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Bioresource Technology 98 (2007) 534538

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

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 NaNO3, 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 recovery, medical purposes, food processing, feeds, chemical industry as well as waste treatment (Kurmar and Tagaki, 1999; Anwar and Saleemuddin, 1998). Proteases also contribute 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 protease in the intestine regulates the use of components in the compound diet and shows the stage of development in

* Corresponding author. Tel.: +86 532 820322266; fax: +86 532 82032266. E-mail address: zhenming@sdu.edu.cn (Z. Chi).

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-producing 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). However, 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 Aureobasidium pullulans (Tobe et al., 1976; Ogrydziak, 1993; Donaghy and McKay, 1993). Especially, among the extracellular 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 optimization of medium and cultivation conditions for alkaline protease production by one of them.

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 2 g of the sediments were suspended in 20 ml 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% chloramphenicol and the plates were incubated at 2025 C for Wve days. DiVerent colonies from the plates were transferred to the double plates with 2.0% casein and incubated at 2025 C 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 transferred to 50 ml of YPD medium prepared with sea water in 250 ml Xask and aerobically cultivated for 24 h. Cell culture (5 ml, OD600 nm D 2.92) was transferred to 45 ml of the production medium (prepared with seawater), which contained 2.5% soluble starch and 2.0% NaNO3, 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 5000 rpm and 4 C for 10 min. The supernatant (0.5 ml) was mixed with 1.0 ml of 0.5% casein solution in glycineNaOH buVer (0.05M, pH 9.0), preincubated at 45 C for 30 min. The mixture was incubated at 45 C for 30 min and 2ml of 10% TCA (trichloroacetic acid) solution was added to the mixture immediately to stop the reaction. The reaction mixture was centrifuged at 10000 rpm and 4 C for 10min. Tyrosine content in the supernatant was determined colorimetrically at 650 nm by using Folinphenol reagent (Lowry et al., 1951). The enzyme activity was deWned as the amount of the enzyme that liberated 1 g 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 isolated and puriWed by using the methods as described by

Sambrook et al. (1989). The common primers for ampliWcation 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 -TCCGTAGGTGAACCTGCGG-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, MgCl2 1.5 mmol/l, P1 or P11 0.5 mol/l, P2 or P21 0.5 mol/l, Taq DNA polymerase 1.25 U, template DNA 1 l and H2O 16.6 l. The conditions for the PCR ampliWcation were as follows: initial denaturation at 94 C 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, Shanghai). 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 plasmids 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 fragment 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 belonging 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 performed 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 determined by incubating the culture supernatant at diVerent pH between 4.0 and 10.0 using the standard assay conditions described in Section 2.4. The buVers used were 0.02 M Na2HPO4citric acid (pH 4.08.0) and 0.05 M glycineNaOH buVer (pH 9.010.0). The optimal temperature for activity of the enzyme was determined at 30, 40, 45, 50, 55 and 60 C in the same buVer as described in Section 2.4.

2.8. Fermentation The fermentation was carried out in a 2-l BIOSTATB bioreactor (B. Braun Biotech International, Germany) with working volume of 2 l of the production medium (prepared with seawater). The bioreactor with 1800 ml of the production medium was sterilized at 121 C for 30 min. After cooling, the medium was inoculated with 200 ml of inoculum to make OD600 nm value of the initial culture be 0.20.3. The fermentation was carried out at 24.5 C, 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 8 h. 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 5 min. Then, cells in the tube were dried at 100 C 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 colony 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, maltose, lactose, raYnose, trehalose. However, it could assimilate 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 temperature 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 suggested 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 concentrations 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 available 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 fructose (Malathis and Chakraborty, 1991; Tsuchiya et al., 1991; Phadatare et al., 1993). The results in Fig. 2 indicated that the optimal concentration of corn starch for the maximum protease production by the yeast strain was 2.0%. Under this condition, the speciWc protease activity reached 398 U/mg protein. In conSpecific protease activity (U/mg protein)

350 300 250 200 150 100 50 0

Soluble starch

Sucrose

Glucose

Lactose

Maltose

Fructose

Carbon sources

Corn starch

Citric acid

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 D 3.

activity (U/mg protein)

Specific protease activity (U/mg protein)

450 Specific protease 400 350 300 250

600 500 400 300 200 100 0


NaNO3 Ammonium citrate KNO3 Urea Peptone Casein Tryptone Protease peptone

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

Nitrogen sources

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 D 3.

460 440 Specific protease activity (U/mg protein) 420 400 380 360 340 320 300 1 1.5 2 2.5 3 3.5 4 4.5 5 Initial concentrations of soluble starch (w/v %)

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 KNO3 0.20 mol/l; NaNO3 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 D 3.

not shown). On the contrary, Banerjee and Bhattacharyya (1992) found that 0.25% of sodium nitrate could be stimulatory 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 highest 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 extracellular 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 protease (447.5 U/mg protein) at initial pH 6.0 of the production 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, 623.1 U/mg protein (7.2 U/ml) of protease activity was reached in the culture of strain 10 within 30 h of the fermentation when the cell growth reached midlog phase (Fig. 5).

Fig. 3. EVects of diVerent initial concentrations of soluble starch on protease production. The cells were cultivated in the production medium. All the data are given as mean SD, n D 3.

trast, the results in Fig. 3 revealed that the optimal concentration 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 soluble 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

700 activity (U/mg protein) 600 500 400 300 200 100 0 0 8 Specific protease

8 7 6 5 4 3 2 1 0 16 24 32 40 48 56 64 72 80 Time (h) Cell dry weight (g/l)

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 D 3.

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. pullulans produced high yield of protease. This is the Wrst report on alkaline protease production by marine yeast A. pullulans. Acknowledgement The authors would like to thank National Natural Science Foundation of China for its Wnancial support. The Grant No. is 30328021. References
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