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Characterization of a mycelial fructosyltransferase from Aspergillus tamarii NKRC 1229 for efficient synthesis of fructooligosaccharides
Accepted Manuscript Characterization of a mycelial fructosyltransferase from Aspergillus tamarii NKRC 1229 for efficient synthesis of fructooligosaccharides Ritumbhara Choukade, Naveen Kango PII: DOI: Reference:
S0308-8146(19)30339-5 https://doi.org/10.1016/j.foodchem.2019.02.025 FOCH 24336
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
10 October 2018 4 February 2019 6 February 2019
Please cite this article as: Choukade, R., Kango, N., Characterization of a mycelial fructosyltransferase from Aspergillus tamarii NKRC 1229 for efficient synthesis of fructooligosaccharides, Food Chemistry (2019), doi: https://doi.org/10.1016/j.foodchem.2019.02.025
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Characterization of a mycelial fructosyltransferase from Aspergillus tamarii NKRC 1229 for efficient synthesis of fructooligosaccharides
Ritumbhara Choukade and Naveen Kango* Enzyme Technology and Molecular Catalysis Laboratory, Department of Microbiology Dr. Harisingh Gour Vishwavidyalaya (A Central University), Sagar (Madhya Pradesh) 470003, India
Authors Information Ritumbhara Choukade Enzyme Technology and Molecular Catalysis Laboratory, Department of Microbiology Dr. Harisingh Gour Vishwavidyalaya (A Central University), Sagar (Madhya Pradesh) 470003, India Phone No. +91 9644430994 e-mail: [email protected] Prof. Naveen Kango Designation: Professor Enzyme Technology and Molecular Catalysis Laboratory, Department of Microbiology Dr. Harisingh Gour Vishwavidyalaya (A Central University), Sagar (Madhya Pradesh) 470003, India Phone No. +91 9425635736 e-mail: [email protected] *Corresponding Author
Supernatant
Fungal Biomass Screening and Identification Fructose
120
% Yield
Lysis and Centrifugation
Intracellular (Supernatant)
Glucose
100
Sucrose
80
Kestose
60
Nystose
40
Mycelial Fractions (Pellet)
20 0
1 2 3 4 5 6 7 8 9 10
RecyclingCycle of m-FTase FOS generation using mycelia in 50% Sucrose
Temperature (°C) 0
10
20
30
40
50
A 60
70
80
110
200
100 Biotransformation Yield (%)
150
FOS (g/L)
Total FOS (GF2+GF3) Sucrose (GF) Fructose (F) Glucose (G)
90
175
125 100 75 50
80 70 60 50 40 30 20 10 0
25
0
0 3
4
5
6
7
8
9
10
11
0.5
1
2
3
4
8
15
24
Incubation Time (h)
pH
FOS Purification using Biogel-P2
Localization of FTase
Optimization of FOS generation and characterization of FOS using DOSY NMR and FTIR
Graphical Abstract
Abstract An efficient system for biotransformation of sucrose to fructooligosaccharides (FOS) was obtained using Aspergillus tamarii NKRC 1229 mycelial fructosyltransferase (m-FTase). Zymographic analysis confirmed mycelial localization of the FTase (36 U/g) and lyophilized fungal pellets were used for bioconversion. m-FTase had molecular weight ~75 kDa with optimum activity at pH 7.0 and 20°C. FOS production after parametric optimization (sucrose 50 % w/v, m-FTase dose - 4.5 % w/v, inoculum age - 48 h and incubation time - 24 h) reached 325 g/L (55 % yield) with 14 % residual sucrose, 25 % glucose and 6 % fructose. FTase activity was enhanced after pre-treatment with organic solvents and SDS. FOS was purified in a single step using gel filtration matrix, Bio-Gel P2. FOS was characterized using Diffusion ordered spectroscopy-Nuclear Magnetic Resonance (1H DOSY-NMR) and Fourier-transform infrared spectroscopy (FTIR). Continuous generation of FOS was achieved using recyclable mycelia upto 10 consecutive cycles.
Keywords:
Fructooligosaccharides
(FOS),
Fructosyltransferase
(FTase),
Prebiotics,
Zymography, Aspergillus, Bio-Gel P2
Abbreviations: m-FTase – Mycelial Fructosyltransferase; FFase – Fructofuranosidase; FOS – Fructooligosaccharide; GF2 – Kestose; GF3 – Nystose; GF4 – 1-Fructosylnystose; TTC – Triphenyl tetrazolium chloride; DOSY –Diffusion Ordered Spectroscopy
1. Introduction Prebiotics are receiving increasing attention because of their multifarious nutraceutical and prohealth benefits such as anti-neoplastic, anti-cariogenic, anti-inflammatory and hypolipidemic effects (Mano, Neri-Numa, da Silva, Paulino, Pessoa, & Pastore, 2017). They also impart antiviral, antibacterial and anti-osteoporotic effects by enriching probiotic microflora in intestine (Kango & Jain, 2011). Most commonly used prebiotics are FOS which consist of a homologous series of oligofructose (GFn), with a characteristic terminal glucose viz. kestose (GF2), nystose (GF3) and 1-fructosylnystose (GF4) (Rawat, Ganaie & Kango, 2015). These are generally obtained by sucrose transfructosylation catalyzed by β-fructofuranosidase (FFase: EC 3.2.1.6) and/or by fructosyltransferases (FTase:
EC 2.4.1.9). FTase kinetics suggests that the
transfructosylation of sucrose takes place via the cleavage of beta-2,1-glycosidic bond and the transfer of
fructosyl moiety onto any acceptor other than water such as sucrose or a
fructooligosaccharide (Vega & Zuniga-Hansen, 2014). Among fungi, Aspergillus spp. and Penicillium spp. are indicated in FOS production by FTase based sucrose transfructosylation (Almeida et al., 2018; Rustiguel, Jorge, Henrique & Guimarães, 2015). Large scale FOS production employs fungal FTases sourced from Aspergillus niger, Aspergillus oryzae and Aureobasidium pullulans. Industrial scale sucrose transfructosylation involves either use of extracellular FTases in batches or immobilized FTases in fixed beds (Zambelli et al., 2014). Use of mycelia bound FTase helps in overcoming problems related to enzyme recovery and product contamination (Nobre, Teixeira & Rodrigues, 2012). In the present study, we report FOS generation using mycelia-bound recyclable Aspergillus tamarii NKRC 1229 FTase under parametric optimized conditions. A one-step FOS purification process and successful recycling of m-FTase for continuous FOS generation was demonstrated.
2. Materials and Methods 2.1 Chemicals Sucrose
(GF),
kestose
(GF2),
nystose
(GF3),
1-fructofuranosylnystose
(GF4),
3,5- dinitrosalicylic acid (DNS), fructose and glucose were purchased from Sigma Aldrich, USA. Bio-Gel P2 was obtained from Bio-Rad, USA. HiPurATM fungal DNA purification kit was obtained from Hi-Media, India. TLC silica gel 60 F254 plates were obtained from Merck, Germany and DNA markers were obtained from Invitrogen, USA. All other reagents and chemicals were of analytical grade. 2.2 Isolation of fungus with transfructosylating activity The fungus was isolated from a concentrated sugar syrup sample. Detection of FTase activity in the isolated fungi entailed indirect colorimetric plate screening assay. 1µL (108 spores/mL) of spore suspension (prepared in Tris-HCl buffer, pH 7.0) was inoculated on Czapek Dox agar (w/v; sucrose 3 %, NaNO3 0.3 %, K2HPO4 0.1 %, MgSO4·7H2O 0.05 %, KCl 0.05 %, FeSO4 0.001 %, CuSO4 0.001 % and agar-agar 2 %). The plate was then incubated at 28°C for 24 h followed by addition of 10 mL glucose oxidase-peroxidase (GOD-POD) reagent coupled with 4aminoantipyrine dye. The pink colored halo around the fungal colony was developed by incubating the plate at 37°C for 10 min (Xie et al., 2017). Fungal strain was maintained on potato dextrose agar (PDA) slants at 4°C. 2.3 FTase assay and molecular identification of the fungus FOS production was done by inoculating four mycelial discs (8 mm) into 50 mL fermentation medium containing (w/v) sucrose 20 %, yeast extract 0.5 %, MgSO4.7H2O 0.05 %, NaNO3 1 %, KH2PO4 0.25 %, NaCl 0.25 % and NH4Cl 0.5 % and incubating at 28°C for 24 h (Ganaie et al., 2013). Cell-free culture filtrate obtained by filtration through Whatman filter paper No.1 was
analyzed for the presence of FOS by thin layer chromatography (TLC) as described earlier by Kango (2008). Isolation of genomic DNA from the fungus was done using HiPurATM fungal DNA isolation kit. Internal transcribed spacer 1 and 2 (ITS 1 and ITS 2) regions between DNA encoding 18S rRNA and
28S
rRNA
were
amplified
using
universal
ITS
primers,
ITS
5F’
(5’-GGAAGTAAAAGTCGTAACAAGG-3’) and ITS 4R’ (5’-TCCTCCGCTTATTGATATGC3’). After Sanger sequencing of the amplified ITS regions, the sequence was aligned with NCBI database using BLAST tool and phylogenetic tree was constructed using MEGA 7.0 software. 2.4 Extracellular, intracellular and mycelial fractionation To localize FTase, extracellular, intracellular and mycelial extracts prepared from the submerged culture were analyzed for transfructosylation activity. The extracellular protein was precipitated using ammonium sulfate (90 % saturation) at 4°C with continuous stirring. The mixture was incubated overnight at 4°C and then centrifuged at 10000 g for 20 min. The precipitate was resuspended in Tris-HCl buffer (pH 7.0) and dialyzed using 10 kDa cut-off membrane to remove impurities. To obtain intracellular fraction, 24 h old mycelia was washed thrice with Tris-HCl buffer (pH 7.0) and then crushed under liquid nitrogen. The crushed mycelial powder was resuspended in the same buffer and centrifuged to remove mycelial fractions. Supernatant was used as intracellular FTase enzyme. Mycelial fractions remaining after intracellular enzyme extraction were washed with Tris-HCl buffer (pH 7.0) and resuspended in the same buffer. Suspended mycelia was sonicated for 30 seconds at 4°C (250W). After sonication, extract was centrifuged at 10000 g for 5 min and the supernatant containing mycelia bound enzyme was used as mycelial FTase. 2.5 SDS-PAGE and Zymography
FTase produced by the strain NKRC 1229 was localized by activity staining. Extracellular, intracellular and mycelial samples were resolved on 12 % SDS-polyacrylamide gel at 4°C by applying constant voltage of 90V. After electrophoresis, the gel was washed with distilled water to remove SDS and then incubated with sucrose (50 % w/v, pH 7.0) at 28°C for 1 h. After incubation, the gel was stained with alkaline solution of 1 % (w/v) triphenyl tetrazolium chloride (TTC) for 5 min (Heyer & Wendenburg, 2001). The staining reaction was stopped by placing the gel in 1 % (v/v) glacial acetic acid. FTase activity was seen as distinct dark red bands on the gel. 2.6 Qualitative and quantitative analysis of FTase activity For assaying extracellular, intracellular and mycelia bound FTase activity, 200 µL of above extracts were incubated with 200µL sucrose (20 % w/v in 50mM Tris-HCl buffer, pH 7.0) at 55°C for 1 h. Transfructosylation was stopped by immersing the reaction mixture in boiling water bath for 10 min. Qualitative determination of biotransformation products was done by thin layer chromatography (TLC). 2µL of diluted samples (1:20 dilution) were spotted on pre-coated silica gel plate. Solvent system containing iso-propanol, ethyl acetate and water in the ratio of 2:2:1 was used as mobile phase. Spots were visualized by spraying 0.5 % (w/v) α-naphthol prepared in ethanol and 5 % (v/v) sulfuric acid (Kango, 2008). FTase activity was estimated by analyzing products by high performance liquid chromatography (HPLC, Waters) using Sugar-Pak column (6.5× 300 mm) at column temperature of 60°C and refractive index detector (RI 2414). 20µL of appropriately diluted and pre-filtered (0.45µm membrane) samples were injected in the column. Water was used as mobile phase at a flow rate of 0.2 mL/min. FOS and other sugars were quantified using suitable standards (glucose, fructose, sucrose, kestose and nystose). The peak areas of carbohydrates were obtained using Empower 2 software (Build 2154, Waters). One FTase unit was defined as the amount of FOS (kestose and
nystose) produced (µmol /mL) per min from sucrose under the assay conditions (Lim, Park, Lee, Park & Kim, 2005). 2.7 Optimization of FOS generation by m-FTase 2.7.1 m-FTase preparation Mycelial pellets obtained after submerged cultivation were washed thrice with distilled water, lyophilized under vacuum and used as m-FTase for FOS generation from sucrose. Qualitative and quantitative analyses were done using TLC and HPLC, respectively. 2.7.2 Effect of pH and temperature on m-FTase Effect of pH on m-FTase activity was determined by conducting FOS production at different pH. Sucrose (20 % w/v) was dissolved in suitable buffers viz. sodium citrate buffer (pH 4.0 and 6.0), Tris-HCl buffer (pH 7.0-9.0) and glycine-NaOH buffer (pH 10.0). 0.5 g freeze dried mycelia (mFTase) was incubated with the pH amended sucrose solution at 20°C and 160 rpm for 5 h. Effect of temperature on m-FTase was determined by conducting transfructosylation at different temperatures. For this, 0.5 g of m-FTase was added to 20 mL sucrose solution (20 % w/v, pH 7.0) and incubated for 5 h at different temperatures (5-70°C) under shaking at 160 rpm. 2.7.3 Effect of mycelial age Effect of mycelial age on FOS production was determined by incubating 0.5 g m-FTase (24-96 h old) with 20 mL sucrose solution (20 % w/v, pH 7.0) at 20°C for 5 h and 160 rpm. 2.7.4 Effect of m-FTase dose and substrate concentration The effect of enzyme dose on FOS production was ascertained by adding different amounts of mycelia (0.5-5 % w/v) to 20 mL of sucrose (50 % w/v, pH 7.0) and incubating for 5 h at 20°C and 160 rpm. Effect of substrate concentration on m-FTase activity was determined by incubating it with different sucrose concentrations (1-80 % w/v, pH 7.0). The reaction mix was
incubated for 5 h at 20°C and 160 rpm and the products were analyzed using TLC and HPLC as described above. Relative FOS concentration (%) was determined and the values represent average of three replicates ± standard deviation. 2.7.5 Effect of modulators on m-FTase activity Effect of metal ions, solvents and detergents was evaluated by pre-incubating m-FTase in appropriate solution (5 % v/v or 5 mM) at 30°C for 30 min and the residual transfructosylating activity was estimated as described above. The residual m-FTase activity was reported as relative activity with respect to the untreated control (100%)/ 2.8 Time course of FOS generation Transformation of sucrose (50 % w/v, pH 7.0) by m-FTase was carried out at 20°C under shaking at 160 rpm for 24 h. Samples were collected at different time intervals and analyzed for FOS content. Biotransformation yield was calculated as described by Huang, Wu, Xu, Mo and Feng (2016): FOS yield (%) = [(Nystose + Kestose) / Total Sugars] X 100
where, total sugars represented nystose, kestose, sucrose, glucose and fructose present in the mixture. 2.8.1 Determination of transfructosylation products by Nuclear Magnetic Resonance (NMR) spectroscopy End products of m-FTase action were elucidated using
13C
1D NMR and 1H-2D diffusion
ordered spectroscopy (DOSY) NMR at 16°C. Samples were prepared by dissolving freeze-dried powder in deuterated water and spectra were recorded using JEOL ECX 500 MHz NMR spectrometer operating at 125.721 MHz resonance frequency with diffusion gradient of 0.17 sec. Chemical shifts were expressed in ppm and the background signals were normalized with tetramethylsilane (TMS) as internal reference.
2.8.2 End-product analysis using Fourier Transform Infrared Spectroscopy (FTIR) End products of sucrose biotransformation were analyzed using FTIR. Samples collected at different time intervals were concentrated by lyophilization and FTIR spectra of concentrated liquid samples were recorded in the range 4000-500 cm-1 on Bruker Alpha II ECO-ATR system with OPUS software. Each sample was recorded for 15 scans with 2 cm-1 spectral resolution on transmission mode. 2.9 Purification of FOS For purification of FOS by size exclusion chromatography, 1mL of transfructosylation products were loaded on Bio-Gel P2 fine column (45cm × 1cm). FOS were eluted by HPLC grade water at room temperature with a flow rate of 0.5 mL/min and purified fractions were analyzed by TLC and HPLC as described above. 2.10 Recycling of m-FTase Enzyme recycling was carried out in Poly-Prep® chromatography column (10 mL) for continuous generation of FOS. Lyophilized mycelia (0.3 g) was packed into the column and 3 mL of pre-sterilized sucrose solution (50 % w/v, pH 7.0) was added. After incubation for 2 h at 20°C, flow-through liquid was collected and the bed was washed twice with 50 mM Tris-HCl buffer (pH 7.0). The procedure was repeated up to ten cycles and the end-products of each cycle were analyzed by TLC and HPLC as described above.
3. Results and Discussion 3.1 Screening and identification of fungus for FTase activity FTase activity in the strain NKRC 1229 was confirmed with the formation of a distinct pink halo around the fungal colony after performing GOD-POD assay (Fig. S1 B). Xie et al. (2017) have also reported Aspergillus tubingensis strains XG11, XG12 and XG21 as FTase producers using similar method. Strain NKRC 1229 produced greenish-brown colored colony with large number of spores on Czapek Dox agar. On the basis of the 99 % homology of ITS sequence, it was identified as Aspergillus tamarii (Genbank accession MH788968). The strain is submitted with National fungal culture collection of India, ARI, Pune (Accession no. NFCCI 4401). 3.2 Localization of fructosyltransferase Extracellular, intracellular and mycelial enzyme fractions were subjected to SDS-PAGE followed by zymography. Activity assay and zymography data substantiated that FTase was localized on mycelia. The zymogram revealed prominent mycelial and the intracellular FTase activity but FTase was not detected in extracellular culture filtrate. A. tamarii NKRC 1229 mycelia exhibited total 36 units of FTase / g mycelia (Table 1, Fig. 1). The m-FTase produced by the strain NKRC 1229 was a 75 kDa protein (Fig. 1). The distribution of intracellular (21.58 U/g mycelia) and mycelial FTase (14.45 U/g mycelia) suggested it to be a transmembrane protein with intracellular catalytic domain. Similar observations were made by Perna, Cunha, Gonçalves, Basso, Silva and Maiorano (2018) in case of Aspergillus oryzae IPT-301 mycelial FTase. Fructosyltransferase from Aspergillus sydowii IAM 2455 conidia had a molecular weight of 55 kDa with calculated mass of 75 kDa suggesting post-transcriptional modifications (Heyer and Wendenburg, 2001). 3.3 Parametric optimization of FOS production by A. tamarii NKRC 1229 m-FTase
3.3.1 Effect of pH and temperature The effect of pH and temperature on m-FTase revealed optimal activity at pH 7.0-8.0 and 20°C (Fig. 2A). In contrast, Aspergillus phoenicis and Aspergillus terreus FTases showed optimal activity at acidic pH 4.5 and 4.6 (Almeida et al., 2018; Rustiguel, Jorge, Henrique & Guimarães, 2015). Production of FOS (77.7 g/L) at 5ºC by m-FTase suggested that the enzyme could be employed at low temperatures. Maximum FOS (167.51 g/L) was produced at 20°C (Fig. 2A). In contrast, fructofuranosidase from Penicillium citreonigrum showed optimum activity at 50ºC (Nascimento, Nobre, Cavalcanti, Teixeira & Porto, 2013). Aspergillus oryzae KB FFase 1 (mycelial) and FFase 2 (extracellular) showed optimum FOS generation at 50°C (Kurakake, Ogawa, Sugie, Takemura, Sugiura & Komaki, 2008). FOS generating β-fructosidase from Aspergillus oryzae FS4 had optimal activity at 55°C (Xu et al., 2014). Sucrose biotransformation at low temperatures may help in avoiding impurities due to contamination and minimize energy consumption (Cavicchioli et al., 2011; Sarmiento et al., 2015; Taskin, Ortucu, Unver, Tasar, Ozdemir & Kaymak, 2016). 3.3.2 Effect of mycelium age Inoculum age of 48 h supported best FOS yield (110.67 g/L) with 17.3 g/L nystose and 93.4 g/L kestose (Fig. 2B). The inoculum age showing optimal FOS production in case of other fungal mycelial fructosyltransferases was found to be 96 h (Zambelli et al., 2014).
3.3.3 Effect of m-FTase dose and substrate concentration on FOS production The effect of m-FTase dose on FOS generation indicated that 4.5 % (w/v) of mycelia supported maximum (325 g/L) FOS production (Fig. 2C). Kestose was predominant (51.8-274.5 g/L) transfructosylation product with low amounts (1.6-60.5 g/L) of nystose (Table S1). Bie and Zhu
(2016) achieved optimal neo-kestose production rate (0.028 mol/L/h) using 1.6 % (w/v) dry cells of immobilized yeast, Phaffia rhodozyma. FOS yield increased with increase in sucrose concentration upto 50 % (w/v) yielding 318.8 g/L FOS (Fig. 2D). Highest concentration of kestose (286.4 g/L) was obtained with 50 % (w/v) sucrose while maximum nystose concentration (48.4 g/L) was achieved with 30 % (w/v) sucrose (Table S2). Low availability of water at high sucrose concentration facilitates sucrose as fructosyl acceptor (Zhang et al., 2016). Extracellular β-fructofuranosidase from Xanthophyllomyces dendrorhous produced 40-168 g/L of neo-FOS at 420 to 600 g/L sucrose concentration (Linde, Rodríguez-colinas, Estévez, Poveda, Plou & Fernández, 2012). Maximum FOS (190 mg/mL) was obtained at 300 g/L sucrose concentration in case of β-FFase from Aspergillus phoenicis biofilms (Aziani, Terenzi, Jorge & Guimaraes, 2012). Recombinant FTase from A. niger YZ59 expressed in P. pastoris yielded 343 g/L FOS using 60 % (w/v) sucrose (Yang, Wang, Zhang & Shen, 2016). 3.3.4 Effect of metal ions and solvents on m-FTase Among the metal ions, mercury (Hg2+) and manganese (Mn2+) strongly inhibited A. tamarii m-FTase activity. Slight enhancement in m-FTase activity was observed with copper ions (Cu2+). FTase from Aspergillus aculeatus M105 showed enhanced activity (130 %) with Cu2+ (Huang et al., 2016). m-FTase activity was enhanced with most organic solvents excepting aniline and methanol. Hexane and ethyl acetate increased m-FTase activity by 18 and 14 %, respectively (Table S3). Gupta (1992) has also described enhancement in activity of several enzymes after treatment in organic environment. Reports suggest considerable conformational modifications in active site of enzymes after pre-incubation with hydrated organic solvents (Gupta, Tyagi, Sharma, Karthikeyan & Singh, 2000). 3.4 Time course of FOS generation by m-FTase
Under optimized conditions (pH : 7.0, temperature : 20°C, sucrose : 50 % w/v, m-FTase dose : 4.5 % w/v, inoculum age : 48 h and agitation speed : 160 rpm), highest FOS production (325.27 g/L; 55.14 %) was reached after 24 h with 14.18 % residual sucrose, 5.68 % fructose and 25 % glucose (Fig. 3A). During catalysis, fructose is transferred to sucrose resulting in kestose and nystose generation while residual glucose remains in the mixture due to competitive nature of hydrolysis and transfructosylation reaction (Jitonnom et al., 2018). Recently, 118 g/L FOS (64 % yield) were achieved in 38 h by Aspergillus ibericus (Nobre et al., 2017) while 56.9 % FOS yield was obtained within 8 h of sucrose transformation by Aspergillus tubingensis XG21 βfructofuranosidase (Xie et al., 2017). Dual-cell system of immobilized Aspergillus japonicus spores and Pichia heimii produced 0.62 g FOS per gram of sucrose from 30 % (w/v) sucrose in a continuous reactor in 25 days (Sheu, Chang, Wang, Wu & Huang, 2013). Industrial scale production of FOS by immobilized Aureobasidium pullulans was demonstrated in a packed bed reactor where 180 g/L h-1 FOS were produced from 77 % (w/v) sucrose at 50°C (Jung, Bang, Oh & Park, 2011). Commercial FFase from Aspergillus aculeatus yielded 55 % g/g FOS after 100 min of biotransformation of 60 % (w/v) sucrose at 50°C (Lorenzoni, Aydos, Klein, Rodrigues & Hertz, 2014). 3.4.1 Analysis of end products by 1H DOSY NMR The composition of end products was analyzed using DOSY NMR. Corroborating with the results of TLC and HPLC, four different diffusion coefficients from 0.25-0.65 (x 10-9 m2 s-1) representing fructose, sucrose, kestose and nystose were obtained with a linear gradient of 0.26 T/m at 16°C (Fig. 3B). Our results of 1H DOSY spectrum were in agreement with those obtained by Paredes, Smiderle, Santana-Filho, Kimura, Lacomini and Sassaki (2017) and suggest applicability of this technique in analyzing mixtures.
3.4.2 End product analysis by FTIR The FTIR analysis of emerging end-products over 24 h of biotransformation revealed considerable changes in the region ranging 900-1200 cm-1. Clear evolution of peaks at 996, 1025 and 1122 cm-1 during 1-24 h of FTase action indicated sucrose consumption and glycosidic bond formation due to transfructosylation (Fig. 3C). Strong band in this region corresponds to C–O–C glycosidic linkage, the mC–C and the dCOH vibrational modes. The gradual increase in transmittance in this region can be attributed to the increase in the concentration of FOS. The region (900-1200 cm-1) is called as “fingerprint region” of sugars (Santos, Araujo-Andrade, Tymczyszyn, & Gómez-Zavaglia, 2014; Mellado-Mojica, & López, 2015). Similar observations regarding FTIR spectra for FOS generation by sucrose transfructosylation using commercial FTase (Viscozyme®L) were reported by Romano, Santos, Mobili, Vega and Gómez-Zavaglia (2016). Gradual and characteristic change in this region indicated the applicability of this technique in the monitoring of FOS production. 3.5 Purification of FOS FOS were purified using Bio-Gel P2 matrix in a single step. Pure nystose (39 mg) and nytosekestose mixture (73.84 mg) was obtained in 48-54 min elution time from Bio-Gel P2 with a flow rate of 0.5 mL/min (Fig. 4). Similarly, FOS generated by sucrose transfructosylation by Aspergillus awamori NBRC 4033 β-FFase were purified by Bio-Gel P2 matrix (Smaali, Jazzar, Soussi, Muzard, Aubry & Marzouki, 2012). Nobre et al. (2012) purified FOS from fermentative broth using activated charcoal column matrix and eluted using ethanol gradient upto 50% (v/v). FOS purification using Bio-Gel P2 with water as eluent is a direct and easy method that may be scaled-up (Table S5).
3.6 Recycling of mycelial FTase for continuous FOS generation m-FTase (10 % w/v) was recycled upto 10 consecutive cycles of 2 h each, wherein it showed efficient production of FOS without any significant loss in activity. Kestose was obtained as the major product throughout and only a slight decrease in FOS concentration was noticed after 6th cycle (Fig. 5). Fructosyltransferase from A. oryzae expressed in Yarrowia lipolytica retained only 55 % of initial activity after 15th cycle (Zhang et al., 2016). β-FFase from Xanthophyllomyces dendrorhous retained activity upto 7 cycles of 26 h each (Míguez et al., 2018). In the present study, continuous use of mycelial FTase for efficient biotransformation of sucrose was demonstrated. This system based on mycelium bound FTase combines merits of both pure and immobilized enzyme and utilizes rapidly reproducible mycelia thus avoiding need of purifying enzyme and any tedious immobilization procedures. 4. Conclusion Zymographic analysis of cellular fractions revealed presence of a mycelial FTase in A. tamarii NKRC 1229. Enhanced production of FOS was achieved by parametric optimization of various factors. m-FTase produced high FOS titres (325 g/L) in 24 h at 20°C. Purification of FOS was achieved using Bio-Gel P2 in a single step. FOS production based on mycelia bound FTase combines merits of both pure and immobilized enzyme that can be exploited at large scale.
Acknowledgements Author RC is grateful to UGC, New Delhi for providing financial assistance as PhD scholarship. Authors are thankful to Dr. K.K. Dey, Department of Physics for help in NMR analysis. Authors acknowledge the instrumentation facilities available under Sophisticated Instrumentation Centre (SIC) and DST-PURSE (II) at Dr. Harisingh Gour Vishwavidyalaya, Sagar, India.
Conflict of interest Authors have no conflicts of interest.
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Table 1: Distribution of FTase in A. tamarii NKRC1229 and fructooligosaccharide generation
FTase (U)
Nystose (mg/ml)
Kestose (mg/ml)
Total FOS (mg/ml)
Intracellular
21.58a
12.46
55.88
68.34
Mycelial
14.45a
11.40
35.11
46.51
ND
ND
ND
ND
Location
Extracellular
ND – Not Detected; a - U/g mycelia
Figure Captions Fig. 1: Zymogram showing localization of fructosyltransferase (FTase). MW: Molecular weight marker, EC: Extracellular fraction, IC: Intracellular fraction, ME: Mycelial extract. Gel was incubated in sucrose solution (50 % w/v, pH 7.0) at 28°C for 1 h and stained with 1 % (w/v) TTC in 0.25M NaOH. Fig. 2: Optimization of FOS generation by Aspergillus tamarii NKRC 1229 mycelial fructosyltransferase (A) Effect of pH (-●-) and temperature (-▲-) on FOS generation by m-FTase. (B) Effect of mycelial age on FOS production. 0.5 g of m-FTase was added to 20 mL sucrose (20 % w/v) and incubated at 20°C for 5 h at 160 rpm. (C) Effect of m-FTase dose on FOS generation. m-FTase (0.5-5 % w/v) was added to 20 mL sucrose (50 % w/v) and incubated at 20°C for 5h at 160 rpm. (D) Effect of sucrose concentration on FOS generation. m-FTase (2.5 % w/v mycelia) was incubated with sucrose solution (1-80 % w/v, pH 7.0) at 20°C for 5 h at 160 rpm. Values represent average of three replicates ± standard deviation. Fig. 3: FOS generation and characterization (A) Time course of FOS generation under optimized conditions (sucrose: 50 % w/v, pH: 7.0, temp.: 20°C) over 24 h. (B) 1H DOSY NMR spectrum of
sucrose biotransformation products showing FOS formation (C) FTIR spectra showing FOS generation in 24 h. Arrows at 996, 1025 and 1122 cm-1 peaks indicate increase in the FOS fingerprint region. Fig. 4: Elution profile of biotransformation products using Bio-Gel P2. FOS were eluted using water at 28°C with a flow rate of 0.5 mL/min. Fig. 5: Recycling of A. tamarii m-FTase for continuous FOS generation. Mycelia (10 % w/v) was used to biotransform sucrose solution (50 % w/v, pH 7.0) at 20ºC and 160 rpm for 10 cycles of 2 h each.
kDa MW 250 150 100 75
EC
50 37
25
Fig. 1
IC
ME
Temperature (°C) 0
10
20
30
40
50
60
70
80
120
200
B
A
175
110
FOS Yield (g/L)
125 100 75
100 90 80
50
70
25
60
0 5
6
7
8
9
10
11
24
48
pH
72
96
Mycelial Age (h)
D
110 100
110
Fig.902 Relative FOS Concentration (%)
4
100
110 Relative FOS Concentration (%)
3
100 90 Biotransformation Yield (%)
FOS (g/L)
150
80 70 60 50
90 80 70 60 50 40 30
80
60 50 40 30 20
20
10 0
0
30
Total FOS (GF2+GF3) Sucrose (GF) Fructose (F) Glucose (G)
70
10
40
C
A
0.5
1
1.5
1
5
2
2.5
10
3
20
3.5
4
30
4.5
40
5
50
60
Sucrose Concentration (% w/v) Mycelia (% w/v)
20 10 0 0
0.5
1
2
3
4
Incubation Time (h)
8
15
24
70
80
B
996 1025
Glucose / Fructose
D/10-9 m2s-1
CFig.
Sucrose
1122 Kestose (DP3) Nystose (DP4)
1H
Chemical Shift / ppm
A
50
Nystose Kestose Sucrose Glucose
Concentration (g/L)
40
30
20
Fig. 4
10
0 40
45
50
55
60
Elution Time (min)
65
70
75
3
Fructose
% Yield
120
Glucose
100
Sucrose
80
Kestose
60
Nystose
40 20 0
1
2
3
4
5 6 Cycle
7
8
9
10
Fig. 5
Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Author RC is grateful to UGC, New Delhi for providing financial assistance as PhD scholarship. Authors have no conflicts of interest.
Highlights
Mycelial fructosyltransferase (~75kDa) from Aspergillus tamarii was characterized
Fructosyltransferase yielded 55% (w/v) oligofructose from sucrose at 20°C in 24 h
Fructooligosaccharides were purified and resolved using 1H DOSY-NMR and FTIR
Enzyme was recycled upto 10 cycles with high productivity (106.12 g/L/h)
S0308-8146(19)30339-5 https://doi.org/10.1016/j.foodchem.2019.02.025 FOCH 24336
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
10 October 2018 4 February 2019 6 February 2019
Please cite this article as: Choukade, R., Kango, N., Characterization of a mycelial fructosyltransferase from Aspergillus tamarii NKRC 1229 for efficient synthesis of fructooligosaccharides, Food Chemistry (2019), doi: https://doi.org/10.1016/j.foodchem.2019.02.025
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Characterization of a mycelial fructosyltransferase from Aspergillus tamarii NKRC 1229 for efficient synthesis of fructooligosaccharides
Ritumbhara Choukade and Naveen Kango* Enzyme Technology and Molecular Catalysis Laboratory, Department of Microbiology Dr. Harisingh Gour Vishwavidyalaya (A Central University), Sagar (Madhya Pradesh) 470003, India
Authors Information Ritumbhara Choukade Enzyme Technology and Molecular Catalysis Laboratory, Department of Microbiology Dr. Harisingh Gour Vishwavidyalaya (A Central University), Sagar (Madhya Pradesh) 470003, India Phone No. +91 9644430994 e-mail: [email protected] Prof. Naveen Kango Designation: Professor Enzyme Technology and Molecular Catalysis Laboratory, Department of Microbiology Dr. Harisingh Gour Vishwavidyalaya (A Central University), Sagar (Madhya Pradesh) 470003, India Phone No. +91 9425635736 e-mail: [email protected] *Corresponding Author
Supernatant
Fungal Biomass Screening and Identification Fructose
120
% Yield
Lysis and Centrifugation
Intracellular (Supernatant)
Glucose
100
Sucrose
80
Kestose
60
Nystose
40
Mycelial Fractions (Pellet)
20 0
1 2 3 4 5 6 7 8 9 10
RecyclingCycle of m-FTase FOS generation using mycelia in 50% Sucrose
Temperature (°C) 0
10
20
30
40
50
A 60
70
80
110
200
100 Biotransformation Yield (%)
150
FOS (g/L)
Total FOS (GF2+GF3) Sucrose (GF) Fructose (F) Glucose (G)
90
175
125 100 75 50
80 70 60 50 40 30 20 10 0
25
0
0 3
4
5
6
7
8
9
10
11
0.5
1
2
3
4
8
15
24
Incubation Time (h)
pH
FOS Purification using Biogel-P2
Localization of FTase
Optimization of FOS generation and characterization of FOS using DOSY NMR and FTIR
Graphical Abstract
Abstract An efficient system for biotransformation of sucrose to fructooligosaccharides (FOS) was obtained using Aspergillus tamarii NKRC 1229 mycelial fructosyltransferase (m-FTase). Zymographic analysis confirmed mycelial localization of the FTase (36 U/g) and lyophilized fungal pellets were used for bioconversion. m-FTase had molecular weight ~75 kDa with optimum activity at pH 7.0 and 20°C. FOS production after parametric optimization (sucrose 50 % w/v, m-FTase dose - 4.5 % w/v, inoculum age - 48 h and incubation time - 24 h) reached 325 g/L (55 % yield) with 14 % residual sucrose, 25 % glucose and 6 % fructose. FTase activity was enhanced after pre-treatment with organic solvents and SDS. FOS was purified in a single step using gel filtration matrix, Bio-Gel P2. FOS was characterized using Diffusion ordered spectroscopy-Nuclear Magnetic Resonance (1H DOSY-NMR) and Fourier-transform infrared spectroscopy (FTIR). Continuous generation of FOS was achieved using recyclable mycelia upto 10 consecutive cycles.
Keywords:
Fructooligosaccharides
(FOS),
Fructosyltransferase
(FTase),
Prebiotics,
Zymography, Aspergillus, Bio-Gel P2
Abbreviations: m-FTase – Mycelial Fructosyltransferase; FFase – Fructofuranosidase; FOS – Fructooligosaccharide; GF2 – Kestose; GF3 – Nystose; GF4 – 1-Fructosylnystose; TTC – Triphenyl tetrazolium chloride; DOSY –Diffusion Ordered Spectroscopy
1. Introduction Prebiotics are receiving increasing attention because of their multifarious nutraceutical and prohealth benefits such as anti-neoplastic, anti-cariogenic, anti-inflammatory and hypolipidemic effects (Mano, Neri-Numa, da Silva, Paulino, Pessoa, & Pastore, 2017). They also impart antiviral, antibacterial and anti-osteoporotic effects by enriching probiotic microflora in intestine (Kango & Jain, 2011). Most commonly used prebiotics are FOS which consist of a homologous series of oligofructose (GFn), with a characteristic terminal glucose viz. kestose (GF2), nystose (GF3) and 1-fructosylnystose (GF4) (Rawat, Ganaie & Kango, 2015). These are generally obtained by sucrose transfructosylation catalyzed by β-fructofuranosidase (FFase: EC 3.2.1.6) and/or by fructosyltransferases (FTase:
EC 2.4.1.9). FTase kinetics suggests that the
transfructosylation of sucrose takes place via the cleavage of beta-2,1-glycosidic bond and the transfer of
fructosyl moiety onto any acceptor other than water such as sucrose or a
fructooligosaccharide (Vega & Zuniga-Hansen, 2014). Among fungi, Aspergillus spp. and Penicillium spp. are indicated in FOS production by FTase based sucrose transfructosylation (Almeida et al., 2018; Rustiguel, Jorge, Henrique & Guimarães, 2015). Large scale FOS production employs fungal FTases sourced from Aspergillus niger, Aspergillus oryzae and Aureobasidium pullulans. Industrial scale sucrose transfructosylation involves either use of extracellular FTases in batches or immobilized FTases in fixed beds (Zambelli et al., 2014). Use of mycelia bound FTase helps in overcoming problems related to enzyme recovery and product contamination (Nobre, Teixeira & Rodrigues, 2012). In the present study, we report FOS generation using mycelia-bound recyclable Aspergillus tamarii NKRC 1229 FTase under parametric optimized conditions. A one-step FOS purification process and successful recycling of m-FTase for continuous FOS generation was demonstrated.
2. Materials and Methods 2.1 Chemicals Sucrose
(GF),
kestose
(GF2),
nystose
(GF3),
1-fructofuranosylnystose
(GF4),
3,5- dinitrosalicylic acid (DNS), fructose and glucose were purchased from Sigma Aldrich, USA. Bio-Gel P2 was obtained from Bio-Rad, USA. HiPurATM fungal DNA purification kit was obtained from Hi-Media, India. TLC silica gel 60 F254 plates were obtained from Merck, Germany and DNA markers were obtained from Invitrogen, USA. All other reagents and chemicals were of analytical grade. 2.2 Isolation of fungus with transfructosylating activity The fungus was isolated from a concentrated sugar syrup sample. Detection of FTase activity in the isolated fungi entailed indirect colorimetric plate screening assay. 1µL (108 spores/mL) of spore suspension (prepared in Tris-HCl buffer, pH 7.0) was inoculated on Czapek Dox agar (w/v; sucrose 3 %, NaNO3 0.3 %, K2HPO4 0.1 %, MgSO4·7H2O 0.05 %, KCl 0.05 %, FeSO4 0.001 %, CuSO4 0.001 % and agar-agar 2 %). The plate was then incubated at 28°C for 24 h followed by addition of 10 mL glucose oxidase-peroxidase (GOD-POD) reagent coupled with 4aminoantipyrine dye. The pink colored halo around the fungal colony was developed by incubating the plate at 37°C for 10 min (Xie et al., 2017). Fungal strain was maintained on potato dextrose agar (PDA) slants at 4°C. 2.3 FTase assay and molecular identification of the fungus FOS production was done by inoculating four mycelial discs (8 mm) into 50 mL fermentation medium containing (w/v) sucrose 20 %, yeast extract 0.5 %, MgSO4.7H2O 0.05 %, NaNO3 1 %, KH2PO4 0.25 %, NaCl 0.25 % and NH4Cl 0.5 % and incubating at 28°C for 24 h (Ganaie et al., 2013). Cell-free culture filtrate obtained by filtration through Whatman filter paper No.1 was
analyzed for the presence of FOS by thin layer chromatography (TLC) as described earlier by Kango (2008). Isolation of genomic DNA from the fungus was done using HiPurATM fungal DNA isolation kit. Internal transcribed spacer 1 and 2 (ITS 1 and ITS 2) regions between DNA encoding 18S rRNA and
28S
rRNA
were
amplified
using
universal
ITS
primers,
ITS
5F’
(5’-GGAAGTAAAAGTCGTAACAAGG-3’) and ITS 4R’ (5’-TCCTCCGCTTATTGATATGC3’). After Sanger sequencing of the amplified ITS regions, the sequence was aligned with NCBI database using BLAST tool and phylogenetic tree was constructed using MEGA 7.0 software. 2.4 Extracellular, intracellular and mycelial fractionation To localize FTase, extracellular, intracellular and mycelial extracts prepared from the submerged culture were analyzed for transfructosylation activity. The extracellular protein was precipitated using ammonium sulfate (90 % saturation) at 4°C with continuous stirring. The mixture was incubated overnight at 4°C and then centrifuged at 10000 g for 20 min. The precipitate was resuspended in Tris-HCl buffer (pH 7.0) and dialyzed using 10 kDa cut-off membrane to remove impurities. To obtain intracellular fraction, 24 h old mycelia was washed thrice with Tris-HCl buffer (pH 7.0) and then crushed under liquid nitrogen. The crushed mycelial powder was resuspended in the same buffer and centrifuged to remove mycelial fractions. Supernatant was used as intracellular FTase enzyme. Mycelial fractions remaining after intracellular enzyme extraction were washed with Tris-HCl buffer (pH 7.0) and resuspended in the same buffer. Suspended mycelia was sonicated for 30 seconds at 4°C (250W). After sonication, extract was centrifuged at 10000 g for 5 min and the supernatant containing mycelia bound enzyme was used as mycelial FTase. 2.5 SDS-PAGE and Zymography
FTase produced by the strain NKRC 1229 was localized by activity staining. Extracellular, intracellular and mycelial samples were resolved on 12 % SDS-polyacrylamide gel at 4°C by applying constant voltage of 90V. After electrophoresis, the gel was washed with distilled water to remove SDS and then incubated with sucrose (50 % w/v, pH 7.0) at 28°C for 1 h. After incubation, the gel was stained with alkaline solution of 1 % (w/v) triphenyl tetrazolium chloride (TTC) for 5 min (Heyer & Wendenburg, 2001). The staining reaction was stopped by placing the gel in 1 % (v/v) glacial acetic acid. FTase activity was seen as distinct dark red bands on the gel. 2.6 Qualitative and quantitative analysis of FTase activity For assaying extracellular, intracellular and mycelia bound FTase activity, 200 µL of above extracts were incubated with 200µL sucrose (20 % w/v in 50mM Tris-HCl buffer, pH 7.0) at 55°C for 1 h. Transfructosylation was stopped by immersing the reaction mixture in boiling water bath for 10 min. Qualitative determination of biotransformation products was done by thin layer chromatography (TLC). 2µL of diluted samples (1:20 dilution) were spotted on pre-coated silica gel plate. Solvent system containing iso-propanol, ethyl acetate and water in the ratio of 2:2:1 was used as mobile phase. Spots were visualized by spraying 0.5 % (w/v) α-naphthol prepared in ethanol and 5 % (v/v) sulfuric acid (Kango, 2008). FTase activity was estimated by analyzing products by high performance liquid chromatography (HPLC, Waters) using Sugar-Pak column (6.5× 300 mm) at column temperature of 60°C and refractive index detector (RI 2414). 20µL of appropriately diluted and pre-filtered (0.45µm membrane) samples were injected in the column. Water was used as mobile phase at a flow rate of 0.2 mL/min. FOS and other sugars were quantified using suitable standards (glucose, fructose, sucrose, kestose and nystose). The peak areas of carbohydrates were obtained using Empower 2 software (Build 2154, Waters). One FTase unit was defined as the amount of FOS (kestose and
nystose) produced (µmol /mL) per min from sucrose under the assay conditions (Lim, Park, Lee, Park & Kim, 2005). 2.7 Optimization of FOS generation by m-FTase 2.7.1 m-FTase preparation Mycelial pellets obtained after submerged cultivation were washed thrice with distilled water, lyophilized under vacuum and used as m-FTase for FOS generation from sucrose. Qualitative and quantitative analyses were done using TLC and HPLC, respectively. 2.7.2 Effect of pH and temperature on m-FTase Effect of pH on m-FTase activity was determined by conducting FOS production at different pH. Sucrose (20 % w/v) was dissolved in suitable buffers viz. sodium citrate buffer (pH 4.0 and 6.0), Tris-HCl buffer (pH 7.0-9.0) and glycine-NaOH buffer (pH 10.0). 0.5 g freeze dried mycelia (mFTase) was incubated with the pH amended sucrose solution at 20°C and 160 rpm for 5 h. Effect of temperature on m-FTase was determined by conducting transfructosylation at different temperatures. For this, 0.5 g of m-FTase was added to 20 mL sucrose solution (20 % w/v, pH 7.0) and incubated for 5 h at different temperatures (5-70°C) under shaking at 160 rpm. 2.7.3 Effect of mycelial age Effect of mycelial age on FOS production was determined by incubating 0.5 g m-FTase (24-96 h old) with 20 mL sucrose solution (20 % w/v, pH 7.0) at 20°C for 5 h and 160 rpm. 2.7.4 Effect of m-FTase dose and substrate concentration The effect of enzyme dose on FOS production was ascertained by adding different amounts of mycelia (0.5-5 % w/v) to 20 mL of sucrose (50 % w/v, pH 7.0) and incubating for 5 h at 20°C and 160 rpm. Effect of substrate concentration on m-FTase activity was determined by incubating it with different sucrose concentrations (1-80 % w/v, pH 7.0). The reaction mix was
incubated for 5 h at 20°C and 160 rpm and the products were analyzed using TLC and HPLC as described above. Relative FOS concentration (%) was determined and the values represent average of three replicates ± standard deviation. 2.7.5 Effect of modulators on m-FTase activity Effect of metal ions, solvents and detergents was evaluated by pre-incubating m-FTase in appropriate solution (5 % v/v or 5 mM) at 30°C for 30 min and the residual transfructosylating activity was estimated as described above. The residual m-FTase activity was reported as relative activity with respect to the untreated control (100%)/ 2.8 Time course of FOS generation Transformation of sucrose (50 % w/v, pH 7.0) by m-FTase was carried out at 20°C under shaking at 160 rpm for 24 h. Samples were collected at different time intervals and analyzed for FOS content. Biotransformation yield was calculated as described by Huang, Wu, Xu, Mo and Feng (2016): FOS yield (%) = [(Nystose + Kestose) / Total Sugars] X 100
where, total sugars represented nystose, kestose, sucrose, glucose and fructose present in the mixture. 2.8.1 Determination of transfructosylation products by Nuclear Magnetic Resonance (NMR) spectroscopy End products of m-FTase action were elucidated using
13C
1D NMR and 1H-2D diffusion
ordered spectroscopy (DOSY) NMR at 16°C. Samples were prepared by dissolving freeze-dried powder in deuterated water and spectra were recorded using JEOL ECX 500 MHz NMR spectrometer operating at 125.721 MHz resonance frequency with diffusion gradient of 0.17 sec. Chemical shifts were expressed in ppm and the background signals were normalized with tetramethylsilane (TMS) as internal reference.
2.8.2 End-product analysis using Fourier Transform Infrared Spectroscopy (FTIR) End products of sucrose biotransformation were analyzed using FTIR. Samples collected at different time intervals were concentrated by lyophilization and FTIR spectra of concentrated liquid samples were recorded in the range 4000-500 cm-1 on Bruker Alpha II ECO-ATR system with OPUS software. Each sample was recorded for 15 scans with 2 cm-1 spectral resolution on transmission mode. 2.9 Purification of FOS For purification of FOS by size exclusion chromatography, 1mL of transfructosylation products were loaded on Bio-Gel P2 fine column (45cm × 1cm). FOS were eluted by HPLC grade water at room temperature with a flow rate of 0.5 mL/min and purified fractions were analyzed by TLC and HPLC as described above. 2.10 Recycling of m-FTase Enzyme recycling was carried out in Poly-Prep® chromatography column (10 mL) for continuous generation of FOS. Lyophilized mycelia (0.3 g) was packed into the column and 3 mL of pre-sterilized sucrose solution (50 % w/v, pH 7.0) was added. After incubation for 2 h at 20°C, flow-through liquid was collected and the bed was washed twice with 50 mM Tris-HCl buffer (pH 7.0). The procedure was repeated up to ten cycles and the end-products of each cycle were analyzed by TLC and HPLC as described above.
3. Results and Discussion 3.1 Screening and identification of fungus for FTase activity FTase activity in the strain NKRC 1229 was confirmed with the formation of a distinct pink halo around the fungal colony after performing GOD-POD assay (Fig. S1 B). Xie et al. (2017) have also reported Aspergillus tubingensis strains XG11, XG12 and XG21 as FTase producers using similar method. Strain NKRC 1229 produced greenish-brown colored colony with large number of spores on Czapek Dox agar. On the basis of the 99 % homology of ITS sequence, it was identified as Aspergillus tamarii (Genbank accession MH788968). The strain is submitted with National fungal culture collection of India, ARI, Pune (Accession no. NFCCI 4401). 3.2 Localization of fructosyltransferase Extracellular, intracellular and mycelial enzyme fractions were subjected to SDS-PAGE followed by zymography. Activity assay and zymography data substantiated that FTase was localized on mycelia. The zymogram revealed prominent mycelial and the intracellular FTase activity but FTase was not detected in extracellular culture filtrate. A. tamarii NKRC 1229 mycelia exhibited total 36 units of FTase / g mycelia (Table 1, Fig. 1). The m-FTase produced by the strain NKRC 1229 was a 75 kDa protein (Fig. 1). The distribution of intracellular (21.58 U/g mycelia) and mycelial FTase (14.45 U/g mycelia) suggested it to be a transmembrane protein with intracellular catalytic domain. Similar observations were made by Perna, Cunha, Gonçalves, Basso, Silva and Maiorano (2018) in case of Aspergillus oryzae IPT-301 mycelial FTase. Fructosyltransferase from Aspergillus sydowii IAM 2455 conidia had a molecular weight of 55 kDa with calculated mass of 75 kDa suggesting post-transcriptional modifications (Heyer and Wendenburg, 2001). 3.3 Parametric optimization of FOS production by A. tamarii NKRC 1229 m-FTase
3.3.1 Effect of pH and temperature The effect of pH and temperature on m-FTase revealed optimal activity at pH 7.0-8.0 and 20°C (Fig. 2A). In contrast, Aspergillus phoenicis and Aspergillus terreus FTases showed optimal activity at acidic pH 4.5 and 4.6 (Almeida et al., 2018; Rustiguel, Jorge, Henrique & Guimarães, 2015). Production of FOS (77.7 g/L) at 5ºC by m-FTase suggested that the enzyme could be employed at low temperatures. Maximum FOS (167.51 g/L) was produced at 20°C (Fig. 2A). In contrast, fructofuranosidase from Penicillium citreonigrum showed optimum activity at 50ºC (Nascimento, Nobre, Cavalcanti, Teixeira & Porto, 2013). Aspergillus oryzae KB FFase 1 (mycelial) and FFase 2 (extracellular) showed optimum FOS generation at 50°C (Kurakake, Ogawa, Sugie, Takemura, Sugiura & Komaki, 2008). FOS generating β-fructosidase from Aspergillus oryzae FS4 had optimal activity at 55°C (Xu et al., 2014). Sucrose biotransformation at low temperatures may help in avoiding impurities due to contamination and minimize energy consumption (Cavicchioli et al., 2011; Sarmiento et al., 2015; Taskin, Ortucu, Unver, Tasar, Ozdemir & Kaymak, 2016). 3.3.2 Effect of mycelium age Inoculum age of 48 h supported best FOS yield (110.67 g/L) with 17.3 g/L nystose and 93.4 g/L kestose (Fig. 2B). The inoculum age showing optimal FOS production in case of other fungal mycelial fructosyltransferases was found to be 96 h (Zambelli et al., 2014).
3.3.3 Effect of m-FTase dose and substrate concentration on FOS production The effect of m-FTase dose on FOS generation indicated that 4.5 % (w/v) of mycelia supported maximum (325 g/L) FOS production (Fig. 2C). Kestose was predominant (51.8-274.5 g/L) transfructosylation product with low amounts (1.6-60.5 g/L) of nystose (Table S1). Bie and Zhu
(2016) achieved optimal neo-kestose production rate (0.028 mol/L/h) using 1.6 % (w/v) dry cells of immobilized yeast, Phaffia rhodozyma. FOS yield increased with increase in sucrose concentration upto 50 % (w/v) yielding 318.8 g/L FOS (Fig. 2D). Highest concentration of kestose (286.4 g/L) was obtained with 50 % (w/v) sucrose while maximum nystose concentration (48.4 g/L) was achieved with 30 % (w/v) sucrose (Table S2). Low availability of water at high sucrose concentration facilitates sucrose as fructosyl acceptor (Zhang et al., 2016). Extracellular β-fructofuranosidase from Xanthophyllomyces dendrorhous produced 40-168 g/L of neo-FOS at 420 to 600 g/L sucrose concentration (Linde, Rodríguez-colinas, Estévez, Poveda, Plou & Fernández, 2012). Maximum FOS (190 mg/mL) was obtained at 300 g/L sucrose concentration in case of β-FFase from Aspergillus phoenicis biofilms (Aziani, Terenzi, Jorge & Guimaraes, 2012). Recombinant FTase from A. niger YZ59 expressed in P. pastoris yielded 343 g/L FOS using 60 % (w/v) sucrose (Yang, Wang, Zhang & Shen, 2016). 3.3.4 Effect of metal ions and solvents on m-FTase Among the metal ions, mercury (Hg2+) and manganese (Mn2+) strongly inhibited A. tamarii m-FTase activity. Slight enhancement in m-FTase activity was observed with copper ions (Cu2+). FTase from Aspergillus aculeatus M105 showed enhanced activity (130 %) with Cu2+ (Huang et al., 2016). m-FTase activity was enhanced with most organic solvents excepting aniline and methanol. Hexane and ethyl acetate increased m-FTase activity by 18 and 14 %, respectively (Table S3). Gupta (1992) has also described enhancement in activity of several enzymes after treatment in organic environment. Reports suggest considerable conformational modifications in active site of enzymes after pre-incubation with hydrated organic solvents (Gupta, Tyagi, Sharma, Karthikeyan & Singh, 2000). 3.4 Time course of FOS generation by m-FTase
Under optimized conditions (pH : 7.0, temperature : 20°C, sucrose : 50 % w/v, m-FTase dose : 4.5 % w/v, inoculum age : 48 h and agitation speed : 160 rpm), highest FOS production (325.27 g/L; 55.14 %) was reached after 24 h with 14.18 % residual sucrose, 5.68 % fructose and 25 % glucose (Fig. 3A). During catalysis, fructose is transferred to sucrose resulting in kestose and nystose generation while residual glucose remains in the mixture due to competitive nature of hydrolysis and transfructosylation reaction (Jitonnom et al., 2018). Recently, 118 g/L FOS (64 % yield) were achieved in 38 h by Aspergillus ibericus (Nobre et al., 2017) while 56.9 % FOS yield was obtained within 8 h of sucrose transformation by Aspergillus tubingensis XG21 βfructofuranosidase (Xie et al., 2017). Dual-cell system of immobilized Aspergillus japonicus spores and Pichia heimii produced 0.62 g FOS per gram of sucrose from 30 % (w/v) sucrose in a continuous reactor in 25 days (Sheu, Chang, Wang, Wu & Huang, 2013). Industrial scale production of FOS by immobilized Aureobasidium pullulans was demonstrated in a packed bed reactor where 180 g/L h-1 FOS were produced from 77 % (w/v) sucrose at 50°C (Jung, Bang, Oh & Park, 2011). Commercial FFase from Aspergillus aculeatus yielded 55 % g/g FOS after 100 min of biotransformation of 60 % (w/v) sucrose at 50°C (Lorenzoni, Aydos, Klein, Rodrigues & Hertz, 2014). 3.4.1 Analysis of end products by 1H DOSY NMR The composition of end products was analyzed using DOSY NMR. Corroborating with the results of TLC and HPLC, four different diffusion coefficients from 0.25-0.65 (x 10-9 m2 s-1) representing fructose, sucrose, kestose and nystose were obtained with a linear gradient of 0.26 T/m at 16°C (Fig. 3B). Our results of 1H DOSY spectrum were in agreement with those obtained by Paredes, Smiderle, Santana-Filho, Kimura, Lacomini and Sassaki (2017) and suggest applicability of this technique in analyzing mixtures.
3.4.2 End product analysis by FTIR The FTIR analysis of emerging end-products over 24 h of biotransformation revealed considerable changes in the region ranging 900-1200 cm-1. Clear evolution of peaks at 996, 1025 and 1122 cm-1 during 1-24 h of FTase action indicated sucrose consumption and glycosidic bond formation due to transfructosylation (Fig. 3C). Strong band in this region corresponds to C–O–C glycosidic linkage, the mC–C and the dCOH vibrational modes. The gradual increase in transmittance in this region can be attributed to the increase in the concentration of FOS. The region (900-1200 cm-1) is called as “fingerprint region” of sugars (Santos, Araujo-Andrade, Tymczyszyn, & Gómez-Zavaglia, 2014; Mellado-Mojica, & López, 2015). Similar observations regarding FTIR spectra for FOS generation by sucrose transfructosylation using commercial FTase (Viscozyme®L) were reported by Romano, Santos, Mobili, Vega and Gómez-Zavaglia (2016). Gradual and characteristic change in this region indicated the applicability of this technique in the monitoring of FOS production. 3.5 Purification of FOS FOS were purified using Bio-Gel P2 matrix in a single step. Pure nystose (39 mg) and nytosekestose mixture (73.84 mg) was obtained in 48-54 min elution time from Bio-Gel P2 with a flow rate of 0.5 mL/min (Fig. 4). Similarly, FOS generated by sucrose transfructosylation by Aspergillus awamori NBRC 4033 β-FFase were purified by Bio-Gel P2 matrix (Smaali, Jazzar, Soussi, Muzard, Aubry & Marzouki, 2012). Nobre et al. (2012) purified FOS from fermentative broth using activated charcoal column matrix and eluted using ethanol gradient upto 50% (v/v). FOS purification using Bio-Gel P2 with water as eluent is a direct and easy method that may be scaled-up (Table S5).
3.6 Recycling of mycelial FTase for continuous FOS generation m-FTase (10 % w/v) was recycled upto 10 consecutive cycles of 2 h each, wherein it showed efficient production of FOS without any significant loss in activity. Kestose was obtained as the major product throughout and only a slight decrease in FOS concentration was noticed after 6th cycle (Fig. 5). Fructosyltransferase from A. oryzae expressed in Yarrowia lipolytica retained only 55 % of initial activity after 15th cycle (Zhang et al., 2016). β-FFase from Xanthophyllomyces dendrorhous retained activity upto 7 cycles of 26 h each (Míguez et al., 2018). In the present study, continuous use of mycelial FTase for efficient biotransformation of sucrose was demonstrated. This system based on mycelium bound FTase combines merits of both pure and immobilized enzyme and utilizes rapidly reproducible mycelia thus avoiding need of purifying enzyme and any tedious immobilization procedures. 4. Conclusion Zymographic analysis of cellular fractions revealed presence of a mycelial FTase in A. tamarii NKRC 1229. Enhanced production of FOS was achieved by parametric optimization of various factors. m-FTase produced high FOS titres (325 g/L) in 24 h at 20°C. Purification of FOS was achieved using Bio-Gel P2 in a single step. FOS production based on mycelia bound FTase combines merits of both pure and immobilized enzyme that can be exploited at large scale.
Acknowledgements Author RC is grateful to UGC, New Delhi for providing financial assistance as PhD scholarship. Authors are thankful to Dr. K.K. Dey, Department of Physics for help in NMR analysis. Authors acknowledge the instrumentation facilities available under Sophisticated Instrumentation Centre (SIC) and DST-PURSE (II) at Dr. Harisingh Gour Vishwavidyalaya, Sagar, India.
Conflict of interest Authors have no conflicts of interest.
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Table 1: Distribution of FTase in A. tamarii NKRC1229 and fructooligosaccharide generation
FTase (U)
Nystose (mg/ml)
Kestose (mg/ml)
Total FOS (mg/ml)
Intracellular
21.58a
12.46
55.88
68.34
Mycelial
14.45a
11.40
35.11
46.51
ND
ND
ND
ND
Location
Extracellular
ND – Not Detected; a - U/g mycelia
Figure Captions Fig. 1: Zymogram showing localization of fructosyltransferase (FTase). MW: Molecular weight marker, EC: Extracellular fraction, IC: Intracellular fraction, ME: Mycelial extract. Gel was incubated in sucrose solution (50 % w/v, pH 7.0) at 28°C for 1 h and stained with 1 % (w/v) TTC in 0.25M NaOH. Fig. 2: Optimization of FOS generation by Aspergillus tamarii NKRC 1229 mycelial fructosyltransferase (A) Effect of pH (-●-) and temperature (-▲-) on FOS generation by m-FTase. (B) Effect of mycelial age on FOS production. 0.5 g of m-FTase was added to 20 mL sucrose (20 % w/v) and incubated at 20°C for 5 h at 160 rpm. (C) Effect of m-FTase dose on FOS generation. m-FTase (0.5-5 % w/v) was added to 20 mL sucrose (50 % w/v) and incubated at 20°C for 5h at 160 rpm. (D) Effect of sucrose concentration on FOS generation. m-FTase (2.5 % w/v mycelia) was incubated with sucrose solution (1-80 % w/v, pH 7.0) at 20°C for 5 h at 160 rpm. Values represent average of three replicates ± standard deviation. Fig. 3: FOS generation and characterization (A) Time course of FOS generation under optimized conditions (sucrose: 50 % w/v, pH: 7.0, temp.: 20°C) over 24 h. (B) 1H DOSY NMR spectrum of
sucrose biotransformation products showing FOS formation (C) FTIR spectra showing FOS generation in 24 h. Arrows at 996, 1025 and 1122 cm-1 peaks indicate increase in the FOS fingerprint region. Fig. 4: Elution profile of biotransformation products using Bio-Gel P2. FOS were eluted using water at 28°C with a flow rate of 0.5 mL/min. Fig. 5: Recycling of A. tamarii m-FTase for continuous FOS generation. Mycelia (10 % w/v) was used to biotransform sucrose solution (50 % w/v, pH 7.0) at 20ºC and 160 rpm for 10 cycles of 2 h each.
kDa MW 250 150 100 75
EC
50 37
25
Fig. 1
IC
ME
Temperature (°C) 0
10
20
30
40
50
60
70
80
120
200
B
A
175
110
FOS Yield (g/L)
125 100 75
100 90 80
50
70
25
60
0 5
6
7
8
9
10
11
24
48
pH
72
96
Mycelial Age (h)
D
110 100
110
Fig.902 Relative FOS Concentration (%)
4
100
110 Relative FOS Concentration (%)
3
100 90 Biotransformation Yield (%)
FOS (g/L)
150
80 70 60 50
90 80 70 60 50 40 30
80
60 50 40 30 20
20
10 0
0
30
Total FOS (GF2+GF3) Sucrose (GF) Fructose (F) Glucose (G)
70
10
40
C
A
0.5
1
1.5
1
5
2
2.5
10
3
20
3.5
4
30
4.5
40
5
50
60
Sucrose Concentration (% w/v) Mycelia (% w/v)
20 10 0 0
0.5
1
2
3
4
Incubation Time (h)
8
15
24
70
80
B
996 1025
Glucose / Fructose
D/10-9 m2s-1
CFig.
Sucrose
1122 Kestose (DP3) Nystose (DP4)
1H
Chemical Shift / ppm
A
50
Nystose Kestose Sucrose Glucose
Concentration (g/L)
40
30
20
Fig. 4
10
0 40
45
50
55
60
Elution Time (min)
65
70
75
3
Fructose
% Yield
120
Glucose
100
Sucrose
80
Kestose
60
Nystose
40 20 0
1
2
3
4
5 6 Cycle
7
8
9
10
Fig. 5
Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Author RC is grateful to UGC, New Delhi for providing financial assistance as PhD scholarship. Authors have no conflicts of interest.
Highlights
Mycelial fructosyltransferase (~75kDa) from Aspergillus tamarii was characterized
Fructosyltransferase yielded 55% (w/v) oligofructose from sucrose at 20°C in 24 h
Fructooligosaccharides were purified and resolved using 1H DOSY-NMR and FTIR
Enzyme was recycled upto 10 cycles with high productivity (106.12 g/L/h)