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Table 4
References and notes
ANOVA for EBA bioreduction by R. rubra immobilized cells
1. Chaubey, A.; Parshad, R.; Koul, S.; Taneja, S. C.; Qazi, G. N. Appl. Microbiol.
Biotechnol. 2006, 73, 598–606.
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5140.
3. Yang, W.; Xu, J. H.; Xie, Y.; Xu, Y.; Zhao, G.; Lin, G. Q. Tetrahedron: Asymmetry
2006, 17, 1769–1774.
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480–485.
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C. Catal. Commun. 2005, 6, 131–133.
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9253–9254.
Factor*
Sum of squares
df
Mean squares
F-ratio
p-value
Cell
diam
EBA
MgCl2
2366.72
115.52
1997.12
23.12
1.62
11.52
544.50
6.56
10.41
1
1
1
1
1
1
1
1
2
2366.72
115.52
1997.12
23.12
1.62
11.52
544.50
6.56
454.85
22.20
383.82
4.44
0.31
2.21
0.002
0.042
0.003
0.170
0.633
0.275
0.009
0.378
G
Cell ꢀ diam
Cell ꢀ EBA
Lack of fit
Pure error
Total (corr.)
104.64
1.26
5.20
5077.08
10
7. Microorganisms, media, growth conditions, and biotransformation with free
cells. Hansenula sp., Kluyveromyces marxianus, Candida sp., Pichia sp., three
strains of Saccharomyces cerevisiae, Rhodotorula rubra, Rhodotorula minuta,
filamentous fungi, Aspergillus niger, Trichoderma harzianum and Mucor
ramannianus, belong to the collection of the ‘Departamento de Engenharia
Bioquímica, Escola de Química, UFRJ’ and are freely available upon request.
Cells were allowed to grow for 48 h, under 150 rpm and 30 °C in a medium
containing 1% glucose, 0.5% yeast extract, 0.5% peptone, 0.1% (NH4)2SO4, and
0.1% MgSO4ꢂ7H2O. After that period, they were harvested by centrifugation, re-
suspended in distilled water and used for the reaction. After centrifugation, the
cells (4 g/L, dried weight) were added to the reduction’s medium containing:
glucose (5%), MgCl2 (0.1%) in a final volume of 100 mL. The initial pH of the
reaction’s medium was 5.5. After 30 min of addition of the microorganisms, the
substrate (0.5%) in aqueous-ethanol was added to the medium. The reaction
was carried out in 500 mL cotton plugged Erlenmeyer flasks for 24 h at 30 °C
and 150 rpm. After 24 h, the cells and the liquid phase were separated by
centrifugation. The liquid phase was extracted with ethyl acetate. The organic
phase was dried (anhydrous Na2SO4), filtered, and concentrated under vacuum.
Products were analyzed by (chiral) gas chromatography (GC), on column BGB-
Response: % conversion (r2 = 0.99, r2 adjusted for df = 0.98).
Cell (biomass concentration—g dry weight/L); diam (diameter of calcium alginate
spheres); G (glucose).
*
A
B
C
176B (25 m ꢀ 0.25 mm ꢀ 0.25 l
m), at 140° C, and confirmed by 1H nuclear
magnetic resonance (NMR) and infrared (IR) spectroscopy.
8. Milagre, C. D. F.; Milagre, H. M. S.; Moran, P. J. S.; Rodrigues, J. A. R. J. Mol. Catal.
B: Enzym. 2009, 56, 55–60.
9. Chênevert, R.; Fortier, G.; Rhlid, R. B. Tetrahedron 1992, 48, 6769–6776.
10. He, C.; Chang, D.; Zhang, J. Tetrahedron: Asymmetry 2008, 19, 1347–1351.
11. Ribeiro, J. B.; Ramos, M. C. K. V.; Aquino Neto, F. R.; Leite, S. G. F.; Antunes, O. A.
C. J. Mol. Catal. B: Enzym. 2003, 24–25, 121–124.
12. Ramos, A. S.; Ribeiro, J. B.; Vazquez, L.; Fiaux, S. B.; Leite, S. G. F.; Cruz, R. A.;
Ramos, M. C. K. V.; Aquino Neto, F. R.; Antunes, O. A. C. Tetrahedron: Asymmetry
2009, 20, 559–561.
13. Nakamura, K.; Yamanaka, R.; Matsuda, T.; Harada, T. Tetrahedron: Asymmetry
2003, 14, 2659–2681.
10
12
14
16
tR (min)
Figure 2. Chiral GC analysis on column BGB-176B (25 m ꢀ 0.25 mm ꢀ 0.25
lm), at
140 °C: (A) EBA; (B) S-HPPE (racemate obtained via NaBH4 reduction); (C)
immobilized R. rubra reduction (reaction conditions selected according fractionary
factorial design).
14. Ema, T.; Moriya, H.; Kofukuda, T.; Ishida, T.; Maehara, K.; Utaka, M.; Sakai, T. J.
Org. Chem. 2001, 66, 8682–8684.
15. Ishihara, K.; Yamaguchi, H.; Nakajima, N. J. Mol. Catal. B: Enzym. 2003, 23, 171–
189.
16. Buque, E. M.; Chin-Joe, I.; Straathof, A. J. J.; Jongejan, J. A.; Heijnen, J. J. Enzyme
Microb. Technol. 2002, 31, 656–664.
glucose concentration had no significative effect on response and it
was used at minimum level.
17. Immobilization of cells (Saccharomyces cerevisiae 40, Kluyveromyces marxianus,
Rhodotorula rubra, Candida sp., Hansenula sp., and Pichia sp.) in calcium alginate
and biotransformation. Cells grown during 48 h in the medium described
before7 were harvest by centrifugation and re-suspended in distilled water to
obtain a cell-suspension. A sodium alginate aqueous solution was added and
this mixture (cell-suspension + 1.5% w/v sodium alginate aqueous solution)
was dropped in a CaCl2 aqueous solution (0.1 M) to form calcium alginate
spheres. Spheres were filtered, washed with distilled water and added to the
medium for reduction. Reduction was carried out in 500 mL cotton plugged
Erlenmeyer flasks, at 30 °C, 150 rpm during 24 h. After that period, medium
was filtered to separate the biocatalyst and the liquid phase was treated as
described above.7
18. Logothetis, N. Managing for Total Quality—From Deming to Taguchi and SPC;
Prentice Hall International Ltd: London, 1992.
19. Lundstedt, T.; Seifert, E.; Abramo, L.; Thelin, B.; Nyström, A.; Pettersen, J.;
Bergman, R. Chemom. Intell. Lab. Syst. 1998, 42, 3–40.
20. Fiaux de Medeiros, S.; Avery, M. A.; Avery, B.; Leite, S. G. F.; Freitas, A. C. C.;
Williamson, J. S. Biotechnol. Lett. 2002, 24, 937–941.
Based on these results, the following conditions were selected
to EBA reduction by immobilized cells of R. rubra: biomass, 6.0 g
dry weight/L; diameter, 3.8 mm; EBA, 26 mM; glucose, 10 g/L.
Experiments, performed in triplicate, were conducted to verify
conversion level and enantiomeric excess in the reaction condi-
tions described above. After 18 h, it was obtained 81% conversion
(SD = 1.3%) and 100% ee. This result matched the model prediction.
The high stereoselectivity achieved in this reaction is shown in Fig-
ure 2. Optimization studies are in progress.
In conclusion, a screening study indicated some wild yeast
strains with excellent enantioselectivity in EBA reduction. Beyond
providing good and easy recovery of product, immobilized cells in
calcium alginate spheres led to higher conversion levels in compar-
ison with free cells used. After a fractionary factorial design to
study some reaction’s conditions, calcium alginate immobilized
cells of R. rubra furnished 81% conversion with 100% ee. There
are few studies with immobilized yeasts in EBA reduction and, to
our knowledge, this is the first Letter on the use of R. rubra for
enantioselective reduction of EBA.
21. Experimental design: A 25-2 fractionary factorial design18 was used to study five
variables in eight runs with three replicates of the central point. Variables and
domain were: biomass concentration (Cell): 2.0–6.0 g dry weight/L; diameter
of calcium alginate spheres (diam): 2.4–3.8 mm; EBA: 26–52 mM; MgCl2: 0–
2 g/L; glucose: 10–50 g/L. Response variables were % conversion and % ee. Cells
of R. rubra, obtained as described above,7 was re-suspended 1.5% sodium
alginate solution (50 mL). Immobilization was performed as already
described.17 After 30 min of immobilization, the substrate in aqueous-
ethanol was added to the medium. Reaction’s medium composition was
according experimental design. The reaction was carried out in 500 mL cotton
plugged Erlenmeyer flasks containing 100 mL of medium for 18 h at 30 °C and
150 rpm. After that period, medium was filtered to separate the biocatalyst and
the liquid phase was treated as described above.7 Statistical analyses were
performed using Statistica 6.0 (Statsoft Inc., Tulsa, OK, USA).
Acknowledgments
Financial support from FAPERJ and CNPq—BRAZIL is acknowl-
edged. Analytical support from DQO-IQ-UFRJ and DQI-IQ-UFRJ is
also acknowledged.
22. Houng, J. Y.; Hsu, H. F.; Liu, Y. H.; Wu, J. Y. J. Biotechnol. 2003, 100, 239–250.