Screening of biocatalysts for transformation of sucrose to fructooligosaccharides

Article abstract of DOI:10.1016/j.molcatb.2013.07.008

Twenty microorganisms comprising of sixteen molds, two yeasts and two bacteria were evaluated for their ability to produce fructosyltransferase (FTase) and generate fructooligosaccharides (FOS) from sucrose. FTase production by these microorganisms was studied over a period of 120 h on medium containing 20% (w/v) sucrose as the sole carbon source. High FTase levels (35-31 U/ml) were observed in culture filtrates of Aspergillus flavus, Aspergillus niger, Aspergillus terreus and Penicillium islandicum. Higher concentrations of FOS were generated from 50% (w/v) sucrose using culture filtrates of A. flavus NFCCI 2364 (63.40%, w/w), A. niger SI 19 (54.94%, w/w), A. flavus NFCCI 2785 (44.61%, w/w), P. islandicum MTCC 4926 (43.56%, w/w), A. terreus NFCCI 2347 (24.17%, w/w) and Fusarium solani NFCCI 2315 (15.25%, w/w). Kestose, nystose and 1-fructofuranosyl nystose were the predominant oligosaccharides as revealed by HPLC analysis.

Full text of DOI:10.1016/j.molcatb.2013.07.008

Journal of Molecular Catalysis B: Enzymatic 97 (2013) 12–17
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Screening of biocatalysts for transformation of sucrose to
fructooligosaccharides
Mohd Anis Ganaie , Uma Shanker Gupta , Naveen Kangob
a,∗
a
a
Microbial Technology Laboratory, Department of Zoology, Dr. Harisingh Gour University, Sagar 470003, MP, India
Department of Applied Microbiology and Biotechnology, Dr. Harisingh Gour University, Sagar 470003, MP, India
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Twenty microorganisms comprising of sixteen molds, two yeasts and two bacteria were evaluated for
their ability to produce fructosyltransferase (FTase) and generate fructooligosaccharides (FOS) from
sucrose. FTase production by these microorganisms was studied over a period of 120 h on medium con-
taining 20% (w/v) sucrose as the sole carbon source. High FTase levels (35–31 U/ml) were observed in
culture filtrates of Aspergillus flavus, Aspergillus niger, Aspergillus terreus and Penicillium islandicum. Higher
concentrations of FOS were generated from 50% (w/v) sucrose using culture filtrates of A. flavus NFCCI
Received 12 January 2013
Received in revised form 11 July 2013
Accepted 13 July 2013
Available online 23 July 2013
Keywords:
2
4
364 (63.40%, w/w), A. niger SI 19 (54.94%, w/w), A. flavus NFCCI 2785 (44.61%, w/w), P. islandicum MTCC
926 (43.56%, w/w), A. terreus NFCCI 2347 (24.17%, w/w) and Fusarium solani NFCCI 2315 (15.25%, w/w).
Fructooligosaccharides
Microorganisms
Fructosyltransferase
Biotransformation
Kestose, nystose and 1-fructofuranosyl nystose were the predominant oligosaccharides as revealed by
HPLC analysis.
©
2013 Elsevier B.V. All rights reserved.
1
. Introduction
(FFase, EC 3.2.1.26) with high transfructosylating activity are the
two enzymes known to catalyze FOS generation from sucrose.
FTase possess only transfructosylating activity, cleave the ␤-1,
2 linkage of sucrose and transfer fructosyl group to an accep-
tor molecule leading to formation of fructooligosaccharides and
release of glucose. FFase catalyze both hydrolytic and transfruc-
tosylating reactions, however, latter is evidenced only with higher
sucrose concentrations [9–11]. Efforts are being made to explore
and identify microorganisms that produce FTase with desired
activity, stability and end products to suit industrial conditions
[12].
Owing to these benefits considerable efforts on the isolation
and screening of microorganisms for FTase production with high
transfructosylating activity are being made. Microorganisms like
Aspergillus niger, Aspergillus oryzae, Aspergillus japonicus, Aureoba-
sidium pullulans are regarded as potential producers of FOS [13–15].
The most investigated microorganisms in this subject are fungi
belonging to genera Aureobasidium, Aspergillus and Penicillium
[1,16]. Fructosyltransferase production from bacteria (Lactobacil-
lus) and yeasts (Rhodotorula, Candida, Cryptococcus sp) has also
been reported to some extent [17,18]. However, there is a strong
need to explore the microbial diversity for identification of strains
with high transfructosylating activity containing more oligosaccha-
rides and less of monomeric sugars [14]. The main objective of the
screening scenario was to find new microbial strains having high
transfructosylating activity for biotransformation of sucrose to FOS
production.
Recent awareness regarding the nutritional and health bene-
fits of prebiotic oligosaccharides has lead to the development of
several biotechnological endeavors for their generation. FOS con-
sists of a mixture of fructo-oligomers with two or three fructose
units bound to the ␤-2, 1 position of sucrose. Conventionally, these
are mainly understood to be composed of 1-kestose, 1-nystose and
1
-fructofuranosyl-nystose [1]. FOS have been particularly studied
because of their specific physiological effects on human health
especially growth stimulation of beneficial bifidobacteria in the
digestive tract by inhabiting growth of pathogens [2,3], protonation
of potentially toxic ammonia and amines [4], relief of constipation
[
5], diminution of total blood cholesterol, triglyceride, phospho-
lipids and general improvement of human health [2,6]. They also
confer secondary beneficial effects like non cariogenicity, safety for
diabetics, absorption of Ca , Mg and Fe ions [7]. In food industries,
FOS are also used as stabilizers or bulking agents for the production
of sweeteners [8].
Commercial production of FOS relies upon enzymatic trans-
formation of sucrose by microbial fructosyltransferase (FTase).
Fructosyltransferase (FTase, EC 2.4.1.9) and ␤-fructofuranosidase
+
+
+
∗ Corresponding author. Tel.: +91 7582 265056; fax: +91 7582 265994.
E-mail addresses: mohd.anis90@yahoo.com, anisurehman.2011@rediffmail.com
(
M.A. Ganaie).
1
381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2013.07.008

M.A. Ganaie et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 12–17
13
◦
2
. Materials and methods
with distilled water and dried at 105 C for 4 h and was used for
biomass determination as dry cell weight per volume (g/l).
2.1. Microorganisms and culture conditions
2.5. Enzyme assays
Twenty microbial strains including sixteen molds, two yeasts
and two bacteria were investigated for transfructosylating activ-
ity. Fifteen microorganisms were obtained from culture collections
viz. National fungal culture collection of India (NFCCI, Pune, India),
American type culture collection (ATCC, USA) and Microbial type
culture collection (MTCC, Chandigarh, India). Five molds were iso-
lated from sugarcane field plantations of Sagar district, Madhya
Pradesh and apple orchids of Wakura Ganderbal Kashmir. Soil
isolates (SI) of Aspergillus flavus (NFCCI 2783, NFCCI 2785) and
Fusarium sp. (NFCCI 2784) were deposited in NFCCI, Pune while
A. niger (SI 19) and Trichoderma sp. (SI 27) are deposited in culture
collection of Department of Applied Microbiology, Dr. Harisingh
Gour University, Sagar. A. flavus (NFCCI 2364), Aspergillus versicolor
Extracellular
fructosyltransferase
(FTase)
and
␤-
fructofuranosidase (FFase) were assessed by incubating 250 ␮l of
enzyme sample with 750 ␮l of sucrose solution as described earlier
19,20]. For transfructosylating activity (Ut) 50% (w/v) sucrose in
[
0
.1 M citrate buffer (pH 5.5) was incubated with enzyme sample
◦
at 55 C for 1 h in a water bath and the reaction was arrested by
keeping the reaction mixture in boiling water for 10 min. FTase
activity was analyzed in appropriately diluted reaction mixture
(
1/50) with commercial GOD-POD kit. The absorbance of released
glucose was read at 505 nm by UV/visible spectrophotometer
Hitachi Techcom India). Hydrolytic activity was determined
following the same procedure except the substrate used was 0.5%
w/v) sucrose. The conversion of sucrose by FFase yields glucose
(
(
NFCCI 2025), Aspergillus fumigatus (NFCCI 2452), A. niger (ATCC
(
2
1
2
601), Fusarium solani (NFCCI 2315), Aspergillus awamori (NFCCI
560), Aspergillus terreus (NFCCI 2347), Rhizopus oryzae (NFCCI
282) and Penicillium purpurogenum (MTCC 1786) and aforesaid
and fructose. However, presence of FTase directs the addition of
fructose for generating fructan polymer [15]. The estimated glucose
(
G) and reducing sugar (R) in the reaction mixture (F = fructose,
five soil isolates were grown and maintained on PDA (Potato dex-
trose agar). Penicillium chrysogenum (MTCC 161) and Penicillium
islandicum (MTCC 4926) were grown on Czapek extract agar (CEA).
Yeasts Candida albicans (ATCC 10231) and Saccharomyces cerevisiae
ꢀ
F = transferred fructose) are explained in Eqs. (1) and (2) below.
R = G + F → F = R − G
Fꢀ = G − F = 2G − R
(1)
(2)
(
ATCC 2601) were grown on SYPA (sucrose, yeast extract, peptone
and agar) whereas bacterial strains, Bacillus cereus (ATCC 117) and
Bacillus subtilis (ATCC 6621) were cultured on nutrient agar media
One unit of FFase is defined as the amount of enzyme required
for the hydrolysis of 1 ␮mole of sucrose per minute. One unit
of transfructosylating activity is defined as amount of enzyme
(
NAM). The temperature for incubation for molds and yeasts was
◦
◦
2
8 C while bacteria were incubated at 35 C.
ꢀ
required to transfer one 1 ␮mole of F per minute.
2.2. Chemicals
2.6. Production and analysis of FOS
FOS standards 1-kestose (GF ), 1-nystose (GF ), 1-
2
3
FOS production was carried out by adding 1 ml of enzyme sam-
fructofuranosylnystose (GF ) and sugar standards sucrose, glucose,
4
ples collected at various time intervals to 3 ml of 50% (w/v) sucrose
fructose, were obtained from Wako Pure Chemicals Japan and
Sigma Aldrich USA. Other analytical grade chemicals and media
ingredients were purchased from Hi-media and Merck (India).
◦
dissolved in 0.1 M citrate buffer (pH 5.5) for period of 24 h at 55 C.
The amount of FOS formation in the samples was analyzed by high
performance liquid chromatography (HPLC, Waters) with sugar-
pak column (6.5 × 300 mm) and refractive index (RI) differential
detector (RI 2414). The samples were filtered using 0.45 ␮m mem-
brane filters (Millipore) before injecting through the 20 ␮l valve.
2
.3. Inoculum development
Inoculum of molds was prepared by transferring full loop of
◦
The temperature of the column was maintained at 70 C by column
spores harvested from five days old cultures of strains grown on
PDA into 100 ml of sucrose-yeast extract (SYE) broth (1% sucrose
◦
oven (Dyna, Mumbai). The RI detector was operated at 30 C and
◦
water was used as the mobile phase at a flow rate of 0.2 ml/min.
The retention times of FOS components were compared with the
standards for identification and their concentration was quantified
from the peak area. Calculations and analysis were performed using
Empower 2 software, Build 2154 (Waters).
and 0.2% yeast extract, pH 5.5) and incubating at 28 C for 24 h on
rotatory shaker (Lark Innovata, Germany) at 200 rpm. Yeast inocu-
lum was prepared by adding a loopful of five days old yeast culture
◦
in the SYE medium and incubating it at 28 C for 24 h at 200 rpm. For
bacterial cultures, loopful of 2–3 days old culture was transferred
◦
in the NA medium and flasks were incubated at 37 C for 24 h at
2
00 rpm.
3. Results and discussion
2.4. Production of fructosyltransferase enzyme
3.1. Change in pH and biomass during fermentation
Cultivation medium used for enzyme production contained
w/v): sucrose 20%, yeast extract 0.5%, NaNO3 1%, MgSO .7 H O
4 2
All the test microorganisms were able to colonize sucrose-
yeast extract medium producing different amounts of biomass
and changes in pH of the medium. Maximum biomass forma-
tion (41.6 g/l) was recorded in native soil isolate, A. niger (SI 19)
after 120 h. Other microorganisms like A. fumigatus (NFCCI 2452),
A. flavus (NFCCI 2785), A. flavus (NFCCI 2783) A. awamori (NFCCI
1560), A. terreus (NFCCI 2347), F. solani (NFCCI 2315), P. islandicium
(MTCC 4926) showed cell mass production in the range of 20–40
(g/l) (Table 1). The rest of molds, yeasts and bacteria showed rela-
tively poor growth and produced biomass only upto 10 (g/l). Least
biomass production 0.63 (g/l) was seen in case of A. versicolor NFCCI
2025 at the end of 120 h. A. flavus 2364 produced 36.2 (g/l) biomass
(
0
.05%, KH PO 0.25%, NH Cl 0.5%, and NaCl 0.25% having an ini-
2 4 4
tial pH of 5.5. Aliquots of 100 ml of this medium were dispensed in
◦
2
50 ml Erlenmeyer flasks and autoclaved at 121 C for 15 min. 10 ml
from 24 h old inoculum was transferred into 100 ml media and the
◦
◦
flasks were incubated at 28 C (molds and yeasts) or 35 C (bacteria)
in shaker incubator at 200 rpm for 120 h. Flasks were withdrawn at
regular time intervals of 48, 72, 96, and 120 h. Contents were fil-
tered through Whatman filter paper No. 2 and the cell-free culture
filtrate was used as a source of extracellular enzyme without fur-
ther purification. The cell mass obtained after filtration was washed

1
4
M.A. Ganaie et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 12–17
Table 1
◦
Changes occurring in pH of medium and biomass (g/l) formation during 120 h of fermentation (culture conditions: 200 rpm, 28 C). Data has been represented as
mean ± standard deviation (average of 3 repetitions).
Microorganism
48 (h)
pH
72 (h)
pH
96 (h)
pH
120 (h)
pH
Biomass
Biomass
Biomass
Biomass
A. versicolor (NFCCI 2025)
A. fumigatus (NFCCI 2452)
A. flavus (NFCCI 2364)
A. flavus SI (NFCCI 2785)
A. flavus SI (NFCCI 2783)
A. awamori (NFCCI 1560)
A. terreus (NFCCI 2347)
A. niger (SI 19)
4.48 ± 0.64
5.87 ± 0.49
5.81 ± 0.11
4.45 ± 0.76
4.28 ± 1.45
4.08 ± 0.23
4.93 ± 0.21
3.35 ± 0.58
2.98 ± 0.67
4.29 ± 1.02
6.35 ± 0.38
3.18 ± 0.55
3.14 ± 0.36
2.15 ± 0.63
3.53 ± 0.53
2.55 ± 0.40
2.34 ± 0.45
3.39 ± 0.60
7.50 ± 0.34
7.90 ± 0.17
0.32 ± 0.10
28.41 ± 0.61
36.24 ± 0.60
4.45 ± 1.12
13.66 ± 1.09
23.74 ± 1.45
15.27 ± 0.67
21.63 ± 1.44
15.74 ± 1.56
11.53 ± 0.56
22.98 ± 1.36
2.17 ± 0.06
17.33 ± 0.70
4.29 ± 0.34
26.53 ± 0.56
1.30 ± 0.62
3.46 ± 0.67
2.15 ± 0.82
0.90 ± 0.28
0.66 ± 0.22
4.96 ± 0.19
6.05 ± 0.49
5.70 ± 0.25
4.64 ± 1.23
4.89 ± 0.78
3.88 ± 0.26
3.81 ± 0.25
2.76 ± 0.73
2.54 ± 1.03
5.50 ± 0.67
5.50 ± 0.40
3.33 ± 0.97
3.04 ± 0.45
2.35 ± 0.27
4.17 ± 0.53
3.14 ± 0.16
2.71 ± 0.33
3.13 ± 0.87
7.39 ± 0.52
7.16 ± 0.59
0.45 ± 0.23
30.19 ± 0.48
31.26 ± 0.80
4.64 ± 1.43
16.99 ± 0.78
26.15 ± 1.70
18.49 ± 1.50
33.81 ± 1.45
20.53 ± 1.50
15.65 ± 0.43
24.15 ± 0.55
3.11 ± 0.01
20.0 ± 0.81
5.98 ± 0.61
27.16 ± 1.02
1.77 ± 0.49
5.59 ± 1.00
2.75 ± 0.79
1.07 ± 0.49
0.67 ± 0.35
5.10 ± 0.37
6.25 ± 0.43
5.62 ± 0.23
5.34 ± 0.89
5.46 ± 0.49
3.54 ± 0.54
3.17 ± 0.16
2.45 ± 0.70
1.98 ± 0.65
5.80 ± 0.76
5.19 ± 0.16
3.32 ± 0.50
3.39 ± 0.49
2.37 ± 0.23
4.23 ± 0.55
3.77 ± 0.62
2.73 ± 0.64
3.11 ± 0.80
7.15 ± 0.60
7.15 ± 0.40
0.57 ± 0.21
30.85 ± 0.51
25.39 ± 1.24
5.34 ± 0.56
22.64 ± 0.86
30.20 ± 2.07
21.82 ± 1.43
37.85 ± 1.01
25.67 ± 0.78
17.72 ± 0.88
26.61 ± 1.18
3.91 ± 0.05
23.79 ± 0.65
6.14 ± 0.71
29.87 ± 0.62
2.26 ± 0.39
5.33 ± 0.79
3.01 ± 0.18
1.17 ± 0.59
0.84 ± 0.25
5.45 ± 0.28
6.46 ± 0.39
5.56 ± 0.16
5.75 ± 1.23
5.86 ± 1.07
3.36 ± 0.47
2.51 ± 0.29
1.82 ± 0.47
1.75 ± 0.98
6.37 ± 0.99
4.63 ± 0.24
3.55 ± 0.61
3.47 ± 0.43
2.52 ± 0.32
4.49 ± 0.41
5.26 ± 0.47
2.93 ± 0.28
2.98 ± 0.78
6.94 ± 0.26
6.85 ± 0.75
0.63 ± 0.19
32.03 ± 0.10
20.46 ± 1.27
5.75 ± 1.00
23.86 ± 1.78
31.86 ± 1.48
17.34 ± 1.09
41.61 ± 0.60
13.67 ± 0.46
19.42 ± 0.53
27.64 ± 0.58
3.52 ± 0.05
27.42 ± 0.61
5.95 ± 0.41
31.22 ± 0.45
2.45 ± 0.44
5.66 ± 1.10
3.82 ± 1.44
0.77 ± 0.57
0.64 ± 0.30
A. niger (ATCC 2601)
Trichoderma sp. (SI 27)
F. solani (NFCCI 2315)
Fusarium sp. SI (NFCCI 2784)
R. oryzae (NFCCI 2282)
P. purpurogenum (MTCC 1786)
P. chrysogenum (MTCC 161)
P. islandicum (MTCC 4926)
C. albicans (ATCC 10231)
S. cerevisiae (ATCC 2601)
B. cereus (ATCC 117)
B. subtilis (ATCC 6621)
in 48 h followed by sharp decrease indicating rapid growth of the
fungus in first two days followed by autolysis.
of incubation while A. niger (SI 19) produced 35.64 U/ml of Ut
in relatively short span of 72 h incubation. This value was not
maintained on subsequent periods due to gradual decrease of
transfructosylating activity which reduced Ut/Uh ratio. Noticeably,
A. terreus, A. awamori and P. islandicum produced approximately
32.0 U/ml of transfructosylating activity in 48 h of incubation but
their enzyme activity was sharply reduced on subsequent fermen-
tation periods. Strains of yeasts and bacteria show significant levels
of FTase activity during course of 120 h fermentation. However
higher hydrolytic activities (Uh) were only noticed in A. terreus
(21.5 U/ml) and A. awamori (17.45 U/ml) which indicate presence
of ␤-fructofuranosidase enzyme. The pattern of FOS formation in
these two microorganisms is different because FFase transforms
sugar to FOS (Fig. 1b and c) and simultaneous releases of monomeric
sugars like glucose and fructose respectively [8]. The Ut/Uh ratio
is preferable for indicating relative intensity of transfructosylating
activity for efficient formation of FOS and this ratio is most impor-
tant parameter for screening of microorganisms for FOS production.
A sharp decrease in pH from initial set value of 5.5–2.0 by
many molds and yeasts was noticed during the cultivable length
of time. This may be due to the production of some organic acids
by molds. However, other strains did not show any major changes
in their medium pH. Since the pH was not controlled during the
whole fermentation process and the enzyme extracted was first
settled by buffer system before use. Previously published results
also indicate a sharp decrease in pH in case of Rhizopus stolonifer
[
19].
3.2. Transfructosylating and hydrolytic activities
Aliquots withdrawn from cultures at regular intervals from
8 h to 120 h of incubation were analyzed for the transfructosy-
4
lating activity (Ut) and Uh activities (Table 2). Higher Ut levels
were noticed in A. flavus NFCCI 2364 (33.73 U/ml) after 120 h
Table 2
Transfructosylating activity (Ut) and hydrolytic activity (Uh) of twenty selected microorganisms grown on medium containing 20% (w/v) sucrose as C-source over a period of
◦
1
20 h. Culture filtrate obtained at different time intervals was used as source of enzyme and was incubated for 1 h at 50 C with 50% (w/v) and 0.5% (w/v) sucrose respectively.
Data represent mean ± standard deviation of three independent experiments.
Microorganism
48 (h)
Ut
72 (h)
Ut
96 (h)
Ut
120 (h)
Ut
Uh
2.16 ± 0.41
Uh
3.10 ± 0.53
Uh
6.76 ± 0.53
Uh
4.97 ± 0.46
A. versicolor (NFCCI 2025)
A. fumigatus (NFCCI 2452)
A. flavus (NFCCI 2364)
A. flavus SI (NFCCI 2785)
A. flavus SI (NFCCI 2783)
A. awamori (NFCCI 1560)
A. terreus (NFCCI 2347)
A. niger (SI 19)
3.97 ± 0.39
7.63 ± 0.87
10.76 ± 0.81
7.40 ± 0.24
28.08 ± 0.83
20.09 ± 1.02
19.86 ± 0.67
21.07 ± 1.64
15.47 ± 0.41
28.05 ± 1.16
17.98 ± 1.22
12.95 ± 0.87
12.78 ± 0.43
5.93 ± 0.84
9.00 ± 0.61
15.56 ± 1.06
24.89 ± 1.22
13.78 ± 0.90
11.83 ± 0.77
15.76 ± 1.91
12.85 ± 0.77
7.29 ± 0.56
11.90 ± 0.68
7.52 ± 0.43
33.73 ± 0.74
23.76 ± 0.56
22.84 ± 1.00
13.32 ± 0.82
16.42 ± 0.39
22.77 ± 1.65
19.63 ± 1.09
14.36 ± 0.33
15.67 ± 0.70
6.02 ± 0.80
11.30 ± 0.78
16.33 ± 0.52
19.78 ± 1.39
15.98 ± 1.16
12.70 ± 1.74
6.30 ± 1.92
8.41 ± 0.50
4.67 ± 0.77
3.67 ± 0.46
18.91 ± 0.97
14.36 ± 0.67
11.87 ± 1.09
20.46 ± 2.41
31.85 ± 0.98
24.76 ± 1.28
12.87 ± 0.56
8.98 ± 0.76
5.97 ± 0.60
4.91 ± 0.47
6.61 ± 1.07
13.48 ± 0.85
32.65 ± 1.42
8.78 ± 0.75
12.81 ± 1.23
13.04 ± 1.25
16.87 ± 0.95
4.05 ± 0.44
1.90 ± 0.25
5.02 ± 0.11
4.56 ± 0.74
4.08 ± 0.28
17.45 ± 1.04
21.51 ± 0.80
11.34 ± 1.03
8.98 ± 0.83
6.63 ± 0.53
2.94 ± 0.77
2.36 ± 0.86
3.20 ± 0.47
8.55 ± 0.50
12.67 ± 0.86
6.22 ± 0.73
9.72 ± 0.84
9.05 ± 1.20
9.14 ± 0.27
2.61 ± 0.40
6.07 ± 0.25
24.09 ± 1.41
18.87 ± 0.99
13.98 ± 0.78
22.60 ± 2.38
12.94 ± 0.44
35.64 ± 1.25
15.76 ± 0.98
10.74 ± 0.54
9.10 ± 0.82
5.11 ± 0.82
6.12 ± 1.15
14.70 ± 0.66
29.07 ± 2.77
10.89 ± 0.44
12.22 ± 0.80
12.97 ± 0.92
13.68 ± 0.48
8.86 ± 0.25
2.97 ± 0.21
5.24 ± 0.23
4.98 ± 0.53
4.21 ± 0.74
14.67 ± 0.73
9.21 ± 0.99
13.05 ± 2.04
9.89 ± 0.57
6.89 ± 0.73
3.40 ± 0.41
2.89 ± 0.85
4.72 ± 0.61
9.26 ± 0.75
11.78 ± 0.62
6.95 ± 0.45
7.42 ± 0.82
8.39 ± 1.59
8.99 ± 0.59
2.59 ± 0.25
3.50 ± 0.24
5.04 ± 0.15
5.01 ± 0.44
6.98 ± 0.65
13.62 ± 0.57
8.44 ± 0.90
13.38 ± 2.66
11.73 ± 1.08
7.98 ± 0.45
5.28 ± 0.50
3.17 ± 0.82
7.44 ± 0.64
8.10 ± 1.30
10.39 ± 1.31
9.03 ± 0.65
9.21 ± 0.79
9.50 ± 2.41
9.53 ± 0.23
2.96 ± 0.54
4.16 ± 0.50
4.79 ± 0.34
5.28 ± 0.33
7.89 ± 0.44
10.53 ± 0.67
8.31 ± 0.41
11.19 ± 1.23
10.93 ± 0.68
8.63 ± 1.06
3.76 ± 0.51
3.18 ± 0.87
9.24 ± 0.96
6.68 ± 0.67
10.60 ± 0.87
11.41 ± 1.25
8.90 ± 0.55
3.39 ± 1.29
3.66 ± 0.58
4.24 ± 0.96
A. niger (ATCC 2601)
Trichoderma sp. (SI 27)
F. solani (NFCCI 2315)
Fusarium sp. SI (NFCCI 2784)
R. oryzae (NFCCI 2282)
P. purpurogenum (MTCC 1786)
P. chrysogenum (MTCC 161)
P. islandicum (MTCC 4926)
C. albicans (ATCC 10231)
S. cerevisiae (ATCC 2601)
B. cereus (ATCC 117)
B. subtilis (ATCC 6621)

M.A. Ganaie et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 12–17
15
Fig. 1. HPLC chromatograms of reaction product (FOS) formed by incubating culture filtrates obtained from different fungi with 20% (w/v) sucrose (incubation time: 24 h,
◦
pH 5.5, 50 C). (a) A. flavus NFCCI 2364, (b) A. awamori NFCCI 1560, (c) A. niger SI 19, (d) A. terreus NFCCI 2347, (e) F. solani NFCCI 2315 and (f) P. islandicum MTCC 4926
(
abbreviations: S = solvent, G = glucose, F = fructose, GF = sucrose, GF2 = kestose, GF3 = nystose, GF4 = fructofuranosylnystose).
However this ratio varies by optimizing cultural parameters like pH
and temperature due to dependence of FTase and FFase [9]. In our
study the maximum Ut/Uh was achieved in three strains of A. flavus
NFCCI (2364, 2785, 2783), A. niger (SI 19), P. islandicum, F. solani, A.
terreus and A. awamori in distinct incubation periods. The value of
FTase are comparable by previously published reports by [19,21]
which accounted maximum activity of FTase in 120 h of incubation
in A. oryzae CFR 202 and R. stolonifer LAU 07 respectively. The corre-
sponding increase of FTase activity from 48–120 h in A. flavus NFCCI
2364 may be releasing of intracellular FTase by disintegration of
Table 3
FOS formation % w/w (weight by weight) of selected microorganisms from enzyme substrate (FTase-sucrose) reaction by culture filtrates of different fermentation time.
Microorganism
48 (h)
0.59 ± 0.12
72 (h)
1.25 ± 0.52
96 (h)
1.38 ± 0.24
120 (h)
1.5 ± 0.25
A. versicolor (NFCCI 2025)
A. fumigatus (NFCCI 2452)
A. flavus (NFCCI 2364)
A. flavus SI (NFCCI 2785)
A. flavus SI (NFCCI 2783)
A. awamori (NFCCI 1560)
A. terreus (NFCCI 2347)
A. niger (SI 19)
0.74 ± 0.11
36.14 ± 1.08
19.89 ± 0.67
11.86 ± 0.43
3.66 ± 0.48
24.38 ± 1.35
26.69 ± 1.26
1.78 ± 0.63
0.98 ± 0.56
4.03 ± 0.63
0.67 ± 0.41
0.68 ± 0.39
4.54 ± 0.59
42.51 ± 1.83
0.49 ± 0.44
2.28 ± 1.75
1.53 ± 0.67
1.88 ± 0.74
0.93 ± 0.22
0.85 ± 0.21
48.20 ± 1.92
33.64 ± 0.99
17.97 ± 0.74
4.34 ± 0.59
21.08 ± 0.91
55.81 ± 1.71
2.35 ± 1.08
1.53 ± 1.08
8.51 ± 0.72
1.08 ± 1.16
1.77 ± 0.74
5.57 ± 0.91
18.19 ± 1.25
1.06 ± 0.29
5.16 ± 1.44
1.45 ± 0.93
2.58 ± 0.43
0.95 ± 0.64
0.89 ± 0.63
55.51 ± 1.54
40.56 ± 0.53
23.86 ± 0.53
7.29 ± 1.31
20.13 ± 1.54
43.83 ± 2.08
4.18 ± 1.35
1.78 ± 0.97
12.45 ± 1.44
1.82 ± 0.66
2.41 ± 1.53
5.84 ± 1.27
14.96 ± 1.31
1.32 ± 0.65
2.67 ± 1.28
2.83 ± 0.70
1.29 ± 0.51
1.42 ± 0.58
1.20 ± 0.22
63.40 ± 2.58
44.61 ± 1.09
28.98 ± 0.97
10.47 ± 0.67
18.07 ± 1.03
37.22 ± 1.52
5.89 ± 0.54
2.10 ± 1.03
15.12 ± 0.71
2.88 ± 0.23
3.55 ± 1.24
8.56 ± 2.41
10.21 ± 1.05
1.15 ± 0.70
3.42 ± 1.67
2.76 ± 0.60
0.78 ± 0.39
0.56 ± 0.31
A. niger (ATCC 2601)
Trichoderma sp. (SI 27)
F. solani (NFCCI 2315)
Fusarium sp. SI (NFCCI 2784)
R. oryzae (NFCCI 2282)
P. purpurogenum (MTCC 1786)
P. chrysogenum (MTCC 161)
P. islandicum (MTCC 4926)
C. albicans (ATCC 10231)
S. cerevisiae (ATCC 2601)
B. cereus (ATCC 117)
B. subtilis (ATCC 6621)

1
6
M.A. Ganaie et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 12–17
Fig. 2. Maximum production of kestose (GF2), nystose (GF3), 1-fructofuranosylnystose (GF4) during production of FOS by microorganisms.
Fig. 3. Reaction scheme of FOS formation by enzyme catlyzed reaction of diaccharide (sucrose) with fructosyltransferase (FTase) and fructofuranosidase (FFase) enzyme.

M.A. Ganaie et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 12–17
17
hypae in consequence of prolonged agitation. While as decrease in
4. Conclusion
enzyme activity after 48–72 h of incubation in other strains may be
releasing of some metabolic by-products such as ethanol and acetic
acid which tend to decline the FTase activity [22].
The present effort of screening programme resulted in
identification of some new microbial strains showing high trans-
fructosylating activity for FOS production. A good attainment of
their favourable yield clearly signifies their vast ability for com-
mercial application in food and feed industry. However more
extensive work is needed to optimize the cultural parameters
and purifying the FTase to enhance the productivity of FOS. The
HPLC analysis deduce A. flavus (NFCCI 2364), A. niger (SI 19)
and P. islandicum (MTCC 4926) the most prosperous strain for
synthesizing high amount of FOS to be captivated on industrial
level.
3.3. Production of FOS using cell-free culture filtrates
Evaluation of FOS formation by cell-free culture filtrate collected
at different time intervals (48, 72, 96, and 120 h) was studied by
incubating enzyme samples with 50% (w/v) sucrose as substrate for
period of 24 h. The results of FOS production expressed as % (w/w) of
the substrate (sucrose 50%, w/v) are summarized in Table 3. Among
2
0 screened microorganisms for FTase activity, A. flavus (2364)
proved to be potent producer of FOS in all fermentation periods.
Its outcome yield was highest quantified 63.40% (w/w) in which
Acknowledgements
kestose (GF ) was the predominant product formed in initial reac-
tion time while high molecular weight oligomers like nystose (GF3)
2
Authors are thankful to the Head, Department of Zoology for
availing facilities to carry out research work. MAG is thankful to
UGC, New Delhi for providing financial support as JRF (UGC-BSR)
fellowship.
and 1-fructofuranosyl nystose (GF ) was formed at later stage of
4
2
4 h of reaction (Fig. 1a). The estimated concentration of kestose
(
GF ) was observed maximum 29.89% (w/w) followed by nys-
2
tose (GF ) 25.26% (w/w) and 1-fructofuranosyl nystose (GF ) 8.25%
3
4
Appendix A. Supplementary data
(
w/w). In case of A. niger (SI 19) highest FOS formation was observed
from 72 h culture filtrate. However, the concentration of nystose
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.molcatb.
(
GF ) 25.31% (w/w) was more than kestose (GF ) 23.74% (w/w) and
3 2
1
-fructofuranosyl nystose (GF ) 5.94% (w/w) (Fig. 1b). High nys-
4
2
013.07.008.
tose (GF ) formation in late reaction period can be attributed to the
3
fact that the newly produced kestose (GF ) serves as acceptor for
further oligomerization. This is in accordance with observation by
2
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