Molecular identification of naturally isolated Candida

International Journal of Agricultural Sciences and Veterinary Medicine Vol. 7 (4), November (2019)

 Molecular identification of naturally isolated Candida
 tropicalis AUN-H100 and optimization of their
 extracellular amylase production
 Hesham Abd El-Latif1*, Gheit Heba1, Abd El-Fatah Bahaa E.S.1, Saleh Fathy1 and Al-Bedak Osama A.2
 1. Genetics Department, Faculty of Agriculture, Assiut University, Assiut 71516, EGYPT
 2. Assiut University Mycological Centre, Assiut University, Assiut, EGYPT
 *hesham_egypt5@aun.edu.eg; hesham_egypt5@yahoo.com; hesham_egypt5@agr.bsu.edu.eg

Abstract manipulated to genetic improvement11,31,32. Microbial
A total of 12 yeast isolates recovered from rice were amylases have almost completely replaced chemical
screened for their amylase activity. All isolates showed hydrolysis of starch in starch processing industry33. Amylase
positive results and according to clear zone has been produced from several fungi, yeasts and bacteria.
 The present investigation aimed to isolation of yeast strains
measurements and Enzyme Activity Index (EAI), 4
 from different rice samples, screening the isolates for their
isolates were recorded as high amylase producers, 3 amylase activity and optimization of some nutritional and
moderate and 5 low. Amylase activity for all isolates environmental conditions for the maximum enzyme
was estimated using the DNS method. A potent strain production from the most potent amylase-producer strain.
AUN-H100 was recorded and identified by sequencing 26S rRNA gene D1/D2 domain sequences were used for
the variable D1/D2 domain of the large subunit (26S) yeast identification.
rDNA region as Candida tropicalis AUN-H100.
 Material and Methods
The strain exhibited an enzyme index of 5.1 on the solid Sampling and strain isolation: A total of 12 composite
medium and showed amylase activity of 6.050 samples of rice collected from different local markets in
IU/ml/min in submerged fermentation. Amylase activity Assiut Governorate, Egypt during October to December
was also optimized at different pH values ranging from 2016 (4 samples per month), were involved in the current
 study. Isolation of yeast strains was performed using dilution
3 to 10 and different nitrogen sources namely yeast
 plate technique on yeast extract peptone dextrose (YPD)
extract peptone, sodium nitrate, sodium nitrite and medium which composed of (g/L): yeast extract, 10;
ammonium chloride at five temperatures of 25, 30, 35, peptone, 20; dextrose, 20 and agar, 20. A 100 µg/ml
40 and 45 °C respectively. C. tropicalis AUN-H100 ampicillin was added after autoclaving to avoid the bacterial
showed its maximum enzyme activity of 21.123 ± 2.060 growth. The dilution plate technique was employed in which
IU/ml/min at pH 5 and 30 °C using ammonium chloride one gram of each sample was individually placed in 10 ml
as nitrogen source in submerged fermentation. of sterile distilled water and shaked at 150 rpm for 30 min.

Keywords: Amylase, 26S rDNA, D1/D2 region, Candida 0.1 mL aliquots of each sample’s suspension were
tropicalis, optimization, submerged fermentation. individually spread on Petri dishes containing the YPD
 medium and the plates were then incubated at 28°C for 3
Introduction days. The developed colonies with distinct morphological
Amylases are the biocatalysts which undergo biodegradation differences such as color, shape and size were picked and
of starch molecule into glucose, maltose, maltotriose and purified by single colony isolation on the same medium.
dextrin through the breakdown of the glycosidic bonds in the Pure culture of each individual strain was transferred to YPD
starch polymer3,11. There are three types of amylase namely agar slants and maintained at 4 °C for further studies.
α amylase (E.C.3.2.1.1), β-amylase (EC 3.2.1.2) and γ-
amylase (EC 3.2.1.3). Amylases are from the most important Screening and selection of amylase-producing yeast
enzymes that comprise approximately 25-33 % of the strains
international enzyme market and have a great role in many 1. Qualitative assay: The abilities of the isolated yeast
industries such as bioethanol production, starch strains to secrete amylase enzyme were screened on
saccharification, food, textile, brewing and paper industries Amylase Activity Medium (AAM) described by Yalcin et
as well as in many other fields such as medicinal and al34. The medium contained (g/L): soluble starch, 5;
pharmaceutical, analytical chemistry and peptone, 5; yeast extract, 5; MgSO4.7H2O, 0.5;
biotechnology10,18,26,27. FeSO4.7H2O, 0.01; NaCl, 0.01 and agar, 15. Five isolates per
 plate were inoculated using sterile needle. The inoculated
Amylases can be obtained from several sources like plants plates were then incubated at 30 °C for 3 days.
and animals; the enzymes from microbial sources have
dominated applications in different industrial sectors After incubation, the plates were logged with 0.25 % iodine
because of their economic feasibility and cells are easily solution. The formation of clear zone around the yeast
 colonies indicates amylase activity. The positive isolates for

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International Journal of Agricultural Sciences and Veterinary Medicine Vol. 7 (4), November (2019)

amylase activity were categorized to high, moderate and low Ethidium bromide was used for gel staining and photographs
producers based on their clear zone and enzyme index were taken under ultraviolet light.
measurements. The high producers were selected for further
investigation. Purification of PCR products and determination of 26S
 rRNA gene D1/D2 domain sequences: PCR products of the
2. Quantitative assay correct size (~600 bp) were purified with a Takara agarose
Determination of amylase activity under submerged gel DNA purification kit and then sequenced in both
fermentation (SmF): The positive yeast isolates were directions using an ABI 3730 automated sequencer by
grown each in Erlenmeyer conical flask containing 50 ml of Macrogen (Seoul, Korea).
sucrose-free Cz broth supplemented with soluble starch as a
sole carbon source. The medium contained (g/l): soluble Phylogenetic analyses and comparisons of 26S rRNA
starch, 10; NaNO3, 2.0; KCl, 0.5; MgSO4.7H2O, 0.5; gene D1/D2 domain sequences: The 26S rRNA gene
K2HPO4, 1.0; FeSO4.7H2O, 0.01; agar, 15.0. The flasks were D1/D2 domain sequences from yeast isolate AUN-H100
inoculated each with 1 ml of spore suspension of 3-day-old were searched in the GenBank and aligned with known 26S
culture in a concentration equal to 0.5 McFarland. The flasks rRNA gene sequences using the basic local alignment search
were incubated at 30 °C on shacking condition at 150 rpm tool (BLAST) at the National Center for Biotechnology
for 3 days. After incubation, the cell-free supernatant was Information (http://www.ncbi.nlm.nih.gov/BLAST/).
obtained by centrifugation at 10000 rpm for 10 min at 4 °C. Percent identity scores were generated to identify yeast
The clear supernatant was used as a source of amylase in isolate. Phylogenetic tree was also constructed using MEGA
determination of amylase activity. version 4.0 with the neighbor-joining algorithm and Jukes-
 Cantor distance estimation with 1,000 bootstrap replicates to
Amylase assay: Amylase assay was carried out according to confirm the taxonomic position of the yeast isolate AUN-
the method described by Miller21. The reaction mixture H100.
composed of 0.9 ml starch solution (in 50 mM citrate buffer;
pH 5) and 0.1 ml of media filtrate. The reaction was Optimization of pH, nitrogen source and temperature on
completed at 50 °C for 10 min4. Afterwards, 2 ml of 3,5- amylase activity
dinitrosalicylic acid (DNS) was added to the tube contents to Optimization of pH: To determine the optimum pH for
eliminate the reaction and the contents were then boiled in amylase production, the potent yeast strain was cultivated in
water bath for 10 min and cooled at room temperature. Erlenmeyer conical flask containing sucrose-free Cz broth
Absorbance of the color developed was measured at 540 nm. supplemented with 1 % starch as the sole carbon source. The
 medium was adjusted at different pH values ranging from 3
The amount of reducing sugars liberated was quantified to 10 in 1.0 of increment. The buffers used for the required
using standard curve of glucose. One unit of amylase activity pH range were citrate buffer (pH 3–6), sodium phosphate
was defined as the amount of enzyme that liberated 1 µmole (pH 7–8) and glycine-NaOH (pH 9–10). The flasks were
of glucose equivalent per minute under the standard assay inoculated with 5 ml spore suspension containing spore
conditions8. The amylase activity was calculated according concentration equal to 0.5 McFarland (1.5 x 108 spore/ml) of
to the following equation: 3-day old cultures.

 1 1 1 1 The flasks were then incubated at 30 °C for 7 days in shaken
Enzyme activity = Absorbance x DF x ( ) ( ) ( ) ( )
 condition at 150 rpm. Amylase activity was determined by
 the reaction of the cell-free supernatant with the starch
DNA extraction and 26S rRNA gene D1/D2 domain substrate at the standard assay conditions of 50 °C, pH 5 and
amplification for amylase producing yeast: Total genomic 10 min. The amount of reducing sugar was estimated
DNA from yeast isolate AUN-H100 with the most promising following the method of Mileer21.
ability to produce -amylase was isolated13. The 26S rDNA
D1/D2 domain region was amplified using the primers NL1 Optimization of nitrogen source: The effect of five
(5′-GCATATCAATAAGCGGAGGAAAAG-3′) and NL4 nitrogen sources namely peptone, yeast extract, sodium
(5′-GGTCCGTGTTTCAAGACGG-3′)19. PCR was nitrate, sodium nitrite and ammonium chloride on amylase
performed in a final volume of 50 μl containing GoTaq green production and activity by C. tropicalis AUM-H100 at the
master mix (Promega, Madison, WI, USA), 1 µl DNA optimized pH was determined. Sodium nitrite (1.621 g/l) and
sample and 1 µl of each primer (at a concentration of 0.5 ammonium chloride (1.257 g/l) were used as sodium nitrate
mM)14. The amplification was carried out under the (2 g/l) equivalent23. After incubation, amylase activity was
following conditions: an initial denaturation at 95 °C for 5 estimated at the standard assay conditions of 50 °C, pH 5 and
min followed by 36 cycles at 94 °C for 2 min, 52 °C for 1 10 min as previously discussed.
min, 72 °C for 2 min; a final extension at 72 °C for 7 min
and then held at 4 °C. A total of 5 μl of PCR products were Optimization of temperature: The effect of five
then analyzed using 1.5% 0.5× TBE agarose gel temperature degrees 25, 30, 35, 40 and 45 ºC on amylase
electrophoresis. A 100-bp DNA ladder was used as a marker. production and activity was determined at the optimal pH

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International Journal of Agricultural Sciences and Veterinary Medicine Vol. 7 (4), November (2019)

and nitrogen source for the tested strain C. tropicalis AUN- phylogenetic position of yeast AUN-H100 isolate.
H100. Amplified 26S rDNA D1/D2 region from the selected isolate
 was (~ 650) bp long which is the expected size (600–650)
Nucleotide sequence accession number: The nucleotide (Figure 1).
sequences of the yeast isolate AUN-H100 reported in this
study have been deposited in the DDBJ Alignment of 26S rRNA gene sequences of the yeast AUN-
(www.ddbj.nig.ac.jp/), EMBL (www.embl.de/) and H100 with published 26S rRNA sequences from GenBank
GenBank nucleotide sequence databases using BLAST shows identity 99% with Candida tropicalis.
(http://www.ncbi.nlm.nih.gov/genbank/) under accession Phylogentic tree was constructed for AUN-H100 isolate
number: MK744048. along with other sequences of the same genus from
 GenBank. As shown in fig. 2, strain AUN-H100 and
Results and Discussion Candida tropicalis share one clade. Therefore, strain AUN-
Isolation and phenotypic characterization of yeast H100 was identified as Candida tropicalis. The 26S rRNA
strains: In this study, twelve yeast strains were isolated from gene D1/D2 domain has gained recognition in yeast
the rice samples collected from Assiut Governorate, Egypt. taxonomy as a valuable identification tool12,15,16. D1/D2
The isolated yeasts were purified using single spore culture domain sequence databases are available for all currently
and identified according to the phenotypic characteristics recognized ascomycetous and basidiomycetous yeasts. This
including colony color (cream and white); colony margin makes species identification much easier and serves as a
(entire, filamentous or undulate); colony surface (smooth, reliable and practical criterion for the identification of most
rough or dry powdery) and elevation (convex and flat). known yeasts1,15,19.

Screening of amylase-producing yeast strains: In the Optimization of amylase production
present investigation, a total of twelve isolates recovered Optimization of pH: In the current study, amylase
from rice were screened for their abilities to produce production by Candida tropicalis AUN-H 100 was
amylase. All isolates could produce amylase but with examined on eight pH values (3-10) at 30 °C. pH 5.0 showed
different degrees. Based on measurements of the enzymatic the maximum (6.819 ± 0.104 U/ml/min) enzyme production
activity index (EAI), the 12 isolates were categorized into (Table 1; Figure 3) while the maximum inhibitory effect
high producers (4 isolates) that registered EAI ranging from (1.527 ± 0.237 IU/ml/min) of amylase activity was recorded
5.3 to 8.33, moderate (3 isolates) giving EAI ranging from at pH 10 (Table 1).
3.17 to 4.0 and low (5 isolates) which harbored EAI ranging
from 1.17 to 2.11. In agreement with the current results, Gupta et al10 reported
 that fungal amylases have optimum pH values ranging from
The enzymatic activity index (EAI) is a semi-quantitative acidic to neutral. Most of the earlier studies revealed that
parameter commonly used for fast estimation of the fungi required slightly acidic pH for optimum growth and
enzymatic activity for microorganisms grown on solid amylase production. In this respect, the production of
media. Some authors recommend an EAI ≥ 2.5 for amylase by some yeast strains was found to be the best at pH
considering a microorganism as a producer of enzymes in 5.5 and below and above this pH, amylase production
solid medium20,25,28. Colonies with the highest EAI are those decreased gradually34. Hostinová17 recorded the optimal pH
with higher extracellular enzyme activity5,25,30. All the 12 for glucoamylase and α-amylase of S. fibuligera as 5.0-6.2.
yeast isolates were further tested for their amylase
production in submerged fermentation to estimate the potent Moreira et al22 found that the amylase production by
strain that can be used for amylase production. Aspergillus tamarii was higher at pH 6 while Nahas et al24
 observed the maximum amylase production by Aspergillus
Production of amylase in submerged fermentation: A ochraceus at initial pH 5.0. Giannesi et al9 obtained
total of 12 yeast isolates were grown individually in amylases from several microbial sources, exhibiting an
Erlenmeyer flasks each containing 50 ml sucrose-free Cz optimum pH from 4.5 to 7.0. Rahardjo et al29 reported pH
broth. The flasks were inoculated with 5 ml spore suspension 7.0 as optimal for amylases produced by Aspergillus oryzae
containing 1.5 x 108 spore/ml of 3-day old culture. After and Figueira and Hirooka7 found optimum pH values of
incubation at 30 °C for 3 days in shaked conditions of 150 around 6.7 for amylases produced by Fusarium moniliforme.
rpm, the enzyme activity was determined using dinitro-
salicylic acid (DNS) in the cell-free supernatant. The Optimization of nitrogen sources: Nitrogen sources play
quantitative assay of amylase activity showed a potent yeast an important role in the growth of the organism and
strain which recorded an enzyme production of 2.471 IU/ml production of enzymes. The effect of organic and inorganic
and enzyme activity of 6.05 IU/ml/min. nitrogen sources on amylase production at the optimized pH
 was tested. In the current study inorganic nitrogen
Identification using 26S rRNA gene D1/D2 Region (ammonium chloride) showed the best amylase activity
Sequencing and Phylogenetic Analyses: Molecular (21.123 ± 2.060 IU/ml/min) over the other sources used
techniques were used to identify and determine the (Table 2; Figure 4) while the highest decrease in amylase

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International Journal of Agricultural Sciences and Veterinary Medicine Vol. 7 (4), November (2019)

activity was obtained when sodium nitrite was used as Optimization of temperature: The tested strain C.
nitrogen source. tropicalis AUN-H100 showed its optimum production and
 activity of amylase at 30 °C (Table 3; Figure 5). Regarding
This decrease in activity may be due to the somewhat toxic the effect of temperature on amylase production,34 test the
effect of sodium nitrite in fermentation medium. In this amylase production by S. fibuligera strains in liquid medium
respect, Alnahdi and Geweely 2 noticed that amylase activity at 25, 30, 35 and 40 °C and it was found that the enzyme
recorded its higher level when potassium nitrate was used activities increased progressively with increase in
and the lowest when ammonium sulfate was used as nitrogen temperature from 25 °C reaching a maximum at 30 °C.
source by A. niger. Similar results were also obtained by De Mot and
 Verachtert6 with Filobasidium capsuligenum.

 Table 1
 Effect of pH on amylase activity by C. tropicalis AUN-H100
 pH Glucose Enzyme concentration (IU/ml) Enzyme activity
 (mg/ml) (IU/ml/min)
 3 1.018 ± 0.082 5.654 ± 0.456 4.207 ± 0.340
 4 1.283 ± 0.061 7.127 ± 0.338 5.303 ± 0.252
 5 1.649 ± 0.025 9.162 ± 0.139 6.819 ± 0.104
 6 1.083 ± 0.049 6.019 ± 0.273 4.478 ± 0.204
 7 0.985 ± 0.046 5.472 ± 0.254 4.071 ± 0.189
 8 0.892± 0.062 4.956 ± 0.343 3.686 ± 0.256
 9 0.818 ± 0.048 4.546 ± 0.265 3.381 ± 0.197
 10 0.370 ± 0.057 2.056 ± 0.319 1.527 ± 0.237
Numbers in table are mean of three replicates, the optimum pH value was shown in bold

 Table 2
 Effect of nitrogen source on amylase activity by C. tropicalis AUN-H 100
 Glucose Enzyme concentration Enzyme activity
 Nitrogen source (mg/ml) (IU/ml) (IU/ml/min)
 Sodium nitrite 2.595 ± 0.405 14.415 ± 2.249 10.731 ± 1.675
 Ammonium chloride 5.106 ± 0.498 28.368 ± 2.766 21.123 ± 2.060
 Peptone 4.398 ± 0.169 24.436 ± 0.941 18.194 ± 0.701
 Yeast extract 4.511 ± 0.319 25.058 ± 1.771 18.658 ± 1.319
Numbers in table are mean of three replicates, the optimum N 2-source was shown in bold

 Fig. 1: Amplified 26S rDNA D1/D2 domain with primer pair NL1 and NL4. Lane L represents Ladder
 from 1500-100 bp.; Lane 1 PCR products amplified from strain AUN-H 100.

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International Journal of Agricultural Sciences and Veterinary Medicine Vol. 7 (4), November (2019)

 Fig. 2: Phylogenetic tree relationships between strain AUN-H 100 and 26S rDNA sequences
 from other published Candida spp.
 GenBank accession numbers are given in parentheses

 8
 7
 Enzyme Activity (u/ml/min)

 6
 5
 4
 3
 2
 1
 0
 3 4 5 6 7 8 9 10
 pH Value

 Figure 3: Effect of pH on amylase activity by C. tropicalis AUN-H 100

 Table 3
 Effect of temperature on amylase activity by C. tropicalis AUN-H 100
 Glucose Enzyme concentration (IU/ml) Enzyme activity
 Temperature °C (mg/ml) (IU/ml/min)
 25 0.889± 0.058 4.941± 0.321 3.675± 0.239
 30 5.106± 0.498 28.368± 2.766 21.123± 2.060
 35 0.671± 0.024 3.726± 0.132 2.770± 0.099
 40 0.564± 0.014 3.134± 0.076 2.329± 0.057
 45 0.359± 0.019 1.995± 0.106 1.481± 0.079
Numbers in table are mean of three replicates, the optimum temperature was shown in bold

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International Journal of Agricultural Sciences and Veterinary Medicine Vol. 7 (4), November (2019)

 25

 Enzyme Activity (IU/ml/min)
 20

 15

 10

 5

 0
 Sodium nitrite Ammonium Peptone Yeast extract
 chloride
 Nitrogen Sources
 Figure 4: Effect of nitrogen source on amylase activity by C. tropicalis AUN-H 100

 25

 20
 Enzyme Activity (U/ml/min)

 15

 10

 5

 0
 25 30 35 40 45
 Temperature

 Figure 5: Effect of temperature on amylase activity by C. tropicalis AUN-H 100

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