Bioresource Technology 98 (2007) 534–538

Optimization of medium and cultivation conditions for alkalineprotease 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 2006Available online 20 March 2006

Abstract

A yeast strain, Aureobasidium pullulans, which could produce the high yield of protease was isolated from sediment of saltern inQingdao, China. Maximum production of enzyme (623.1 U/mg protein; 7.2 U/ml) was obtained in a medium containing 2.5 g solublestarch and 2.0 g NaNO3, 100 ml seawater, initial pH 6.0, after fermentation at 24.5 °C for 30 h. The protease had the highest activity at pH9.0 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 animportant bioresource for microorganisms includingyeasts (Chi and Liu, 2005). However, little is known aboutbiodiversity and production of bioactive substances frommarine yeasts. Proteases have been shown to have manyapplications in detergents, leather processing, silver recov-ery, medical purposes, food processing, feeds, chemicalindustry as well as waste treatment (Kurmar and Tagaki,1999; Anwar and Saleemuddin, 1998). Proteases also con-tribute to the development of high-added applications orproducts by using the enzyme-aided digestion of proteinsfrom diVerent sources (Kurmar and Tagaki, 1999). Inrecent years, many results also have shown that alkalineproteinase in the intestine of marine animals can helpdigest protein in the feed and the activity of alkaline prote-ase in the intestine regulates the use of components in thecompound diet and shows the stage of development in

* Corresponding author. Tel.: +86 532 820322266; fax: +86 53282032266.

E-mail address: zhenming@sdu.edu.cn (Z. Chi).

0960-8524/$ - see front matter © 2006 Elsevier Ltd. All rights reserved.doi:10.1016/j.biortech.2006.02.006

marine animals. Therefore, alkaline protease in the guts ofmarine animals has received much attention in recent years(Chong et al., 2002; Fu et al., 2005). So far, it has beenfound that microorganisms are the most suitable resourcesfor industrial production of protease as protease-produc-ing microorganisms are easily cultivated in a large scale,protease yields from microorganisms are very high anddiVerent proteases produced by microorganisms havediVerent biochemical and physical characteristics andphysiological functions (Kurmar and Tagaki, 1999). How-ever, to our knowledge, marine yeast is still an untouchedbioresource for enzyme production.

Terrestrial yeasts reported to produce alkaline proteasesinclude 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 couldreach several grams per liter under optimised conditions(Barth and Garlardin, 1996). However, very few studiesexist on the alkaline protease-producing marine yeasts (Chiand Liu, 2005). This study aimed at screening and isolationof marine yeasts with high protease activities and optimiza-tion of medium and cultivation conditions for alkaline pro-tease production by one of them.

Z. Chi et al. / Bioresource Technology 98 (2007) 534–538 535

2. Methods

2.1. Sampling

DiVerent samples of seawater and sediments in SouthernSea of China and the PaciWc Ocean were collected duringthe Antarctic exploration in 2004 and hypersaline sea waterand sediments of the salterns around the coastal line ofQingdao were also collected.

2.2. Screening and isolation of marine yeasts

Two milliliters of the seawater or 2 g of the sediments weresuspended in 20 ml of YPD (2.0% glucose, 2.0% polypeptoneand 1.0% yeast extract) medium prepared with seawater andsupplemented with 0.05% chloramphenicol immediately aftersampling and cultivated at natural temperature on the shipfor Wve days. After suitable dilution of the cell cultures, themedium was plated on YPD plates with 0.05% chloramphen-icol and the plates were incubated at 20–25°C for Wve days.DiVerent colonies from the plates were transferred to thedouble plates with 2.0% casein and incubated at 20–25 °C forWve days and the strains, which showed big clear zone aroundthe 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 in250 ml Xask and aerobically cultivated for 24 h. Cell culture(5 ml, OD600 nmD 2.92) was transferred to 45 ml of the pro-duction medium (prepared with seawater), which contained2.5% soluble starch and 2.0% NaNO3, pH 6.0 and grown byshaking at 180 rpm and 24.5 °C for two days.

2.4. Determination of protease activity

The cell culture was centrifuged at 5000rpm and 4°C for10min. The supernatant (0.5ml) was mixed with 1.0ml of0.5% casein solution in glycine–NaOH buVer (0.05M, pH 9.0),preincubated at 45°C for 30min. The mixture was incubatedat 45°C for 30min and 2ml of 10% TCA (trichloroacetic acid)solution was added to the mixture immediately to stop thereaction. The reaction mixture was centrifuged at 10000rpmand 4°C for 10min. Tyrosine content in the supernatant wasdetermined colorimetrically at 650nm by using Folin–phenolreagent (Lowry et al., 1951). The enzyme activity was deWnedas the amount of the enzyme that liberated 1�g of tyrosineper minute under the conditions used in this study. ThespeciWc protease activity was units per mg of protein. Proteinconcentration was measured by the method of Bradford andbovine 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 primerP1:5�-ATCTGGTTGATCCTGCCAGT-3� and the reverseprimer P2:5�-GATCCTTCCGCAGGTTCACC-3� (Thanhet al., 2002) and the common primers for ampliWcation ofITS in yeasts were used, the forward primer P11:5�-TCCG-TAGGTGAACCTGCGG-3� and the reverse primerP21:5�-TCCTCCGCTTATTGATATGC-3� (Josefa et al.,2004). The reaction system (25 �l) was composed of 10£buVer 2.5 �l, dNTP 0.8 �mol/l, MgCl2 1.5 mmol/l, P1 or P110.5�mol/l, P2 or P21 0.5 �mol/l, Taq DNA polymerase1.25 U, template DNA 1 �l and H2O 16.6 �l. The conditionsfor the PCR ampliWcation were as follows: initial denatur-ation at 94 °C for 10 min, denaturation at 94 °C for 1 min,annealing temperature at 53 °C for 1 min, extension at 72 °Cfor 2 min, Wnal extension at 72 °C for 10 min. PCR was runfor 32 cycles and PCR cycler was GeneAmp® PCR System2400 made by Perkin–Elmer. PCR products were separatedby agarose gel electrophoresis and recovered by usingUNIQ-column DNA gel recovery kits (BIOASIA, Shang-hai). The recovered PCR products were ligated intopGEM-T easy vector and transformed into competent cellsof Escherichia coli JM109. The transformants were selectedon plates with ampicillin. The plasmids in the transformantcells were extracted by using the methods as described bySambrook et al. (1989). In order to conWrm that the PCRproducts had been ligated into the vector, the puriWed plas-mids were used as templates for ampliWcation of 18S rDNAand ITS in yeast strain 10, respectively. The reaction systemand the conditions for PCR ampliWcation were the same asdescribed above. The 18S rDNA fragment and ITS frag-ment inserted on the vector were sequenced by ShanghaiSangon Company.

2.6. Phylogenetic analysis and identiWcation of the yeast

The sequences obtained above were aligned by usingBLAST analysis (http://www.ncbi.nlm.nih.gov/BLAST).For comparison with currently available sequences, 20sequences 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 usingBioedit 7.0. The routine identiWcation of the yeasts was per-formed by using the methods as described by Kurtzmanand 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 diVerentpH between 4.0 and 10.0 using the standard assay condi-tions described in Section 2.4. The buVers used were0.02 M Na2HPO4–citric acid (pH 4.0–8.0) and 0.05 M gly-cine–NaOH buVer (pH 9.0–10.0). 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) 534–538

2.8. Fermentation

The fermentation was carried out in a 2-l BIOSTAT®Bbioreactor (B. Braun Biotech International, Germany) withworking volume of 2 l of the production medium (preparedwith seawater). The bioreactor with 1800 ml of the produc-tion medium was sterilized at 121 °C for 30 min. After cool-ing, the medium was inoculated with 200 ml of inoculum tomake OD600 nm value of the initial culture be 0.2–0.3. Thefermentation was carried out at 24.5 °C, aeration rate of8.0 l/min and agitation speed of 150 rpm. Samples for thedetermination of the enzyme activity and cell dry weightwere withdrawn at interval of 8 h.

2.9. Determination of cell dry weight

The yeast cells from 5.0 ml of culture were harvested andwashed three times with distilled water by centrifugation at5000 rpm for 5 min. Then, cells in the tube were dried at100 °C until the cell dry weight was constant (Chi andZhao, 2003).

3. Results and discussion

3.1. Screening and isolation of marine yeasts with protease activities

Total 327 yeast strains from seawater and sedimentswere obtained but only 12 strains among them could formclear zone around the colonies on the double plates with2.0% casein (results not shown). Except strain 6 that wasisolated from seawater, other strains were obtained fromsediment of the saltern in Qingdao. Saltern has been used toproduce sea salts in this area for over 50 years. The resultsindicated that protease activity of strain 10 was the highest,which grew better in YPD medium prepared with sea waterthan 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 buddingin liquid YM medium. Pseudomycelia occurred. The yeaststrain 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, solublestarch, D-xylose, L-arabinose, D-arabinose and L-rhamnose(data not shown). The results of the routine identiWcationof 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 weredeposited in NCBI (Accession Nos. DQ 242509 and DQ309591). Phylogenetic analysis of 20 18S rDNA and ITSsequences with over 97% similarity belonging to 20 diVer-

ent genera from NCBI showed that the similarities between18S rDNA sequences and between ITS sequences of yeaststrain 10 and A. pullulans were 100%. Therefore, the yeaststrain 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 notshown). Results on the eVect of pH on the enzyme activityshowed that the maximum activity of protease wasobserved at pH 9.0 (data not shown). These results sug-gested that the enzyme was alkaline protease (Anwar andSaleemuddin, 1998).

3.4. EVect of diVerent carbon sources on protease production

There are several reports showing that diVerent carbonsources have diVerent inXuences on extracellular enzymeproduction 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. pullulanswere examined. The results in Fig. 1 showed that solublestarch and corn starch were the best carbon sources forprotease production. The speciWc protease activity in theculture supernatant was 321 U/mg protein. This meant thatstrain could secrete extracellular amylase to hydrolyzestarch in the medium and used starch as sole carbon sourcefor protease production (Fig. 1). It is thought that starch isthe best carbon source for fermentation industry due to itslow cost and easily obtained material (Chi and Zhao, 2003).Fig. 1 also showed that in the presence of other carbonsources, there was a reduction in protease production. Thiscould be due to catabolite repression by high glucose avail-able in the medium. However, increased yields of alkalineproteases were reported by several other workers who useddiVerent 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, thespeciWc protease activity reached 398 U/mg protein. In con-

Fig. 1. EVects of diVerent carbon sources on protease production. The cellswere cultivated in the production medium. All the data are givenmean § SD, n D 3.

0

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Sucrose Glucose Lactose Maltose Fructose Cornstarch

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Z. Chi et al. / Bioresource Technology 98 (2007) 534–538 537

trast, the results in Fig. 3 revealed that the optimal concen-tration of soluble starch for the maximum proteaseproduction was 2.5%. Under this condition, the speciWcprotease 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 grownin the presence of corn starch.

3.5. EVects of diVerent nitrogen sources on protease production

It has been reported that eVects of a speciWc nitrogensupplement on protease production diVer from organism toorganism although complex nitrogen sources are usuallyused for alkaline protease production (Kurmar and Tagaki,1999). Fig. 4 showed that sodium nitrate was stimulatoryfor alkaline protease production by the yeast strain andsubstitution of sodium nitrate in the medium with othernitrogen sources including organic nitrogen sourcesdecreased greatly the enzyme production. SpeciWc proteaseactivity in the presence of 2.0% sodium nitrate reached485.6 U/mg protein. The speciWc protease activity in thesupernatant of the yeast strain reached the highest when theproduction medium contained 2.0% sodium nitrate (data

Fig. 2. EVects of diVerent initial concentrations of corn starch on proteaseproduction. The cells were cultivated in the production medium. All thedata are given as mean § SD, nD 3.

250

300

350

400

450

1 2 3 4Initial concentrations of corn starch (w/v %)

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cific

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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. Allthe data are given as mean § SD, n D 3.

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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 criticalparameters that has to be controlled in bioprocess (Chi andZhao, 2003). The results on the eVect of temperaturerevealed 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 mostmicroorganisms is their strong dependence on the extracel-lular pH for cell growth and enzyme production (Kurmarand Tagaki, 1999). The results of pH studies showed thatthe yeast strain produced the highest yields of alkaline pro-tease (447.5 U/mg protein) at initial pH 6.0 of the produc-tion medium (data not shown). However, for increasedprotease yields from alkalophilic microorganisms, the pHof the medium must be maintained above 7.5 throughoutthe fermentation period (Aunstrup, 1980).

3.7. Time course of protease production and cell growth during the fermentation

During the fermentation, diVerent dissolved oxygen levelin the fermentation broth of the bioreactor can be obtainedby variations in the aeration rate and the agitation speed(Chi and Zhao, 2003), which can inXuence greatly cellgrowth of the yeasts, thus production of extracellularenzymes. A agitation speed 150 rpm and 8 l/min of aerationrate in the fermentor were the most suitable for proteaseproduction by this yeast strain (data not shown). Under theoptimal conditions, 623.1 U/mg protein (7.2 U/ml) of prote-ase activity was reached in the culture of strain 10 within30 h of the fermentation when the cell growth reached mid-log phase (Fig. 5).

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; casein0.091 mol/l; tryptone 0.099 mol/l; proteose peptone 0.109 mol/l; urea0.094 mol/l. Inorganic nitrogen concentrations were KNO3 0.20 mol/l;NaNO3 0.24 mol/l; ammonium citrate 0.095 mol/l, respectively. The cellswere cultivated in the production medium. All the data are given asmean § SD, nD 3.

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600

NaNO3 Ammoniumcitrate

KNO3 Urea Peptone Casein Tryptone Proteasepeptone

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538 Z. Chi et al. / Bioresource Technology 98 (2007) 534–538

4. Conclusions

Microbial alkaline proteases have many applicationsand marine yeast is an untouched bioresource for enzymeproduction. Alkaline protease-producing marine yeasts canbe applied to maricultural industry. Yeast strain A. pullu-lans produced high yield of protease. This is the Wrst reporton alkaline protease production by marine yeast A. pullu-lans.

Acknowledgement

The authors would like to thank National NaturalScience Foundation of China for its Wnancial support. TheGrant 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 productionmedium. All the data are given as mean § SD, nD 3.

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