type of oxidation requires cofactors such as NAD, etc. For efficiency and economy reasons, it is preferable that there is a regeneration system of cofactors in the alcohol dehydrogenase type of oxidation. However, alcohol oxidase is an unidirectional (mostly flavin dependant) redox enzyme. If a cell suspension of the microorganisms having alcohol oxidase activity is used, regeneration by endogenous production relieves one of exogenous addition. This type of oxidation seems more practical. It has been reported that methylotrophic yeasts such as Candida boidinii (7), Pichia pastoris (2) and Candida maltosa (3) show alcohol oxidase activity and can oxidize alcohols effectively to the corresponding aldehydes. Candida boidinii SA051 (4) used in this study was derived from Candida boidinii AOU-1 by UV irradiation and had the highest alcohol oxidase activity producing formaldehyde among the mutants. In our previous work, this oxidation showed feasibility for the preparative scale production of flavor aldehydes (5). In this paper, substrate specificity of this system and chemistry of this microbial oxidation was examined. Methods and Materials Organisms. Candida boidinii SA051 was generously donated by Prof. Y. Tani and Prof. Y. Sakai (Kyoto University). Biomass production. The cultures of the yeasts were carried out in a 70 L jar fermentor with a working volume of 40 L. Seed cultures for the fermentor were started from slant cultures. The slant cultures were inoculated directly into 100 mL of the basal medium containing 3% of glucose in a 500 mL Sakaguchi flask. In lOOOmL of the basal medium, there were NH C1 7.63g, K H P 0 2.81g,MgS04/7H O 0.59g, EDTA/2Na 0.45g, CaCl /2H 0 55.0mg, FeCl3/6H 0 4
2
2
0097-6156/96/0637-0188$15.00/0 © 1996 American Chemical Society
2
2
4
2
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Microbial Oxidation of Alcohols £y Candida boidinii
189
37.5mg, ZnSO4/7H 0 22mg, MnS0 17mg, Thiamine HC1 17mg, H B 0 4mg, CoCl /2H 0 2.8mg, Na Mo0 2.6mg, KI 0.6mg and Biotin 0.05mg. The cultures were incubated at 28°C with shaking (150 rpm). After 2 days growth, the cultures were used to start the 40 L scale fermentation. The volume of inoculum used was 800 mL. It was cultivated on the basal medium containing 0.3% (W/V) glucose as the carbon source to accelerate the initial growth. Methanol is required to induce the alcohol oxidase; no enzyme is formed during growth on glucose. However, if methanol is added at the beginning of fermentation, it inhibits the initial growth of the microorganisms. The impeller speed was 200 rpm and the aeration rate was 0.5 vvm. The temperature was kept at 28°C. After 16 hours, the pH of the cultured broth was pH statically adjusted to 5.0 by addition of 5N-NaOH solution. At this point the first aliquot of methanol (1% of the broth) was added. After the consumption of the initially added methanol, a second amount of methanol was added. This addition-consumption technique was repeated and showed a typical fedbatch fermentation profile. This gave a high biomass concentration (60 g dry cell weight base/L) after six feeding cycles (Figure 1). 2
2
2
4
2
3
4
4
Figure 1. Biomass production. The grown cells were harvested by centrifugation. They were washed with 0.1M potassium phosphate buffer (pH 7.5), resuspended in the buffer and stored at -20°C until use. Conversion of alcohols. Grown cells having high alcohol oxidase activity were harvested. The cells and substrate alcohols were added to potassium phosphate buffer under pure oxygen atmosphere. The biomass concentration used was 30 g dry cell weight base /L and that of substrates was from 30 g/L. The reaction mixtures were kept at 25°C. The reaction time varied depending on substrates and conversion ratio. The reaction was then terminated by removing the cells from the reaction mixture by centrifugation. The supernatant was distilled under atmospheric pressure or extracted with ethyl acetate and concentrated at 40°C/20mmHg. This gave the crude products. The crude products were subjected to further purification (distillation or silica gel chromatography) to afford the products. The products by this microbial oxidation were analyzed by TLC, GLC and GC/MS. Some were analyzed by NMR. Conversion was calculated from GLC area ratio.
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Results Chemo-selectivity Oxidation of Primary Alcohols. Candida boidinii SA051 oxidized primary alcohols to the corresponding aldehydes effectively. In the case of 3-methyl-lbutanol oxidation, generated isovaleraldehyde was up to 50 grams per liter medium. Thus high yield of the aldehyde was obtained but was the equimolar amount of H 0 produced? The strain had strong catalase activity and therefore H 0 accumulation was not observed. The oxidation proceeded smoothly even at high substrate concentration. Primary alcohols used and the relative conversion ratio to the corresponding aldehydes are shown in Table I. In this system, the corresponding acids were not detected. 2
2
2
2
Table I. Oxidation of primary alcohols. Relative activity (%)
Substrate methanol ethanol 1-propanol 1-butanol 1-pentanol 1-hexanol 1-heptanol 1-octanol 1-nonanol
2-methyl-1 -propanol 3-methyl-1-butanol 2-methyl-1-butanol allyl alcohol (E)-2-pentenol (E)-2-hexenol (E)-2-heptenol (E)-2-octenol benzyl alcohol cinnamyl alcohol
80 100 60 80 64 42 23 19 5
68 33 15 65 61 59 52 37 11 4
substrate: 3 % w/v SA051:30g/L time:2hrs
Table I suggests that shorter chain alcohols were oxidized faster than longer chain ones. In the case of alcohols with the same carbon number, the straight chain alcohols were oxidized faster than branched chain ones and the oc,p unsaturated alcohols were oxidized faster than straight chain ones. These observations were applied to the selective oxidation of C6 alcohols. The oxidation profile of C6 alcohols by Candida boidinii SA051 whole cells is shown in Figure 2.
-•-
hexanal
-#-
(E)-2-hexenal
—A—
(Z)-2-hexenal (E)-3-hexenal
—o—
1 time
2 (hrs)
(Z)-3-hexenal
biomass 33g/L substrate 30g/L
Figure 2. Oxidation profile of C6 alcohols by Candida boidinii SA051.
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Microbial Oxidation of Alcohols by Candida boidinii
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This figure demonstrates that among the C6 alcohols, (E)-2 hexenol was oxidized fastest, followed by hexanol, (Z)-2 hexenol, (E)-3 hexenol and (Z)-3 hexenol. Figure 3 shows that the oxidation profile of the C6 alcohols mixture composed of equal volumes of C6 alcohols. hexanal - • - (E)-2-hexenal -A—
(Z)-2-hexenal (E)-3-hexenal
— D — (Z)-3-hexenal biomass
33g/L
C-6 alcohol mixture 30 g/L
time (hrs)
Figure 3. Oxidation profile of C-6 alcohols mixture by Candida boidinii SA051.
In this system, surprisingly only (E)-2 hexenol was oxidized. Thus this observation was applied to developing natural green notes. The natural C6 alcohols mixture used was composed of (E)-2-hexenol, (Z)-3-hexenol and hexanol and was isolated as the top fraction of mint oils from distillation. In terms of green note, (E)-2-hexenol is less valuable. On other hand, its aldehyde form (E)-2-hexenal is quite an important component of green notes. Figure 4 shows Candida boidinii SA051 could selectively oxidize (E)-2-hexenol in the mixture to desirable (E)-2-hexenal.
hexanal (E)-2-hexenal hexanol (Z)-3-hexenol (E)-2-hexenol
0
0.5 time (hrs)
1
Figure 4. Oxidation profile of natural C-6 alcohols fraction.
Regio-selective Oxidation of Alcohols Oxidation of a,co-diols. In the field of organic synthesis, it is known that it is difficult to oxidize oc,co-diols to the corresponding co-hydroxy aldehydes since oxidizing agents used in organic synthesis can not differentiate both terminal hydroxy groups. However, it was expected that biocatalysts could distinguish the a
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hydroxy group from the co-one. Therefore, selective oxidation of oc,co-diols by Candida boidinii SA051 was investigated. Concerning the oxidation of 1,4butanediol and 1,5-pentanediol, this microbial oxidation gave the corresponding lactones in the broth at yields of 24% and 34%, respectively . It is known that 4hydroxybutanal and 5-hydroxypentanal transform themselves into the corresponding lactols easily. However, in the course of this oxidation, the lactols were not detected. This means that the lactols were rapidly oxidized to the corresponding lactones or that this oxidation afforded directly the lactones from the diols. Considering that Candida boidinii SA051 did not give acids from alcohols, these lactones should be produced by the microbial oxidation of the lactols. In the case of 1,6-hexanediol, this gave no product (Figure 5). This was caused by the substrate specificity of Candida boidinii SA051.
substrate: 3 % w/v SA051:30g/L, 6hrs.
Figure 5. Oxidation of a,(0-diols 1.
In the case of 1,7-heptanediol, 1,8-octanediol and 1,9-nonanediol, these only gave the corresponding ©-hydroxy aldehydes in the broth at yields of 52%, 75% and 60%, respectively (Figure 6). The corresponding lactols were not found.
yield1*1
yield*
52 %
14%
8
75
18
5.50
9
60
13
3.96
n=7
2
m a x
- production 4.12 g/L
1
2
substrate: * 0.1 w/v, * 3% SA051 :* 30 g/L, * 90g/L 1
2
Figure 6. Oxidation of a,co-diols 2.
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Microbial Oxidation of Alcohols by Candida boidinii
Neither dialdehydes nor dicarboxylic acids were detected and it was shown that this microbial oxidation had excellent regioselectivity . These generated co-hydroxy aldehydes could be used as starting materials for macrocyclic musk related compounds. The yields varied depending on the substrate concentration. Lower concentrations gave better yields. However, even at 3% concentration, the conversion ratio reached about 15% and ©-hydroxy aldehydes were produced at about 5g/L medium. This figure was sufficient for production. Stereoselective Oxidation of Alcohols Alkane Diols Having Primary and Secondary Alcohol Groups. This microbial oxidation was also applied to alkanediols having one terminal hydroxy group. With 1,2 alkanediols, this oxidation did not proceed. With 1,3 alkanediols, the oxidation proceeded and gave the corresponding keto alcohols. This showed that the yeasts preferred the secondary hydroxy group to the primary one. The oxidation of 1,3-butanediol gave 4-hydroxy-2-butanone at 75% yield. The oxidation of 1,3pentanediol gave 5-hydroxy-3-pentanone at 80% yield (Figure 7).
substrate:3 % w/v
SA051:30 g/L 6hrs.
Figure 7. Oxidation of alkane 1,3-diols. In the case of 1,3-butanediol, the remaining diol had slight optical activity. This showed (S)-configuration at 27% optical purity. This was determined by comparison of optical rotation with a reference (6). The yeasts could differentiate the (R)- and (S)-alcohol and oxidize the (R)-alcohol predominantly. With 1,4pentanediol, the yeasts preferred the primary hydroxy group to the secondary one and gave pentane-l,4-olide and its lactol at 6.3% yield. The afforded lactone showed slight optical activity and was the (R)-form at 5% optical purity. This was determined by comparison of optical rotation with a reference (7). This showed that the yeasts preferred (R)-l,4-pentanediol as the substrate. Oxidation of Meso Diols. Asymmetric induction of meso and prochiral diols by lipases is very successful in the field of organic synthesis. Also it is well known that selective oxidation of prochiral or meso diols by HLADH provides oxidized products with a significant degree of enantioselectivity. However, it has not been reported that alcohol oxidases were applied to such types of oxidation. The microbial oxidation of meso diols by Candida boidinii SA051 was carried out and gave optically active hydroxy ketones (Figure 8).
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yield
o.p.
61 %
72%
63%
93%
OH meso-form
substrate: 3 % w/v SA051:30 g/L 40hrs
Figure 8. Oxidation of meso-diols. Oxidation of me so 2,3-butanediol gave (S)-acetoin at 61% yield and its optical purity was 72%. This was determined by comparison of optical rotation with a reference (8). This is the antipode of the product into which meso 2,3-butanediol was transformed by lactic acid bacteria. Oxidation of meso and a racemic mixture of 2,4-pentanediol afforded 4-(R)-hydroxy-2-pentanone at 63% yield and its optical purity was 93%. This was determined by comparison of optical rotation with a reference (9). These results proved the feasibility of the stereoselective oxidation of alcohols by the yeasts. Oxidation of Prochiral Diol. The oxidation of prochiral diol 3-methyl-l,5pentanediol did not give the corresponding ©-hydroxy aldehyde but gave the corresponding lactol at 22% yield. To determine stereochemistry of the lactol, this was chemically oxidized by Ag 0 and afforded 3-(R)-methyl-pentan-l,5-olide at 38% optical purity. (Figure 9). This was determined by comparison of optical rotation with a reference (70). 2
yield
o.p.
22 %
38%
Figure 9. Oxidation of 3-methyl-l,5-pentanediol. Conclusion The methylotropic yeast Candida boidinii SA051 showed excellent ability for oxidation of alcohols to aldehydes or ketones. In the production of isovaleraldehyde, the generated aldehyde was up to 50 grams/L. Also this oxidation showed reaction selectivities. It was an example of chemoselectivity that the yeasts preferred (E)-2hexenol among various C6 alcohols and oxidized it selectively to the desired (E)-2-
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hexenal. It was an example of regioselectivity that a,co-alkanediols were oxidized to the corresponding co-hydroxy aldehydes. It was an example of stereoselectivity that some diols were stereochemically distinguished as substrates and that some meso or prochiral diols were oxidized and gave one enantiomer predominantly. These results mentioned above showed that this microbial oxidation is useful for production of flavor chemicals and related compounds. Acknowledgment We thank Prof. Y . Tani and Prof. Y . Sakai for generous gift of Candida boidinii SA051. Literature Cited 1.
2. 3. 4. 5
(a)Sakai, Y.; Tani ,Y. Agric. Biol. Chem. 1987, 51(9), 2617-2620. (b)Shachar-Nishri, Y.; Freeman, A. Appl. Biochem. Biotechnol. 1993, 39/40, 387-399 (c) Clark, D.S., et al. Bioorganic & Medicinal Chem. Lett., 1994, 4, 1745-1748. Murray, W.D.; Duff, S.J.B.; Lanthler, P.H. Production of natural flavor aldehydes from natural source primary alcohols C2-C7, USP, 4871669, 1989. Mauersberger, S. et al. Appl. Microbiol. Biotechnol. 1992, 37, 66-73. Sakai, Y.; Tani, Y . Agric. Biol. Chem. 1987, 51(8), 2177-2184.
Nozaki, M . ; Suzuki, N.; Washizu, Y . In Bioflavour95; Étiévant, P.; Schreier, P.,Eds.; INRA editions: France, 1995; pp 255-260. 6 Dictionary of Organic Compounds, 5th ed., Chapman and Hall. 7. Mori, K. Tetrahedron 1975, 31, 3011. 8. Taylor, M.B.; Juni, E. Biochim. Biophys. Acta. 1960, 39, 448. 9. Ohta, H., et al. Agric. Biol. Chem. 1986, 50, 2499. 10. Jones, J. B. J. Am.Chem. Soc. 1977, 99, 556.
