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Amylase production from the yeast Saccharomycopsis fibuligera and its potency
for glucose production from raw starch

Conference Paper · October 2008

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 S. Ishmayana et al. Proceeding of The International Seminar on Chemistry 2008 (pp. 688-691)
ISBN 978-979-18962-0-7 Jatinangor, 30-31 October 2008

Amylase production from the yeast Saccharomycopsis fibuligera and

its potency for glucose production from raw starch

Safri Ishmayana*, Dian S. Kamara, Saadah D. Rachman,

Idar Kardi, Muhammad Fadhlillah
Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran
Jl. Raya Bandung-Sumedang km. 21 Jatinangor 45363 Sumedang, West Java, Indonesia
*e-mail: ishmayana@unpad.ac.id

Abstract

Raw starch digesting amylolytic enzyme is a potential enzyme that can be used in bioethanol
production, since it can save energy requirement of starch processing. S. fibuligera R-64 produces
two types of amylolytic enzyme, i.e. α-amylase and glucoamylase, which are capable to digest raw
starch. This research is aimed to investigate the effect of different carbon sources on amylase
production by the yeast S. fibuligera and to investigate the enzyme ability in digesting raw starches.
The media that used for enzyme production media were 1% of yeast extract and 1% of raw starch
(sago, corn, cassava, and rice). The media was shaked (180 rpm) in room temperature for 72 hours.
The crude extract was used for digesting raw starches. The crude extract was added to 5% (w/v) raw
starch solution in 25 mM phosphate-citrate buffer pH 5.8. The mixture was then shaked in room
temperature for 24 hours. After 24 hours, the sample was taken, and the reducing sugar was
determined using colorimetric method using alkaline potassium ferry cyanide reagent. The results
showed that sago is the best carbon source, which gave the highest amylolytic activity (305.77
units/mg). We also found that sago was the easiest starch digested by the enzyme, followed by rice,
corn and cassava.

Keywords: amylase, glucose, raw starch, Saccharomycopsis fibuligera

Introduction other only produces one of the enzyme (Hostinova,

In recent years, there are high demands for highly
efficient starch processing. This type of processing
require starch acting enzyme that has capability in
digesting raw starch (Hostinova, 2002). Raw starch
digesting enzyme has important application in various
industries, such as foods, beverages, textile,
pharmaceuticals and also bioethanol (Nigam & Singh,
1995).
Bioconversion of raw starch using raw starch
acting enzyme has been reported represent economical
advantages compared to conventional process which
uses pregelatinised starch as substrate. While
conventional process require high amount of energy
for gelatinization process, raw starch processing does
not require this step for the hydrolysis process (van
der Maarel, 2006).
Therefore, exploration has been conducted to find
amylolytic enzyme that highly active towards raw
starches. Many researchers found this type of enzyme,
and some of these types of enzyme have been well
studied (Hayashida et al., 1988; Hostinova et al.,
2003; Goyal et al., 2005).
S. fibuligera is a food borne yeast that has high
amylolytic activity. Previous studies showed that some
S. fibuligera strains produces two types of amylolytic
enzyme which are α-amylase and glucoamylase while
2002).
Our group has worked on S. fibuligera R-64 and
previous studies showed that this strain produces
glucoamylase and α-amylase. Interestingly, while
glucoamylase highly adsorbed onto raw starch, the α-
amylase can digest raw starch without adsorption
mechanism (Hasan et al., 2008).
Optimizing of production of these enzyme need to
be done. One of factors affecting the production of
enzyme is nutrient in growth medium (de Oliveira et
al., 2007). Previous studies showed that optimum
nutrient concentration for amylase production by S.
fibuligera R-64 is 1% of starch and 1% of yeast
extract (Soemitro et al., 1996). But, influence of
different starches on amylase production has not been
investigated. Therefore in present study, we
investigate the influence of different starches as
carbon sources on amylolytic activity produced by the
yeast.
We also examine the effectiveness of the enzyme
in hydrolyzing different type of raw starches.

688
 S. Ishmayana et al.
Materials and Methods
Microorganism
S. fibuligera is routinely grown at room temperature
for 48 hours on the agar plate contain 6% sucrose,
1.5% bacto agar, and 10% tauge extract.
Production of the enzyme
The enzyme was produced, in the medium containing
1% starch (cassava, sago, rice and corn) and 1% yeast
extract as described by Ismaya et al. (2003), and
cultivated for 3 days at room temperature. After
removal of cells by centrifugation at 4oC, the
supernatant was used as crude extract of enzyme
which was used for hydrolyzing raw starch.
Amylase activity assay
The enzyme activity was determined using Fuwa
(1951) method. Briefly, 100 µL of enzyme solution
was added to 100 µL 0.1% soluble starch solution,
and incubated for 10 minutes in 50°C. Reaction was
stopped by addition of 100 µL HCl 0.1 N. To
visualize remaining starch in solution, 100 µL iodine
solution was added and the total volume brought to 2
mL by water addition. The absorbance was recorded
on 600 nm wavelength. To calculate unit activity of
the enzyme, following equation was used:
Ac - As 1 sago starch as sole carbon source, followed by rice
AU = × 10 × × f
Ac Ve
Where : AU = Activity Units
Ac = Control Absorbance
As = Sample absorbance
Ve = Volume of enzyme used in assay
f = dilution factor
Hydrolysis of raw starches
Hydrolysis of raw starch was conducted as described
by Hostinova et al. (2003) with slight modification.
Raw starch (cassava, corn, rice and sago) suspension
was made with concentration of 5% (w/v) in
phosphate-citrate buffer pH 5.8 (25 mM). To 20 mL
of the suspension 1 mL of crude enzyme extract (2000
units/mL) was added. This mixture was than shaken
(180 rpm) in room temperature for 24 hours.
Reducing sugar after 24 hour of digestion was
determined using method described by Walker &
Harmond (1994).
Determination of reducing sugar
To 1 mL of sample solution, 0.8 mL of alkaline ferry
cyanide (7.5 mM potassium ferry cyanide in 0.3 M
sodium carbonate) was added. The mixture was than
heated for 10 minutes. After cooling, 4 mL of water
was added and the absorbance at 420 nm was
recorded.
689
Proceeding of The International Seminar on Chemistry 2008 (pp. 688-691)
Jatinangor, 30-31 October 2008
Results and Discussion
One of factors affecting enzyme production is
availability of nutrition in growth media, including
carbon source. However, carbon sources such as
dextrin, fructose, glucose, lactose, maltose and starch
are very expensive for commercial production of these
enzymes (Gupta et al., 2003). These expensive carbon
sources can be replaced in the medium with
economically available agricultural by-products (de
Oliveira et al., 2007; Abu et al., 2005). To investigate
the influence of different carbon source on the
amylase production, we examine some starch as
carbon source in relation to amylase production.
There were no significant differences of
extracellular protein produced by the yeast (Figure 1).
This finding is different with previously reported
finding (de Oliveira et al., 2007). Even though same
nitrogen source was used, de Oliveira et al. (2007)
found that extracellular protein produced by the same
rhizobia can differ significantly. However, this result
is very influenced by intrinsic properties of the
microorganism. For S. fibuligera, it might be that total
extracellular protein production is highly affected by
the nitrogen source in the media. This hypothesis
needs to be confirmed by more experimental data.
Figure 2 show the influence of different starches as
carbon source on amylase activity. High activity of
amylase (~305 units/mg) was found in the presence of
(~210 units/mg), cassava (~127 units/mg) and maize
(~76 units/mg) (Figure 2). These differences also
found in production of amylase by rhizobia (de
Oliveira et al., 2007) and Bacillus strain (Aiyer,
2004). This finding suggests that the same behavior
also true for yeast.
High activity of raw starch digesting enzymes is
desired for more economical process. Therefore we
examine the activity of our enzyme towards several
raw starches, namely corn, cassava, rice and sago.
From the experiment, it can be suggested that sago
gave the highest glucose production (1935 µg/ g raw
starch), followed by rice (924µg/ g raw starch),
cassava (751 µg/ g raw starch), and maize (720 µg/ g
raw starch) (Figure 3). This result have similar pattern
as amylase activity, therefore high amylase production
might be related to high digestability of the substrate
in the growth media.
The physicochemical properties of partially
hydrolyzed starch are interesting thing to be
elucidated. Different treatment during hydrolysis
process can influence the final product. Therefore,
optimization for hydrolysis process and finally
products that formed during hydrolysis also still needs
to be investigated.
 S. Ishmayana et al. Proceeding of The International Seminar on Chemistry 2008 (pp. 688-691)
Jatinangor, 30-31 October 2008

Protein Concentration (mg/mL)

4

0
Sago Cassava Rice Maize
Type of Carbon Source

Figure 1 Extracellular protein produced by the yeast S. fibuligera R-64 in growth media composed of 1% starch
and 1% yeast extract

350.00
Specific Activity (Units/mg)

300.00

250.00

200.00

150.00

100.00

50.00

0.00
Sago Cassava Rice Maize
Type of Carbon Source

Figure 2 Amylase production by the yeast produced by the yeast S. fibuligera R-64 in growth media composed of
1% starch and 1% yeast extract

2500
Glucose Production (µg/g)

2000

1500

1000

500

0
Sago Cassava Rice Maize
Type of Raw Starch

Figure 3 Glucose produced using crude extract of amylase produced by S. fibuligera R-64

690
 S. Ishmayana et al. Proceeding of The International Seminar on Chemistry 2008 (pp. 688-691)
Jatinangor, 30-31 October 2008

Conclusions Hostinova E., Solovicova A., Dvorsky R. &

Gasperik J. 2003. Molecular cloning and 3D
S. fibuligera R-64 produces high activity of structure prediction of the first raw-starch-
amylase in the presence of sago starch as sole degrading glucoamylase without a separate
carbon source in the growth medium. The amylase starch binding domain. Arch. Biochem.
can be used for direct glucose production from raw Biophys. 411: 189–195.
starch. However, efforts still need to be done for Hostinova, E. 2002. Amylolytic enzymes produced
optimizing the process for higher yield of enzyme by the yeast Saccharomycopsis fibuligera.
activity by modification of growth media or genetic Biologia, Bratislava. 57/Suppl. 11: 247-251.
engineering of the enzyme. Ismaya, W. T., Setiana, T., Natalia, D. & Soemitro,
S. 2003. Peningkatan kestabilan amilase
melalui rekayasa protein. Laporan Hibah
Acknowledgements Bersaing IX. Departemen Pendidikan Nasional.
Jakarta.
This research is funded by DIPA Universitas Nigam, P. & Singh, D. 1995. Enzymes and
Padjadjaran budget year 2007 based on contract microbial system involved in starch processing.
No. 251D/JO6.14.LP/PL/2007. Therefore we are Enzyme Microbiol. Technol., 17, 770-778.
very grateful to research institute of Universitas Soemitro, S., Bahti, H. H., Adi, T. P., Thaurhesia,
Padjadjaran. S., Hardjito, L., Niloperbowo, W. & Wenten I.
G. 1996. Produksi enzim pemecah pati.
Laporan RUT I. Kementrian Riset dan
References Teknologi.
van der Maarel, M.J.E.C. 2006. Bioethanol
Abu, E.A., Ado, S.A. & James, D.B. 2005. Raw production from starch and cellulose. The
starch degrading amylase production by mixed Gruber Soedigdo Lecture. ITB Bandung 20
culture of Aspergillus niger and June 2006.
Saccharomyces cerevisae grown on sorghum Walker, J.A. & Harmon, D.L.. 1996. Technical
pomace. Afr. J. Biotechnol. 4: 785-790. note: a simple, rapid assay for α-amylase in
Aiyer, P.V.D. 2004. Effect of C:N ratio on alpha bovine pancreatic juice. J. Anim. Sci. 74:658-
amylase production by Bacillus licheniformis 662.
SPT 27. Afr. J. Biotechnol. 3 : 519-522.
de Oliveira, A.N., de Oliveira, L.A., Andrade, J.S.
& Chagas Junior, A.F. 2007. Rhizobia amylase
production using various starchy substances as
carbon substrates. Braz. J. Microbiol. 38:208-
216.
Fuwa, H., 1951. A new method for micro
determinations of α-amylase activity by the use
of amylose as substrate. J. Biochem. 41: 583-
603.
Goyal, N., Gupta, J. K. & Soni, S. K. 2005. A novel
raw-starch digesting thermostable α-amylase
from Bacillus sp. I-3 and its use in the direct
hydrolysis of raw potato starch. Enz. Microb.
Technol. 37:723-734.
Gupta, R., Gigras, P., Mohapatra, H., Goswami,
V.K., & Chauhan, B. (2003). Microbial α-
amylases: a biotechnological perspective.
Process Biotechem., 38, 1599-1616.
Hasan, K., Ismaya, W.T., Kardi I., Andiyana Y.,
Kusumawidjaya S., Ishmayana, S., Subroto, T.,
& Soemitro, S. 2008. Proteolysis of α-amylase
from Saccharomycopsis fibuligera:
characterization of digestion products.
Biologia. 63: 1044 – 1050.
Hayashida, S., Teramoto, Y. & Inoue, T. 1988.
Production and characteristics of raw potato
starch digesting α-amylase from Bacillus
subtilis 65. App. Envir. Microb. 54:1516-1522.

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