Bioresource Technology 98 (2007) 534538

0960-8524/$ - see front matter 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2006.02.006
Optimization of medium and cultivation conditions for alkaline
protease production by the marine yeast Aureobasidium pullulans
Z. Chi

, C. Ma, P. Wang, H.F. Li
UNESCO Chinese Center of Marine Biotechnology, Ocean University of China, Yushan Road, No. 5, Qingdao, China
Received 10 November 2005; received in revised form 1 February 2006; accepted 6 February 2006
Available online 20 March 2006
Abstract
A yeast strain, Aureobasidium pullulans, which could produce the high yield of protease was isolated from sediment of saltern in
Qingdao, China. Maximum production of enzyme (623.1 U/mg protein; 7.2 U/ml) was obtained in a medium containing 2.5 g soluble
starch and 2.0 g NaNO
3
, 100 ml seawater, initial pH 6.0, after fermentation at 24.5 C for 30 h. The protease had the highest activity at pH
9.0 and 45 C.
2006 Elsevier Ltd. All rights reserved.
Keywords: Marine yeasts; Alkaline protease; Fermentation; Optimal conditions
1. Introduction
The oceans covering 71% of the planet represent an
important bioresource for microorganisms including
yeasts (Chi and Liu, 2005). However, little is known about
biodiversity and production of bioactive substances from
marine yeasts. Proteases have been shown to have many
applications in detergents, leather processing, silver recov-
ery, medical purposes, food processing, feeds, chemical
industry as well as waste treatment (Kurmar and Tagaki,
1999; Anwar and Saleemuddin, 1998). Proteases also con-
tribute to the development of high-added applications or
products by using the enzyme-aided digestion of proteins
from diVerent sources (Kurmar and Tagaki, 1999). In
recent years, many results also have shown that alkaline
proteinase in the intestine of marine animals can help
digest protein in the feed and the activity of alkaline prote-
ase in the intestine regulates the use of components in the
compound diet and shows the stage of development in
marine animals. Therefore, alkaline protease in the guts of
marine animals has received much attention in recent years
(Chong et al., 2002; Fu et al., 2005). So far, it has been
found that microorganisms are the most suitable resources
for industrial production of protease as protease-produc-
ing microorganisms are easily cultivated in a large scale,
protease yields from microorganisms are very high and
diVerent proteases produced by microorganisms have
diVerent biochemical and physical characteristics and
physiological functions (Kurmar and Tagaki, 1999). How-
ever, to our knowledge, marine yeast is still an untouched
bioresource for enzyme production.
Terrestrial yeasts reported to produce alkaline proteases
include Candida lipolytica, Yarrowia lipolytica and Aure-
obasidium pullulans (Tobe et al., 1976; Ogrydziak, 1993;
Donaghy and McKay, 1993). Especially, among the extra-
cellular enzymes of Y. lipolytica, alkaline protease could
reach several grams per liter under optimised conditions
(Barth and Garlardin, 1996). However, very few studies
exist on the alkaline protease-producing marine yeasts (Chi
and Liu, 2005). This study aimed at screening and isolation
of marine yeasts with high protease activities and optimiza-
tion of medium and cultivation conditions for alkaline pro-
tease production by one of them.
*
Corresponding author. Tel.: +86 532 820322266; fax: +86 532
82032266.
E-mail address: zhenming@sdu.edu.cn (Z. Chi).
Z. Chi et al. / Bioresource Technology 98 (2007) 534538 535
2. Methods
2.1. Sampling
DiVerent samples of seawater and sediments in Southern
Sea of China and the PaciWc Ocean were collected during
the Antarctic exploration in 2004 and hypersaline sea water
and sediments of the salterns around the coastal line of
Qingdao were also collected.
2.2. Screening and isolation of marine yeasts
Two milliliters of the seawater or 2g of the sediments were
suspended in 20ml of YPD (2.0% glucose, 2.0% polypeptone
and 1.0% yeast extract) medium prepared with seawater and
supplemented with 0.05% chloramphenicol immediately after
sampling and cultivated at natural temperature on the ship
for Wve days. After suitable dilution of the cell cultures, the
medium was plated on YPD plates with 0.05% chloramphen-
icol and the plates were incubated at 2025C for Wve days.
DiVerent colonies from the plates were transferred to the
double plates with 2.0% casein and incubated at 2025C for
Wve days and the strains, which showed big clear zone around
the colonies were selected for the subsequent investigation.
2.3. Cultivation of marine yeasts
Two loops of the cells of the puriWed strains were trans-
ferred to 50 ml of YPD medium prepared with sea water in
250 ml Xask and aerobically cultivated for 24 h. Cell culture
(5 ml, OD
600 nm
D2.92) was transferred to 45ml of the pro-
duction medium (prepared with seawater), which contained
2.5% soluble starch and 2.0% NaNO
3
, pH 6.0 and grown by
shaking at 180 rpm and 24.5 C for two days.
2.4. Determination of protease activity
The cell culture was centrifuged at 5000rpm and 4C for
10min. The supernatant (0.5ml) was mixed with 1.0ml of
0.5% casein solution in glycineNaOH buVer (0.05M, pH 9.0),
preincubated at 45C for 30min. The mixture was incubated
at 45C for 30min and 2ml of 10% TCA (trichloroacetic acid)
solution was added to the mixture immediately to stop the
reaction. The reaction mixture was centrifuged at 10000rpm
and 4C for 10min. Tyrosine content in the supernatant was
determined colorimetrically at 650nm by using Folinphenol
reagent (Lowry et al., 1951). The enzyme activity was deWned
as the amount of the enzyme that liberated 1g of tyrosine
per minute under the conditions used in this study. The
speciWc protease activity was units per mg of protein. Protein
concentration was measured by the method of Bradford and
bovine serum albumin served as standard (Bradford, 1976).
2.5. DNA extraction and PCR
The total genomic DNA of the yeast strain 10 was iso-
lated and puriWed by using the methods as described by
Sambrook et al. (1989). The common primers for ampliWca-
tion of 18S rDNA in yeasts were used, the forward primer
P1:5-ATCTGGTTGATCCTGCCAGT-3 and the reverse
primer P2:5-GATCCTTCCGCAGGTTCACC-3 (Thanh
et al., 2002) and the common primers for ampliWcation of
ITS in yeasts were used, the forward primer P11:5-TCCG-
TAGGTGAACCTGCGG-3 and the reverse primer
P21:5-TCCTCCGCTTATTGATATGC-3 (Josefa et al.,
2004). The reaction system (25 l) was composed of 10
buVer 2.5 l, dNTP 0.8 mol/l, MgCl
2
1.5 mmol/l, P1 or P11
0.5mol/l, P2 or P21 0.5 mol/l, Taq DNA polymerase
1.25 U, template DNA 1l and H
2
O 16.6 l. The conditions
for the PCR ampliWcation were as follows: initial denatur-
ation at 94C for 10 min, denaturation at 94 C for 1 min,
annealing temperature at 53 C for 1 min, extension at 72 C
for 2 min, Wnal extension at 72 C for 10 min. PCR was run
for 32 cycles and PCR cycler was GeneAmp

PCR System
2400 made by PerkinElmer. PCR products were separated
by agarose gel electrophoresis and recovered by using
UNIQ-column DNA gel recovery kits (BIOASIA, Shang-
hai). The recovered PCR products were ligated into
pGEM-T easy vector and transformed into competent cells
of Escherichia coli JM109. The transformants were selected
on plates with ampicillin. The plasmids in the transformant
cells were extracted by using the methods as described by
Sambrook et al. (1989). In order to conWrm that the PCR
products had been ligated into the vector, the puriWed plas-
mids were used as templates for ampliWcation of 18S rDNA
and ITS in yeast strain 10, respectively. The reaction system
and the conditions for PCR ampliWcation were the same as
described above. The 18S rDNA fragment and ITS frag-
ment inserted on the vector were sequenced by Shanghai
Sangon Company.
2.6. Phylogenetic analysis and identiWcation of the yeast
The sequences obtained above were aligned by using
BLAST analysis (http://www.ncbi.nlm.nih.gov/BLAST).
For comparison with currently available sequences, 20
sequences were retrieved with over 98% similarity belong-
ing to 20 diVerent genera from NCBI (http://www.ncbi.
nlm.nih.gov) and performed multiple alignment by using
Bioedit 7.0. The routine identiWcation of the yeasts was per-
formed by using the methods as described by Kurtzman
and Fell (1998).
2.7. EVects of pH and temperature on protease activity
The eVects of pH on the enzyme activity were deter-
mined by incubating the culture supernatant at diVerent
pH between 4.0 and 10.0 using the standard assay condi-
tions described in Section 2.4. The buVers used were
0.02 M Na
2
HPO
4
citric acid (pH 4.08.0) and 0.05 M gly-
cineNaOH buVer (pH 9.010.0). The optimal tempera-
ture for activity of the enzyme was determined at 30, 40,
45, 50, 55 and 60 C in the same buVer as described in Sec-
tion 2.4.
536 Z. Chi et al. / Bioresource Technology 98 (2007) 534538
2.8. Fermentation
The fermentation was carried out in a 2-l BIOSTAT

B
bioreactor (B. Braun Biotech International, Germany) with
working volume of 2 l of the production medium (prepared
with seawater). The bioreactor with 1800ml of the produc-
tion medium was sterilized at 121C for 30 min. After cool-
ing, the medium was inoculated with 200 ml of inoculum to
make OD
600 nm
value of the initial culture be 0.20.3. The
fermentation was carried out at 24.5C, aeration rate of
8.0 l/min and agitation speed of 150 rpm. Samples for the
determination of the enzyme activity and cell dry weight
were withdrawn at interval of 8h.
2.9. Determination of cell dry weight
The yeast cells from 5.0 ml of culture were harvested and
washed three times with distilled water by centrifugation at
5000 rpm for 5min. Then, cells in the tube were dried at
100C until the cell dry weight was constant (Chi and
Zhao, 2003).
3. Results and discussion
3.1. Screening and isolation of marine yeasts with protease
activities
Total 327 yeast strains from seawater and sediments
were obtained but only 12 strains among them could form
clear zone around the colonies on the double plates with
2.0% casein (results not shown). Except strain 6 that was
isolated from seawater, other strains were obtained from
sediment of the saltern in Qingdao. Saltern has been used to
produce sea salts in this area for over 50 years. The results
indicated that protease activity of strain 10 was the highest,
which grew better in YPD medium prepared with sea water
than in that prepared with distilled water (data not shown).
Therefore, strain 10 was utilized for the subsequent studies.
3.2. IdentiWcation of yeast strain 10
On YM (yeast extract and malt) medium, the single col-
ony was pale at the beginning and became brown to black.
Single cells were oval producing daughter cells by budding
in liquid YM medium. Pseudomycelia occurred. The yeast
strain could not ferment glucose, galactose, sucrose, malt-
ose, lactose, raYnose, trehalose. However, it could assimi-
late glucose, galactose, L-sorbose, sucrose, maltose,
cellobiose, trehalose, melibiose, raYnose, inulin, soluble
starch, D-xylose, L-arabinose, D-arabinose and L-rhamnose
(data not shown). The results of the routine identiWcation
of the yeast strain showed that it was closely related to A.
pullulans (Kurtzman and Fell, 1998).
18S rDNA and ITS sequences of yeast strain 10 were
deposited in NCBI (Accession Nos. DQ 242509 and DQ
309591). Phylogenetic analysis of 20 18S rDNA and ITS
sequences with over 97% similarity belonging to 20 diVer-
ent genera from NCBI showed that the similarities between
18S rDNA sequences and between ITS sequences of yeast
strain 10 and A. pullulans were 100%. Therefore, the yeast
strain 10 was Wnally identiWed as a strain of A. pullulans
(Kurtzman and Fell, 1998).
3.3. EVects of temperature and pH on protease activity
The protease activity measured as a function of tempera-
ture from 30 to 60 C showed highest at 45 C (data not
shown). Results on the eVect of pH on the enzyme activity
showed that the maximum activity of protease was
observed at pH 9.0 (data not shown). These results sug-
gested that the enzyme was alkaline protease (Anwar and
Saleemuddin, 1998).
3.4. EVect of diVerent carbon sources on protease production
There are several reports showing that diVerent carbon
sources have diVerent inXuences on extracellular enzyme
production by diVerent strains (Chi and Zhao, 2003).
Therefore, eVects of soluble starch, sucrose, glucose, lactose,
fructose, maltose, corn starch and citric acid at the concen-
trations of 2.0% on protease production by A. pullulans
were examined. The results in Fig. 1 showed that soluble
starch and corn starch were the best carbon sources for
protease production. The speciWc protease activity in the
culture supernatant was 321 U/mg protein. This meant that
strain could secrete extracellular amylase to hydrolyze
starch in the medium and used starch as sole carbon source
for protease production (Fig. 1). It is thought that starch is
the best carbon source for fermentation industry due to its
low cost and easily obtained material (Chi and Zhao, 2003).
Fig. 1 also showed that in the presence of other carbon
sources, there was a reduction in protease production. This
could be due to catabolite repression by high glucose avail-
able in the medium. However, increased yields of alkaline
proteases were reported by several other workers who used
diVerent sugars such as lactose, maltose, sucrose and fruc-
tose (Malathis and Chakraborty, 1991; Tsuchiya et al.,
1991; Phadatare et al., 1993).
The results in Fig. 2 indicated that the optimal concen-
tration of corn starch for the maximum protease produc-
tion by the yeast strain was 2.0%. Under this condition, the
speciWc protease activity reached 398U/mg protein. In con-
Fig. 1. EVects of diVerent carbon sources on protease production. The cells
were cultivated in the production medium. All the data are given
mean SD, n D3.
0
50
100
150
200
250
300
350
Soluble
starch
Sucrose Glucose Lactose Maltose Fructose Corn
starch
Citric
acid
Carbon sources
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/
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Z. Chi et al. / Bioresource Technology 98 (2007) 534538 537
trast, the results in Fig. 3 revealed that the optimal concen-
tration of soluble starch for the maximum protease
production was 2.5%. Under this condition, the speciWc
protease activity in the culture reached 434 U/mg protein,
suggesting that the yeast cells grown in the presence of solu-
ble starch could produce more protease than those grown
in the presence of corn starch.
3.5. EVects of diVerent nitrogen sources on protease
production
It has been reported that eVects of a speciWc nitrogen
supplement on protease production diVer from organism to
organism although complex nitrogen sources are usually
used for alkaline protease production (Kurmar and Tagaki,
1999). Fig. 4 showed that sodium nitrate was stimulatory
for alkaline protease production by the yeast strain and
substitution of sodium nitrate in the medium with other
nitrogen sources including organic nitrogen sources
decreased greatly the enzyme production. SpeciWc protease
activity in the presence of 2.0% sodium nitrate reached
485.6 U/mg protein. The speciWc protease activity in the
supernatant of the yeast strain reached the highest when the
production medium contained 2.0% sodium nitrate (data
not shown). On the contrary, Banerjee and Bhattacharyya
(1992) found that 0.25% of sodium nitrate could be stimula-
tory for alkaline protease production by Rhizopus oryzae.
3.6. EVects of temperature and pH on protease production
It is known that temperature is one of the most critical
parameters that has to be controlled in bioprocess (Chi and
Zhao, 2003). The results on the eVect of temperature
revealed that the speciWc protease activity reached the high-
est when the strain was grown at 24.5 C (data not shown).
It has been noted that the important characteristic of most
microorganisms is their strong dependence on the extracel-
lular pH for cell growth and enzyme production (Kurmar
and Tagaki, 1999). The results of pH studies showed that
the yeast strain produced the highest yields of alkaline pro-
tease (447.5U/mg protein) at initial pH 6.0 of the produc-
tion medium (data not shown). However, for increased
protease yields from alkalophilic microorganisms, the pH
of the medium must be maintained above 7.5 throughout
the fermentation period (Aunstrup, 1980).
3.7. Time course of protease production and cell growth
during the fermentation
During the fermentation, diVerent dissolved oxygen level
in the fermentation broth of the bioreactor can be obtained
by variations in the aeration rate and the agitation speed
(Chi and Zhao, 2003), which can inXuence greatly cell
growth of the yeasts, thus production of extracellular
enzymes. A agitation speed 150 rpm and 8 l/min of aeration
rate in the fermentor were the most suitable for protease
production by this yeast strain (data not shown). Under the
optimal conditions, 623.1U/mg protein (7.2 U/ml) of prote-
ase activity was reached in the culture of strain 10 within
30h of the fermentation when the cell growth reached mid-
log phase (Fig. 5).
Fig. 2. EVects of diVerent initial concentrations of corn starch on protease
production. The cells were cultivated in the production medium. All the
data are given as mean SD, n D3.
250
300
350
400
450
1 2 3 4
Initial concentrations of corn starch (w/v %)
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(
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p
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Fig. 3. EVects of diVerent initial concentrations of soluble starch on prote-
ase production. The cells were cultivated in the production medium. All
the data are given as mean SD, n D3.
300
320
340
360
380
400
420
440
460
1 1.5 2 2.5 3 3.5 4 4.5 5
Initial concentrations of soluble starch (w/v %)
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a
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Fig. 4. EVects of diVerent nitrogen sources on protease production.
Organic nitrogen concentrations [determined by Kjehldahl method
(Strickland and Parsons, 1972)] used were peptone 0.11 mol/l; casein
0.091 mol/l; tryptone 0.099 mol/l; proteose peptone 0.109 mol/l; urea
0.094 mol/l. Inorganic nitrogen concentrations were KNO
3
0.20 mol/l;
NaNO
3
0.24 mol/l; ammonium citrate 0.095 mol/l, respectively. The cells
were cultivated in the production medium. All the data are given as
mean SD, n D3.
0
100
200
300
400
500
600
NaNO
3
Ammonium
citrate
KNO
3
Urea Peptone Casein Tryptone Protease
peptone
Nitrogen sources
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538 Z. Chi et al. / Bioresource Technology 98 (2007) 534538
4. Conclusions
Microbial alkaline proteases have many applications
and marine yeast is an untouched bioresource for enzyme
production. Alkaline protease-producing marine yeasts can
be applied to maricultural industry. Yeast strain A. pullu-
lans produced high yield of protease. This is the Wrst report
on alkaline protease production by marine yeast A. pullu-
lans.
Acknowledgement
The authors would like to thank National Natural
Science Foundation of China for its Wnancial support. The
Grant No. is 30328021.
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Fig. 5. The time course of protease production () and cell growth ()
during the fermentation. The cells were cultivated in the production
medium. All the data are given as mean SD, n D3.
0
100
200
300
400
500
600
700
0 8 16 24 32 40 48 56 64 72 80
Time (h)
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1
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