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Diversity of Aspergillus isolates and selection of an isolate with high β-fructofuranosidase activity that is native to the Peruvian coast
Diversity of Aspergillus isolates and selection of an isolate with high β-fructofuranosidase activity that is native to the Peruvian coast
In order to obtain a native isolate with high -fructofuranosidase activity (FFase), a preliminary
selection of 44 isolates, predominantly formed by Aspergillus niger (72%), was performed among 167
isolates of Aspergillus native to the Peruvian coast (Casa grande, Cartavio, Paramonga and Tacama). In
addition, genetic diversity was studied using molecular markers, Inter Simple Sequence Repeats (ISSR).
The FFase activity of these 44 isolates was compared and the isolate which showed the highest
fructosyltransferase (FTase) activity was identified molecularly at the species level by DNA sequencing.
Cluster analysis indicated 16 sub-genetic groups of which 11 of them were morphologically identified
as Aspergillus niger. Samples from Paramonga showed the highest genetic diversity as explained by
the Shannon diversity index (I= 0.21). No positive association was found between the genetic diversity
of populations and FFase activity. Isolates PR-151, PR-144 and PR-142 showed the highest FFase
activity in 5 consecutive evaluation generations, PR-142 being the most active with a total FFase
-1
activity of 11,248 (U.L ) and 6.17 g/L of biomass indicating a great biotechnological potential for the
synthesis of prebiotics. The molecular identification at the species level confirmed that PR-142 belongs
to A. niger lineage.
Key words: -Fructofuranosidase, fructosyltransferase, Aspergillus, Inter Simple Sequence Repeat (ISSR),
fructooligosaccharides.
INTRODUCTION
The Aspergillus species belong to the group of environment, some of which are associated with mycotic
filamentous fungi that are widely disseminated in the diseases while others are of great economic importance.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution
License 4.0 International License
1222 Afr. J. Biotechnol.
Figure 1. Map showing the locations of the four Aspergillus populations used in the present study.
Thus, these are used in the production of amylases for the case of Aspergillus, as in other microorganisms, both
the production of biofuels (Zheng et al., 2012), or in the activities are performed by a single enzyme. The study of
synthesis of metallic nanoparticles that are used in the genetic diversity of Aspergillus with respect to
medicine and electronics (Juraifani and Ghazwani, 2015). biotechnological capacity in Peru has implications for the
In the pharmaceutical and cosmetic industries, it has creation of local bio-industries.
been found that they can be used as an acidulant and
dissolving agent of active ingredients (Pau et al., 2015).
In the functional food industry, fructooligosaccharides MATERIALS AND METHODS
(FOS) are produced by enzymatic synthesis catalyzed by
Sampling and isolation of Aspergillus
microbial enzymes, such as those produced by species
of the Aspergillus genus. FOS is highly valued for its Soil samples were taken from sugarcane fields in 4 coastal regions
beneficial properties, such as selective increases in of Peru: North Coast (Casa Grande and Cartavio), Central Coast
beneficial gut bacteria (bifidobacteria), prevention of (Paramonga), and South Coast (Tacama) (Figure 1). From a series
colon cancer, reduction of cholesterol levels and of dilutions, filamentous fungi were isolated using widely-used
triglycerides in the blood, and can be considered as a microbiological techniques with Czpeck medium. Monosporic
cultures were preserved in 10% glycerol and stored at -80C. Using
calorie-free and non-cryogenic sweetener (Maiorano et microscopic techniques, only Aspergillus species were identified
al., 2008). FOS-producing enzymes are classified as - based on the typical form of the conidiophore, the presence of
fructofuranosidases (FFase, EC 3.2.1.26) with high filiades and the standing cells that are attributed to this genus (Pitt
fructosyltransferase activity (FTase, EC 2.4.1.9) which in and Hocking, 1985).
Castillo et al. 1223
Morphological identification FTase activity linked to mycelium and extracellular activities was
determined. For this purpose, the above-described methodology
Identification of the species was performed in Czapeck solid was used. The reaction products were analyzed by liquid
medium using the adhesive tape methodology. Morphological chromatography with refractive index detector (LC-RID) on an
identification was performed using macro and microscopic features Agilent Technologies 1220 Infinity LC system-1260 RID equipment
following the taxonomic keys of Thom and Raper (1945) and (Boeblingen, Germany). Separation was performed on a HPLC
Watanabe (2002). column Kromasil (100-NH2) (Akzo Nobel, Brewster, NY, USA)
(250 x 4.6 mm i.d., 5 m particle size) using 70:30 (v/v)
acetonitrile/water as the mobile phase and isocratic elution with a
Molecular identification flow rate of 1.0 ml min-1 for 30 min. One unit of fructosyltransferase
activity was defined as the amount of enzyme producing 1 mole of
To enable molecular identification, genomic DNA extraction was FOS (1-kestose plus nystose) per min, under the assay conditions.
performed using the methodology developed by Liu et al. (2000).
The DNA was amplified using the following primers: The ITS1
universal primer (5-TCCGTAGGTGAACCTGCGG-3) and ITS4 Molecular diversity based on Inter Simple Sequence Repeat -
(5-TCCTCCGCTTATTGATATGC-3) (White et al., 1990). The Random Amplified Polymorphic DNA (ISSR-RAPD) markers
primers Bt2a (5-GGTAACCAAATCGGTGCTGCTTTC-3) and Bt2b
(5-ACCCTCAGTGTAGTGACCCTTGGC-3) of the partial Genomic DNA extraction was performed using the methodology
sequence of the -tubulin gene (Glass and Donaldson, 1995) and developed by Liu et al. (2000) for filamentous fungi. The PCR
the primers Cmd5 (5 CCG AGT ACA AGG AGG CCT TC 3) and reaction was undertaken in a Mastercycler Nexus Gradient
Cmd6 (5 CCG ATA GAG GTC ATA ACG TGG 3) of the calmodulin Thermal Cycler (Eppendorf, Germany) using 6 polymorphic primers
gene (Hong et al., 2006). The sequences obtained were analyzed (Table 4). The thermal amplification conditions as well as the
using the BLAST program (http://www.ncbi.nlm.nih.gov). concentrations of each component of the PCR were taken from
Neal et al. (2011). The PCR products were resolved on 1.5%
agarose gels and stained with 0.5 mg mL-1 ethidium bromide. The
Preliminary selection inter simple sequence repeat (ISSR) band patterns were assigned
values of 1 (band presence) and 0 (band absence). An array of
The preliminary selection included the evaluation of the isolates similarities was constructed using the Jaccard Similarity Index from
according to their ability to hydrolyze sucrose (invertase activity or which the dendrogram was constructed by using the neighbor-
FFase), using the methodology described by Guilarte et al. (2009) joining or UPGMA method with the NTSYS-PC program version
which consists of taking circular portions of 7 mm mycelium grown 2.0. The reliability of the dendogram was verified by performing a
in potato dextrose agar (PDA) as a source of enzyme. The cophenetic correlation analysis derived from the Mantel test using
enzymatic reaction was in 50 ml conical tubes with 1.2 ml of 0.2 M the same program. For the genetic analysis, populations were
tris-acetate buffer at pH 5.5, using 3.7 ml of 64% (w/v) sucrose as a subdivided in two ways by their geographical origin (4 populations)
substrate. The control consisted of circular fragments of the same and by the value of the absorbance reached in three levels: High
medium but without mycelium. Triplicate tubes were placed in a (OD 540 > 1), medium (0.5 <OD540 < 1), and low (OD540 < 0.5).
shaking water bath GFL-1083 (Biovendis Ltd.) at 100 rpm for 60 Genetic diversity was estimated from the Shannon Indices (I), from
min at a temperature of 50C. The amount of released reducing the average heterozygosity (h), genetic differentiation between
sugars was quantified by the 3.5-Dinitrosalicylic acid (DNS) method populations (GST), as well as genetic flow (Nm) by using the
(Miller, 1959), while considering the mean absorbance values (OD POPGENE program version 1.32. The distribution of diversity
540 nm) as the selection factor (Table 1). Subsequently, the among populations and within populations was calculated using the
isolates that presented the highest values were evaluated in the analysis of molecular variance (AMOVA) within the GENALEX 6.5
same way for 5 consecutive generations in order to eliminate program.
possible errors and to verify the genetic stability of the isolates so
as to maintain the FFase activity.
RESULTS
FFase activity assay in shaken flasks
Preliminary selection
With the most representative isolates from the previous stage,
FFase activity assays were performed. For this purpose, 10 7 spores From a total of 167 Aspergillus lineages, only 44 isolates
per ml-1 were cultured in 250 ml flasks containing 50 ml of
fermentative medium whose composition was (g/L): 3.0 sucrose, showed a higher capacity for invertase activity (Table 1)
0.3 NaNO3, 0.2 KH2PO4, 0.05 MgSO4.7H2O, 0.02 MnCl2.4H2O, and than the control and were grouped according to their
0.001 FeSO4.7H2O, with pH of 5.5. These were incubated on a optical density (OD) at 540 nm as follows: High: 9
rotary shaker at 30C and 200 rpm for 72 h. The mycelia were isolates; median: 14 isolates; and low: 21 isolates. The
obtained using a polysulfone (PSF) filtration kit (Nalgene Ltd.) and evaluation of the 9 isolates in the 5 consecutive
Whatman quantitative filter paper grade N 1 in a vacuum and
then washed 3 times with distilled water. The FFase activity of the
generations (Figure 2) showed that isolates PR-142, PR-
mycelia was obtained using approximately 0.02 g mass of enzyme 152 and PR-144 presented the highest values of
source, while the extracellular activity was performed with 0.3 ml of absorbance and were the only isolates that were
the fermentation broth. The FFase activity linked to mycelium was considered for the next stage of analysis in shaking
defined as that catalysing the formation of 1 mol reducing sugar flasks.
per minute under the above conditions.
With the most active isolate selected in the previous steps, the Following the taxonomic keys, 32 isolates were identified
1224 Afr. J. Biotechnol.
Table 1. List of the 44 isolates with invertase activity measured at 540nm and their species identification.
Table 2. Parental mycelial and extracellular FFase activities of the 3 most representative native isolates.
-1 -1 -1 -1
Isolate Intracellular activity (U.L ) Extracellular activity (U.L ) Total activity (U.L ) Biomass (g/L )
b ab c
PR-151 1,066.7 179.3 6,350.00 23 7416.7 3.17 0.55
c b b
PR-144 112.4 58.3 4,140.00 80 4252.4 4.47 1.12
a a a
PR-142 3,528.2 49.8 7,720.00 77 11248.2 6.17 0.74
1
Mean of six samples (n=6) SD. The different letters indicate significant differences (p < 0.05).
3.00
Absorbance (OD 540 nm)
2.50
2.00
1.50
1.00
0.50
0.00
Isolates
Figure 2. Average absorbance values (3 replicates) in 5 consecutive generations of 9
Aspergillus isolates.
activity that represents 1.5 times the activity of the PR- Determining of FTase activity
144 isolate. It also presents the highest biomass among
all the isolates and was therefore considered the most LC-RID analysis of isolate PR-142 yielded a typical FOS
representative (Table 2). chromatogram (Figure 3) consisting of 86.6% GF2 (1-
1226 Afr. J. Biotechnol.
Table 3. GenBank accession numbers and similarity of ITS region, calmodulin and -tubulin gene sequences of PR-142.
Figure 4. ISSR band patterns generated from some Aspergillus isolates using primer UBC-834. Gene Ruler 1 kb DNA ladder (M)
(numbers represent size in bp).
kestose) and 13.4% GF3 (1-nystose). FOS production partial sequence of the -tubulin gene, and 97% similarity
-1
was 18.4 g/L, mycelial activity was 11,500 (U.L ), and with the sequence of the calmodulin gene were observed
-1
extracellular activity was 7400 U.L (p<0.05). The range (Table 3).
of fructosyltransferase and hydrolytic activities (At/Ah)
was 1.8.
ISSR analysis
Table 5. Measures of genetic diversity of 44 isolates of Aspergillus according to their geographic origin.
Population N PL% h I
Casa Grande 13 46.59 0.1253 0.173 0.1967 0.249
Cartavio 14 47.73 0.1120 0.153 0.1818 0.228
Paramonga 10 55.68 0.1252 0.142 0.2082 0.216
Tacama 7 40.91 0.1167 0.170 0.1820 0248
Total population GST: 0.135; Nm: 3.18%; PL: Percentage of polymorphic loci; N: Number of isolates. h: Genetic
diversity of Nei (1973); I: Shannon Diversity Index (Lewontin, 1972).
to 2000 bp. The first, UBC 817 and UBC834, were those three coastal regions of Peru showed potential for
that generated the highest polymorphism indexes biotechnological use in the fruit industry including the
(polymorphism information content, PIC values) (Table production of fructooligosacharides (FOS). The PR-142
4). Analysis of the grouping (Figure 5) allowed us to isolate had a fairly similar mycelial activity as compared
separate 6 groups, one of them being composed of A. to another native isolate, Aspergillus oryzae IPT-301
niger (Group A) and another being composed of other (Cuervo et al., 2007) which exhibited a mycelial activity
-1
species (B, C, D, E and F). The A. niger formed 11 sub- value of 3,599 (UL ). However, the extracellular activity
groups (32 isolates). The isolates belonging to other of PR-142 isolate was lower than that of IPT-301, whose
-1
species formed 5 subgroups: Group XII was formed of reported value was 14,190 (UL ) and the ratio of At/Ah
two A. fumigatus isolates (PR147 and PR154), Group XIII was also lower than that of IPT-301 which had an activity
was formed of two isolates of A. flavus (CG103 and value of 5.2. This means that this isolate has a lower rate
CG104), Group XIV was formed of the CG106 isolates of of FOS formation (Cuervo et al., 2007). These innate
A. flavus, Group XV was formed of the CR86 isolates of characteristics of the native enzyme can be enhanced by
A. versicolor and the TA115 isolates of A. ochraceus, and mutagenesis techniques (Guilarte et al., 2009) or
Group XVI was formed of 6 A. fumigatus isolates recombinant DNA technologies (Zhang et al., 2015). The
(CR106, CR108, PR146, CR107, PR141). It was FFase capacity of A. niger has been known for a long
determined that pairs of isolates CG29/CG30, time and is used in the production of FOS (Maiorano et
CG64/CR87, CR106/CR108 are duplicates. The al., 2008). However, there are few reports on FOS
polymorphism in the evaluated populations ranged from synthesis capacity of A. flavus (Praveen et al., 2010), A.
40.91 to 55.68% (Table 5). The Paramonga population versicolor (Belorkar et al., 2015) and A. ochraceus
presented the greatest polymorphism among all (Guimares et al., 2007). It is noteworthy that the same
populations, while the Tacama population had the lowest microorganism can produce different enzymes with
polymorphism. Genetic diversity (h) in these populations different characteristics via transfructosylation activity
ranged from 0.11 (Cartavio) to 0.12 (Casa Grande), the (Yoshikawa et al., 2007).
latter being similar to the Paramonga population (0.12) This study has shown that it is possible to know the
(Table 5). The Shannon Index (I) presented very similar genetic diversity of 44 isolates of Aspergillus having the
values and was in the range of 0.18 to 0.20, being in the ability to hydrolyze sucrose through the use of ISSR
locality of Paramonga which presented a greater value markers. The efficiency of ISSR markers in the study of
(0.20). The genetic diversity attributable to genetic Aspergillus diversity has been evidenced in different
differentiation between populations (Gst) was 0.13 on all reports (Zhang et al., 2013; Neal et al., 2011). The
loci, hence a low differentiation of these populations current results show poor overall diversity and low
could be considered as having similar results to the genetic differentiation which would demonstrate these
obtained AMOVA (p<0.001) which attributed 80% all populations are closely related despite the geographic
variation between isolates within populations and only distance that separates them. Similar results were
20% between different populations. The populations of obtained in black Aspergillus originating in 6 European
Cartavio and Paramonga were genetically the closest countries (Perrone et al., 2006). In addition, no
(0.02), while the most distant populations were those of relationship between the high FFase activity shown by
Tacama and Casa Grande (Figure 6). Gene flow among the strains from A. niger and their geographical origin
these populations was low (Nm = 0.13) among these 4 was found, which could be attributable to the fact that
populations. The cophenetic correlation between these microorganisms are derived from soils intensively
similarity matrix and the dendrogram showed a good fit used in the production of sugar cane. Similar results were
with a correlation coefficient (r) of 0.95. reported by using other Aspergillus strains with high
capacity of synthesizing FOS which was also isolated
DISCUSSION from this type of soils (Aziani et al., 2012; Cuervo et al.,
2007). The ISSR dendrogram (Figure 5) did not provide
Native isolates of A. niger that were isolated from the any correlation to the hydrolytic capacity due to the most
1228 Afr. J. Biotechnol.
Figure 5. Dendogram obtained from 44 isolates with FFase activity using the Jaccard coefficient and the
UPGMA algorithm. The vertical lines on the right-hand side indicate the formation of 14 genetic groups
with a coefficient of 0.53.
active strains were grouped in different genetic groups. ability of producing silver nanoparticles (AgNPs).
This behaviour could indicate that the ISSR fragments On the other hand, the genetic diversity observed in
are not included in the genes associated with sucrose populations categorized according to their hydrolysis
metabolism. Similar data in A. parasiticus and A. terreus capacity had even lower Gst and AMOVA indexes,
were reported by Juraifani and Ghazwani (2015) who stressing, thus, the inadequacy of classifying populations
also used ISSR to identify potential microorganisms with based on their hydrolytic capacity.
Castillo et al. 1229
CONFLICT OF INTERESTS Miller GL (1959). Use of dinitrosalicylic acid reagent for determination of
reducing sugar. Anal. Chem. 31:426-428.
Neal CO, Richardson AO, Hurst SF, Tortorano AM, Viviani MA, Stevens
The authors have not declared any conflict of interests. DA, Balajee SA (2011). Global population structure of Aspergillus
terreus inferred by ISSR typing reveals geographical subclustering.
BMC Microbiol. 11:203.
Nei M (1973). Analysis of gene diversity in subdivided populations. Proc
ACKNOWLEDGEMENTS
Natl. Acad. Sci. USA. 70:3321-3323.
Pau LS, Kehinde OO, Qi YS, Fitri AAZ, John CWL, Tau CL (2015).
This work was financially supported by the Programa Overview of citric acid production from Aspergillus niger. Front. Life
Nacional de Innovacin para Competitividad y Sci. 8(3):271-283.
Perrone G, Susca A, Epifani F, Mule G (2006). AFLP characterization of
Productividad, Innvate Per (FINCYT-IA- Contrato N Southern Europe population of Aspergillus Section Nigri from grapes.
2013). The authors thank the Agro Industrial Paramonga Int. J. Food Microbiol. 111(1):S22-S27.
SAA for allowing us to take samples in their sugarcane Pitt JI, Hocking AD (1985) Interfaces among genera related to
fields. Aspergillus and Penicillium. Mycologia 77(5):810-824.
Praveen P, Reddy GSN, Sulochana MB (2010). Screen of -
Fructofuranosidase Producers with High Transfructosylation Activity
and its 32 Experimental Run Studies on Reaction Rate of Enzyme
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