CHAPTER III: ENZYMATIC FRUCTOSYLATION OF FLAVONOIDS USING β-
B. Results and discussion
B.4. Cloning and expression of the new β-fructosidase from Rhodotorula mucilaginosa for
B.4. Cloning and expression of the new β-fructosidase from Rhodotorula mucilaginosa for
Figure 16. Electrophoresis gel of analysis of restriction of pUC57-RhInv, Lane 1 ladder 10 kbp, lane 2, 3 digest of pUC57-RhInv (β-fructosidase from R. mucilaginosa) using EcoRI and SalI.
Figure 17. Electrophoresis gel for the purification of β-fructosidase gene Lane 1: ladder 10 kpb, Lane 2 and 3:
Gene of β-fructosidase from R. mucilaginosa.
10,000 bp
1500 1000 700
500
250 2000 3000
1 2 3
1 2 3 10,000 bp
1500 1000 700 500 250 2000 3000
Next, RhInv was introduced into the pGAPZB vector using a T4 ligase (figure 18). Electrophoresis gel shows that at initial time of this reaction, two bands were observed (Lanes 2 and 3 in Figure 18), which corresponded with empty pGAPZB vector (3,100 bp) and RhInv gene (1,600 bp). After 12 h of reaction with T4 ligase at 16 ºC, electrophoresis gel showed the disappearance of the band corresponding to RhInv and only one band can be observed per lane with an increase of the weight around 4,500 bp, showing that it was possible to link RhInv into the expression vector (pGAPZB- RhInv). The construct pGAPZB-RhInv was then propagated into competent cells of E. coli DH5α by electroporation. Figure 19 presents low-salt LB plates with zeocin (25 μg/μL) with positive colonies in pGAPZB-RhInv and pGAPZB, also in the negative control was not observed any colonies.
Figure 18. Electrophoresis gel for RhInv ligation reaction into expression vector (pGAPZB). Lane 1: ladder 10 kbp, lane 2, 3, ligation at initial time of pGAPZB and RhInv, lane 4, 5 at final time of pGAPZB-RhInv.
Figure 19. Propagation of the construct into E. coli DH5α in low-salt LB plates with zeocin (25 μg/μL) a) pGAPZB-RhInv, b) pGAPZB and c) negative control.
a) b) c)
1 2 3 4 5
10,000 bp
1500
1000 700 2000 4000
500
250
The construct pGAPZB-RhInv and the empty vector pGAPZB were linearized and they were introduced into competent Pichia pastoris X-33 cells by electroporation. Figure 20 shows zeocin resistant Pichia transformants colonies containing pGAPZB-RhInv, the empty vector pGAPZB and the negative control. Then, some colonies were subjected to the verification of the insert using PCR method, thus it is expected the weight of PCR product for the empty vector of 275 bp and for the construct (pGAPZB-Rh) a value of 1,928 bp (see Figure 21). As can be observed in line 3, a PCR product with an approximate size of 250 bp, which corresponds to the size of the amplification between the AOXI region and pGABZB of the empty vector; in line 2 a band with a size close to 2000 bp is observed (Figure 21), which suggests that the insert is found in the construction pGAPZB-Rh.
Therefore, it can be suggested that zeocin resistant colonies have the pGAPZB-RhInv construct integrated into the genome.
Figure 20. YPDS plates with 100 μg/mL of zeocin resistant Pichia pastoris X-33 transformants containing a) pGAPZB-RhInv, b) empty pGAPZB and c) negative control.
Figure 21. Electrophoresis gel for PCR products. Lane 1: ladder 10 kbp, lane 2: pGAPZB-RhInv and RhInv, lane
a) b) c)
10,000 pb
500 1000 15002000 3000
1 2 33
50008000
4
Subsequently, the zeocin resistant colonies that resulted from the integration of plasmid pGAZB-RhInv and pGABZB were evaluated by phenotypic screening (Figure 22) using a colorimetric method. A change in the color of the medium from initial green (pH 6.5) to yellow (pH ≤ 6.0) indicates an acidification of the medium due to the formation of organic acids during the catabolism of glucose and fructose released in the sucrose utilization. A blue color (pH ≥ 7.5) suggest an increase of the pH as a consequence of the ammonia released by the catabolic oxidation of the nitrogen containing carbon sources yeast extract and peptone (Menéndez et al. 2013). Thus, as expected cells electroporated with the empty vector (pGAZB) and the negative control (Picha pastoris X-33) showed the non- saccharolytic phenotype revealed by blue color in the growth medium. Yellow color in the growth medium indicates colonies expressing active RhInv (Figure 22). Therefore, the colonies that contained active RhInv were selected in order to produce the enzyme by liquid fermentation.
Figure 22. Phenotypic screening of zeocin resistant colonies transformed with a) negative control (P. pastoris X- 33), b) pGAPZB and c) pGAPZB-RhInv and d) pGAPZB-RhInv (lane 2 in blue is empty).
Table 22 shows the results obtained from the fructosidase activity in the supernatant from P. pastoris X-33, Pichia transformed with pGAPZB and pGAPZB-Rh. As expected only the supernatant from Pichia transformed with the construction pGAPZB-RhInv showed fructosidase activity with a value 28.0 ± 2.3 U mL-1. No fructosidase activities were detected from the supernatant from P. pastoris X-33 and Pichia transformed with the empty vector pGAPZB. As expected these results are in accordance to the previous results observed in the phenotypic screening.
a) b) c) d)
Table 22. Fructosidase activity in the supernatant from P. pastoris X-33, zeocin resistant Pichia transformed with pGAPZB and pGAPZB-Rh.
ND: Non detected
In summary, it may be inferred from the results obtained from the cloning and expression of the β- fructosidase from Rhodotorula mucilaginosa (RhInv) that it was possible to express an active protein with fructosidase activity using P. pastoris as expression host.
Strain Fructosidase activity (U mL-1) P. pastoris X-33 ND
pGAPZB ND
pGAPZB-RhInv 28.0 ± 2.3
B.5. Enzymatic fructosylation of flavonoids by a recombinant β-fructosidase from
Rhodotorula mucilaginosa (RhInv)The following section describes the results obtained from the enzymatic fructosylation of flavonoids using the recombinant β-fructosidase of Rhodotorula mucilaginosa in order to know its capacity to fructosylate different acceptors, such as puerarin, coniferyl alcohol and mangiferin.
Figure 23 shows the chromatograms obtained from the enzymatic fructosylation of puerarin, where the presence of a new peak (P1) can be observed with a retention time of 6.7 min, which does not appear at the beginning of the reaction. The mass spectrum of this peak reveals that the value of this peak is [M- H]- [m/z] = 577.2, which corresponds to the mass of fructosyl puerarin. Moreover, when coniferyl alcohol was tested as acceptor, the chromatogram revealed after 10 h of reaction a new peak (C1) just before the peak corresponding to coniferyl alcohol (C), which has a retention time of 8.3 min. The molar mass determined by mass spectrometry corresponds to a value of [M-H]- [m/z] = 341, which suggest the addition of a fructose moiety onto coniferyl alcohol (Figure 24). In addition, the enzymatic fructosylation of mangiferin was carried out. The results are shown in figure 25, and a small peak (M1) appears at the end of the reaction with a molar mass value of [M-H]- [m/z] = 583.2 and a retention time of 5.9 min. These results suggest the fructosylation of mangiferin. Therefore, it may be inferred that RhInv is able to fructosylate the tested acceptors. However, in the case of these three acceptors, only the synthesis of new mono fructosides was observed as result of the synthesis.
The substrate conversion for puerarin, coniferyl alcohol and mangiferin are summarized in table 23. As observed, the highest substrate conversion percentage was obtained with coniferyl alcohol (20 % ± 3), while mangiferin had the lowest value around 1.5 % ± 0.7 and puerarin obtained only 10 % ± 1.9. In the literature, Dudíková et al. (2007) reported that coniferyl alcohol could be fructosylated. They reported the fructosylation of coniferyl alcohol using Cryptococcus laurentii cell walls containing β- fructosidases; the reaction was carried out in the presence of 30% of acetone (v/v) used as co-solvent and 200 mM coniferyl alcohol, thus around 2% of conversion was achieved (Dudíková et al. 2007).
However, the advantage of our method is the absence of a co-solvent. Moreover, the quantity of the mono fructosyl coniferyl alcohol was 2.5 mM using RhInv and 4 mM using Cryptococcus laurentii cell walls. In both cases the conversion is low; the ability to fructosylate this acceptor are similar using these β-fructosidases.
The synthesis of puerarin fructosides using β-fructosidases has been reported in the literature. Wu et al.
(2013a) reported that mono- and di-fructosides were synthetized using a β-fructosidase from Arthrobacter nicotianae at 265.5 mM of puerarin after a reaction time of 72 h in the presence of 25%
of DMSO as co-solvent with a substrate conversion of 91.0%. Indeed, this percentage of conversion is higher than the one obtained using RhInv (10 %). It suggests that β-fructosidase from Rhodotorula mucilaginosa (RhInv) is less efficient to carry out this reaction. In addition, the mono fructosyl puerarin was the only product; no traces of the di fructosides were detected as in the case of the work of Wu et al. (2013a).
In the case of mangiferin, percentage of conversion was much lower (1.5 %) than the value reported by Wu et al. (2013b). According to the literature, they achieved a percentage of conversion of 67.5%
from 112 mM of acceptor using the β-fructosidase from Arthrobacter arilaitensis in DMSO (20% v/v), indicating that the β-fructosidase from Arthrobacter arilaitensis recognizes mangiferin more efficiently than β-fructosidase from Rhodotorula mucilaginosa (RhInv).
Table 23. Percentage of substrate conversion of different acceptors using RhInv at 1 U mL-1, 14 mM of acceptor, 877mM of sucrose (100 mM acetate buffer pH 5.5) during 10 h at 40 ºC and 600 rpm.
*The conversion rate was calculated from the amount of acceptor remaining. % conversion = 100*([acceptor] initial – [acceptor] final)/ [acceptor] initial.
Compound Structure % Conversion
Puerarin 10 ± 1.9*
Coniferyl alcohol 20 ± 3*
Mangiferin 1.5 ± 0.7*
O
O O HO
OH OH
OH
OH HO
O
HO
OH
O
O O
HO
HO
OH OH
OH OH HO
OH
Figure 23. a) LC Chromatograms from screening of enzymatic fructosylation of puerarin using RhInv at 1 U mL-
1, 14 mM puerarin, 877mM of sucrose (100 mM acetate buffer pH 5.5) during 10 h at 40 ºC and 600 rpm. b) ESI- MS spectra in negative mode of new peak. (P) Puerarin; P1, mono-fructosyl puerarin.
a)
b)
Time (min)
Absorbance (mAu)
P P1
t=0 h t= 10 h
m/z=415.2 22
Relative abundance (%)
P
m/z=577.0 P1
Relative abundance (%)
Figure 24. a) LC Chromatograms from screening of enzymatic fructosylation of coniferyl alcohol using RhInv at 1 U mL-1, 14 mM coniferyl alcohol, 877mM of sucrose (100 mM acetate buffer pH 5.5) during 10 h at 40 ºC and a)
b)
t=10 h t=0 h C
C1
Time (min)
Absorbance (mAu)
C
Relative abundance (%) m/z=179.2
m/z=342.1 C1
Relative abundance (%)
Figure 25. a) LC Chromatograms from screening of enzymatic fructosylation of mangiferin using RhInv at 1 U mL-1, 14 mM mangiferin, 877mM of sucrose (100 mM acetate buffer pH 5.5) during 10 h at 40 ºC and 600 rpm.
b) ESI-MS spectra in negative mode of new peak. (M) Mangiferin; (M1), mono-fructosyl mangiferin.
a)
b)
M M1
Time (min)
Absorbance (mAu)
t=0 h t=10 h
m/z=421.3 M
Relative abundance (%)
m/z= 583.2 M1
Relative abundance (%)
In summary, it may be inferred from the results of the reactions of the enzymatic fructosylation of flavonoids using the β-fructosidase from Rhodotorula mucilaginosa (RhInv) that it is possible to fructosylate acceptors other than sucrose but with low yields around 20%. The best yield was obtained in the case of coniferyl alcohol. Finally, it is necessary to biochemically characterize the enzyme and then perform the optimization of the enzymatic fructosylation reactions of flavonoids to further improve the conversion yields.