CHAPTER III: ENZYMATIC FRUCTOSYLATION OF FLAVONOIDS USING β-
C. Experimental methods
C.3.2. Fructosyltransferase activity
Fructosyltransferase activity was measured in the enzymatic extracts for selected yeast strains in order to confirm the capacity to perform the synthesis of fructooligosaccharides, which can be performed when the equilibrium of the reaction is displaced to a high concentration of sucrose and a low concentration of water. The reaction was carried out in 1 mL that contains 900 μL of 600 g L-1 of sucrose in acetate buffer pH 5 and 100μL of each enzymatic extract at 45 ºC, 100 rpm during 120 min.
Then, the microtubes were heating at 95 ºC during 5 min, in order to stop the reactions. Then the samples were diluted 1:100 and analyzed by HPLC. It was used a DIONEX Ultimate 3000 equipped with a Shodex refractometer IR. The column was a Biorad Carbohydrate Analysis column (Animex HPX-87K, 300 x 7.8 mm, USA), the mobile phase was ultrapure water and the flow rate was 0.65 mL min-1; the temperature of the oven was 65 ºC. Glucose, fructose and sucrose were used as standards.
Data acquisition and processing were performed with Chromeleon 6.8 version. For each reaction, the moles of sucrose, glucose and fructose were calculated. The fructosyltransferase activity was calculated considering the difference between moles of glucose and fructose (equivalent to the moles of fructose transferred, moles of sucrose transfructosylated). One unit of enzyme activity was defined as the amount of enzyme transferring 1 μmol of sucrose per minute (Arrizon et al. 2012).
C.4. Screening of enzymatic fructosylation of different flavonoids by enzymatic extracts from non-Saccharomyces yeasts
The screening of enzymatic fructosylation of different flavonoids was carried out with 5 different types of flavonoids such as quercertin, (+)-Catechin, Luteolin, Puerarin and Hesperidin. The final reaction volume of the mixture was 1 mL with a flavonoid concentration of 5 mM in 20 % of DMSO and 408 mM of sucrose (100 mM acetate buffer pH 5) at 45 ºC, during 24 h and 600 rpm. For all the enzymatic extract, the fructosidase enzymatic activity was 0.5 U.mL-1. Then, in order to stop the reaction, the microtubes were boiled at 95 ºC during 5 min.
C.5. Quantification of percentage of conversion of flavonoids by Liquid Chromatography-Mass Spectrometry (LC-MS)
Flavonoids and fructosylated products were analyzed by LC-MS using a Dionex Ultimate 3000 series
formic acid 0.05%) and solvent B (Acetonitrile with formic acid 0.05 %), the gradient started from 10
% B to 50 % over 20 min and from 50 % B to 95% B during 5 min, the flow rate was 1 mL min-1. The wavelengths in the UV detector were 254 and 350 nm. The mass spectrometer was in negative and positive mode with a voltage cone of 50 and 110 V and the range of mass was between 100 and 1000.
Temperature of the electrospray was 450 ºC and the gas carrier was nitrogen.
C.6. Search of a new β-fructosidase gene sequence in silico from available genomes
In this section, the methodology for the search of a new of β-fructosidase gene sequence is described,it was carried out in silico from available genomes (2017). Figure 26 shows the general diagram of this procedure. The considerations for the search of a new sequence in silico were the following: Biochemical evidence of enzymatic fructosylation of puerarin in the enzymatic extracts
No reported fructosidase sequence at CAZy or KEGG database
Genome sequenced available in a database
Then, it was performed a dendrogram in PhiloT: a tree generator (http://phylot.biobyte.de) with the selected species yeast with fructosidase reported sequences in order to have a reference sequence for the in silico search in the genome. Thus, it was download the genome and the sequences of reported fructosidases from NCBI (https://www.ncbi.nlm.nih.gov). Afterwards, it was performed using BLAST tool in CLC Genomics Workbench 9.5 a BLAST in genome; it was carried out with β-fructosidase sequence close to the yeast according to the phylogenetic tree.
Thus, the alignment of the gene in the genome in a in a scaffold or counting were analyzed by Softberry (http://www.softberry.com) in order to gene prediction using the tool of FGENESH+
(Solovyev, 2007). Finally, in order to confirm if the predicted gene correspond to a β-fructosidase, it was carried out a bioinformatics analysis of the sequence.
Figure 26. General schema of in silico search of β-fructosidase gene in yeast genome.
Download genome from NCBI
Dendrogram of yeast species with fructosidases reported
Download sequences of fructosidases reported from
NCBI
Blast gene in genome in CLC Genomics Workbench 9.5
GenePredictionbySoftberry
Predicted gene Blast NCBI, Pfam, Interpro
Yeast Selection
Search in predicted gen GH32
Motif, catiltyc site and signal peptide Molecular model, pI,
molecular mass
by SWISS-MPDEL Boxshade
Analysis
C.7. Cloning and expression of β-fructosidase from Rhodotorula mucilaginosa in Pichia
pastoris X-33Cloning and expression of β-fructosidase from R. mucilaginosa was carried out using all the protocols reported in pGAPzα A, B, & C and pGAPZ A, B, & C Pichia expression vectors for constitutive expression and purification of recombinant proteins manual from Invitrogen.
β-Fructosidase gene from R. mucilaginosa (RhInv) was optimized in its codon usage for P. pastoris and synthetized into a puc57 plasmid (GenScript, USA) with flanking EcoRI and SalI. Then, E. coli DH5α was used as a cloning host for plasmid propagation and Pichia pastoris X-33 (wild-type) was used as the expression host. PUC57-RhInv plasmid was introduced into competent E. coli strain DH5α (Invitrogen) by electroporation (2100 V, 100 Ω, 25 μF). Then, E. coli DH5α transformants were grown at 37 ºC in low-salt LB (10 g L-1 peptone, 5 g L-1 yeast extract and 5 g L-1 NaCl) plates with ampicillin (1μg/μL) during an overnight. Ampicillin resistant transformants were inoculated in 2 mL of low-salt LB medium with ampicillin (1μg/μL) at 37ºC and 250 rpm for 14 h. Later, the DNA plasmid was isolated by miniprep (QUIAGEN plasmid miniprep Kit) for restriction analysis using EcoRI and SalI (Thermofisher). Afterwards, the purification of the gene was carried out by gel purification (QUIAGEN Gel purification kit).
Ligation of β-fructosidase gene to expression vector pGAPZB (Invitrogen) was carried out using T4 ligase (Sigma-Aldrich) at 16 ºC during 12 h of reaction. The E. coli DH5α was transformed with ligation reaction and inoculated in low-salt LB plates with Zeocin (25 μg/μL). Zeocin resistant transformant colonies were chosen for isolation of plasmid DNA by miniprep (QUIAGEN plasmid miniprep Kit) and for subsequent restriction analysis. Then, it was prepared 5-10 μg of plasmid DNA and BspHI was used to linearize the pGAPZB, pGAPZB-RhInv plasmids were introduced into competent P. pastoris X-33 cells by electroporation (1500 V, 200 Ω, 25 μF). Then, transformants cells were inoculated in YPDS plates (10 gL-1 yeast extract, 20 g L-1 peptone, 20 g L-1 dextrose and 1 M sorbitol) with Zeocin (100 μg/mL) and incubated at 30 ºC during 48 h. Zeocin resistant Pichia transformant colonies were subjected to the verification of the insertion using the PCR method. The primers were pGAP forward (5’-GTCCCTATTTCAATCAATTGAACAAC-3’) and 3'AOX1 (5’- GCAAATGGCATTCTGACATCC- 3’) as recommended in the invitrogen manual.
Then, it was carried out a phenotypic screening was carried out in Zeocin resistant Pichia transformants expressing active RhInv. It was used a solid SPY medium (5 % (w/v) sucrose, 2 % (w/v) peptone, 1 % (w/v) yeast extract, and 0.025 % (w/v) bromothymol blue, 1.5 % (w/v) agar, pH 6.5) reported by Menéndez et al. (2013).
Thus, positive Zeocin resistant Pichia transformants expressing active RhInv were inoculated in the expression media (10 g L-1 yeast extract, 20 g L-1 peptone, 0.5 g L-1 glycerol and 50 g L-1 sucrose) at 30 º C, 200 rpm for 72 h (Hernández et al. 2018). Then, the culture was centrifuged at 6000 rpm and 4 ºC during 20 min. Finally, the fructosidase activity was measured (see section C.3.1) in the supernatant.
The supernatant containing invertase activity was directly used in order to evaluate its capacity to fructosylate flavonoids.
C.8. Enzymatic fructosylation of flavonoids by a recombinant β-fructosidase (RhInv) from Rhodotorula mucilaginosa
The fructosylation of different flavonoids is carried out using a recombinant β-fructosidase (RhInv) from Rhodotorula mucilaginosa in order to know the capacity of this enzyme to fructosylate different acceptors. The reactions were performed using 877 mM of sucrose (300 g L-1), 14 mM of acceptor (puerarin, phlorizin, mangiferin and coniferyl alcohol) in 50 mM 100 mM acetate buffer pH 5.5, in presence of 1 U mL-1 of RhInv during 10 h at 40 ºC. The reactions mixtures were stopped by boiling at 95 ºC during 10 min. Then the samples were diluted with DMSO and analyzed by HPLC-MS (see section C.5). Percentage of conversion is calculated, taking into account the difference between initial and final concentration of acceptor.
% Conversion = ([Acceptortinitial]– [Acceptortfinal]/ [Acceptortinitial]) x100