ELSEVIER BioresourceTechnology69 (1999) 155-159

blOl lSOIl (l II(IlOLOGT

Optimization of alkaline protease productivity by Bacillus licheniformis ATCC 21415

S.S. Mabrouk, A.M. Hashem, N.M.A. E1-Shayeb, A.-M.S. Ismail, A.F. Abdel-Fattah Department of Natural and Microbial Products Chemistry, National Research Centre, Dokki, Cairo, Egypt

Received 27 May 1998; revised 20 August 1998; accepted 15 September 1998

Abstract

The production of alkaline proteases by Bacillus licheniformis ATCC 21415 was studied. The highest yield of alkaline protease was achieved using a mixture of lactose (4%) and glucose (1.5%) as carbon source. An alkaline extracted soybean (6%) and ammonium phosphate (1.2%) mixture was the best nitrogen source. Addition of CaCI2 from 0.01 to 0.07% optimized the production of the enzyme. Adding 1% corn oil to the medium as surfactant led to a dramatic increase of the activity to 20379 U m1-1. In addition, the activity reached 29554 U ml -I when the agitation was increased from 250 to 400 rpm. B. licheniformis 21415 could produce the same amount of protease whether sodium lauryl sulphate (SLS) was added to the medium at 0.15% concentration or not. The enzyme was stable at 50C for 15 min and lost 48.8% of its activity after 1 h. Polyphosphate slightly inhibited the enzyme activity (3%), but EDTA caused a loss of 22% of the original activity. 1999 Elsevier Science Ltd. All rights reserved.

Keywords: Alkaline protease; Microbial enzymes; Enzyme production; Bacillus licheniformis; Properties

1. Introduction

Many bacteria belonging to the genus Bacillus excrete large amounts of enzymes into the culture medium. The alkaline serine protease subtilisin carls- berg, one of the most important enzymes, is excreted into the medium by strains of Bacillus licheniformis or B. pumilus in the early stationary phase (Ward, 1983). On the other hand, Bacillus species also produce extra- cellular proteases during the post-exponential and stationary growth phases (Dawson and Kurz, 1969; Schaeffer, 1969; Kole et al., 1987).

B. licheniformis strains are listed in the third edition of Food Chemicals Codex (1981) as sources of carbohy- drase and protease enzyme preparations used in food processing (Boer et al., 1994). Till now, the exact mechanisms responsible for the cellular control of protease synthesis have been unknown and the produc- tivity of proteases can be inhibited by both nitrogen and carbon sources (Levisohn and Aronson, 1967; May and Elliott, 1968; Schaeffer, 1969).

In view of its uses, e.g. in the food industry, leather tanning and processing, fiber industry and preparations for food or pharmaceutical uses (Van-Kessel et al., 1991; Ming Chu et al., 1992), alkaline protease should

be produced commercially in high yields by a low-cost method.

The aim of the present work was to identify the culture conditions that supported protease production by the strain B. licheniformis 21415 using inexpensive materials, and some properties of the enzyme produced were determined.

2. Methods

2.1. Microorganism

The organism used was Bacillus licheniformis 21415, obtained from American Type Culture Collection, USA. The culture was maintained on nutrient agar medium at 30C for 7 days and stored at 4C.

2.2. Inoculum culture media and enzyme

2.1.1. Inoculation and fermentation media The following inocula and fermentation media were

used with the compositions (final concentrations in g litre-1).

0960-8524/99/$ -- see front matter 1999 Elsevier Science Ltd. All rights reserved. PII: S0960-8524(98)00165-5

156 s.s. Mabrouk et al./Bioresource Technology 69 (1999) 155-159

Inoculum media 1, 2 and 3 (used with fermentation media 4, 5 and 6 respectively):

1. Tryptic casein hydrolysate, 17; peptone, 3.0; glucose, 2.5; NaCI, 5.0; KH2PO4, 2.5 in 0.1 M NazCO3 solution.

2. Peptone, 1; (NH4)2SOa, 1; KHzPO4, 0.5; MgSO4.7H20, 0.3; CaCO3, 1.0; NaC1, 1.0 and glycerol, 20 ml.

3. (Soybean alkaline extracted medium; Fukumoto et al., 1974). Extracted soybean cake, 50; dextrin, 30; ammonium phosphate, 10.0; KC1, 0.3 and MgSO4.7H20, 0.2. pH was adjusted to 7.0 and autoclaving was for 15 min at 121C.

Fermentation media 4, 5 and 6: 4. (Vroemen, 1974) a. Casein, 9.4; yeast extract, 2.0 and KHzPO4, 10.0 in

700 ml distilled H20. pH was adjusted to 8.5. b. Sucrose, 25; citric acid, 1.4; CaClz.6H20, 0.16;

MgC12.6H20, 0.58; Mn C12.4H20, 0.01 and FeCI3.6H20, 0.005 in 200 ml distilled H20. pH was adjusted to 6.0.

c. NazCO3, 10.6 in 100 ml distilled H20.

a, b and c were autoclaved separately for 20 min at 121C and mixed just before culturing; initial pH 9.5.

Cultivation was in a rotary shaker at 220 rpm and 35C.

5. Peptone, 1; dextrin, 30; (NH4)2804, 1.0; KH2PO4, 1.0; MgSO4.7H20, 0.3 and NaC1, 1.0. Cultivation was at 220 rpm and 35C.

6. Had the same composition as medium 3 and cultiva- tion was at 180 rpm and 37C.

addition of various carbohydrates and organic nitrogen sources were evaluated in relation to enzyme yield.

The experiments were conducted in triplicate, and the results are the average of these three independent trials.

At the end of the fermentation period, the culture medium was centrifuged to obtain the culture liquid that was used as the enzyme source.

2.2.3. Assay of enzyme activity Caseinase activity was estimated according to the

method of Bergkvist (1963), by determining the rate of hydrolysis of 1 ml of 1.5% (w/v) casein solution in Tris-HC1 buffer, 0.05 M (at different pH values of 7, 8, 9, 10) by 1 ml diluted enzyme after incubation for 10 min at 37C.

One unit (U) of enzyme activity was taken as the amount of enzyme that liberated 1 #g of tyrosine per ml per minute.

3. Results and discussion

3.1. Culture conditions for alkaline protease production

3.1.1. Effect of culture media The data in Table 1 show that through the produc-

tion time course (1-6 days), the pH of the culture filtrates changed between acidic and alkaline. Of the three media investigated, medium 6 was the most suitable for the production of active alkaline protease, giving 3000 Um1-1 enzyme at pH 10.0 after 5 days incubation, therefore it was used as the culture medium in the following studies.

In all cases, each 250-ml Erlenmyer flask contained 50 ml of medium, except for medium 3 where each flask contained 25 ml, and in all cases a 5% (v/v) 2-day-old inoculum was used.

Fermentation was also performed in a laboratory 14-1itre fermentor (New Brunswick, USA) with a working volume of 2 litre. The cultivation was carried out for 1-7 days at 37C with agitation at 300 rpm and aeration at 0.8 litre min-1 2 litre-1.

Alkali-extracted soybean was prepared by stirring 100 g of ground soybean in 1 litre 0.1 N NaOH at 37C for 30 min. The mixture was filtered off and soybean was washed several times until it became neutral, then dried at 50C.

Corn flour and maize flour were obtained from the local market, potato starch and maize starch were obtained from Fluka, AG; Buchs SG, Switzerland.

2.2.2. Optimization studies Incubation periods ranged from 1 to 6 days and

growth temperature ranged from 30 to 55C. Effects of

3.1.2. Effect of incubation temperature At 37C B. licheniformis 21415 produced the

maximal enzyme activity (3000Uml 1) after 5 days incubation and a higher temperature (45C) caused more than 40% loss of the enzyme activity, with a greater loss at 55C where the activity did not exceed 107 U m1-1.

3.1.3. Effects of different carbon sources The effect of replacement of dextrin (30 g litre l) in

the basal medium by various carbon sources, on protease synthesis is shown in Table 2. Both lactose or fructose enhanced the enzyme productivity. On the other hand, maize starch reduced the enzyme activity to 75% of the control. Each of sucrose, potato starch or corn flour reduced the enzyme productivity, whereas maltose and molasses were completely unsuitable for enzyme production.

Since lactose or fructose were the most favourable carbon sources for the enzyme production, it was necessary to study the effect of their concentrations on

S.S. Mabrouk et al./Bioresource Technology 69 (1999) 155-159

Table 1 Effects of different media on the production of alkaline proteases from Bacillus licheniformis ATCC 21415

157

Medium" Incubation period Final pH Enzyme activity (U ml--~) no. (days) pH of reaction

7 8 9 10

1 8.50 18.7 18.8 19.1 18.9 2 8.00 89.5 99.1 103.2 105.7 3 7.60 109.1 107.1 115.0 120.5 4 7.10 75.0 79.0 99.5 101.1 1 7.53 182.4 182.4 184.3 84.9 2 7.44 1073.0 1073.0 1073.0 1085.8 3 7.52 2076.0 2076.0 2125.7 2072,6 4 7.50 1056.0 1056.0 907.9 772.6 1 5.60 110.1 114.2 110.8 100.1 2 5.74 140.0 141.3 116.0 114.2 3 6.80 908.1 911.0 905.9 827.9 4 7.15 1850.1 2053.9 2106.5 2226.3 5 7.50 2500.0 2711.0 2960.9 3000.0 6 8.00 2410.2 2482.4 2663.6 2482.4

"For media compositions see Methods section. In this and the following tables, the results are the averages of three independent experiments.

the enzyme productivity. Different concentrations of lactose or fructose instead of 3% dextrin (w/v) were tested (Table 3). Three per cent fructose enhanced the enzyme synthesis whereas higher concentrations repressed synthesis. On the other hand, 4% lactose caused the highest enzyme productivity (6850 U ml-I). The combination of 4% lactose and glucose led to a definite improvement in the enzyme production and the maximal value (10655 U ml 1) was obtained using 1.5% glucose, above which the enzyme productivity gradually decreased (Table 4). Similar results have been reported by Malathi and Chakraborty (1991) who found that lactose was the best carbon source for enzyme production by Aspergillus flavus, whereas starch, fructose, dextrin and maltose severely repressed the enzyme synthesis. On the other hand, Fujiwara and

Table 2 Effects of different carbon sources on the production of alkaline protease by Bacillus licheniformis ATCC 21415

Carbon source (3%)

Alkaline protease activity (U ml ~) pH of the reaction

8 9 10

Dextrin (control) 2711 2611 3000 Lactose 4314 4466 4503 Fructose 4114 4171 4285 Ma~e starch 1851 2051 2249 Sucrose 1589 1586 1537 Potato starch 1406 1300 1371 Corn flour 1351 1246 1214 Molasses 171 183 217 Maltose 274 126 91

Table 3 Effects of different concentrations of lactose and fructose on alkaline protease production by Bacillus licheniJormis ATCC 21415

Concentration Alkaline protease activity (% w/v) (U/ml ~ ~)

Lactose 2.5 2420 3.0 4500 4.0 6850 5.0 5586 Fructose 2.5 3332 3.0 4285 4.0 2961 5.0 2565

In this and the following tables enzyme pH 10.

activity was measured at

Table 4 Effects of adding different concentrations of glucose to a lactose medium on the production of alkaline protease by Bacilh~s lichen# formis ATCC 21415

Concentration of glucose Alkaline protcase activity (% w/v) (U/ml ~)

None (control) a 6850 +0.5 8316 + 1.0 9729 + 1.5 10655 + 2.0 10 548 + 2.5 9669 +3.0 8183

a4% Lactose.

158 S.S. Mabrouk et al./Bioresource Technology 69 (1999) 155-159

Table 5 Effects of different nitrogen sources on the production of alkaline protease by Bacillus licheniformis ATCC 21415

Nitrogen source Alkaline protease activity (U/ml i)

Control" (alkali-extracted soybean 10655 + ammonium phosphate)

Alkali-extracted soybean 7820 Milk-whey 3111 Urea 1331 Corn steep liquor 45 Wheat bran 29

"Carbon source was a mixture of lactose (4%) and glucose (1.5%).

Yamamoto (1987) found that glucose and starch were the best carbon sources for enzyme production by Bacillus B 21-2, whereas lactose, sucrose and glycerin were ineffective. Xiaohang and Guanchui (1990) found that the simple carbon sources such as glucose and fructose inhibited protease production.

Table 7 Effects of different salts on the production of alkaline protease by Bacillus licheniformis ATCC 21415

Salt (% w/v) Alkaline protease activity (U/ml t)

None (control) 14922 ZnCI2 0.01 3815 0.03 4194 0.05 3954 CaC12 0.01 14922 0.03 15195 0.05 15 312 0.07 18899 0.09 14 976 SPP 0.01 6824 0.03 8081 0.05 6543 SLS 0.15 14900

higher concentrations considerably repressed the enzyme synthesis.

3.1.4. Effect of various nitrogen sources This was tested by eliminating alkali-extracted

soybean and ammonium phosphate from the culture medium and using (on equivalent N-basis) untreated soybean, alkali extracted soybean, corn steep liquor, wheat bran, urea or milk-whey, separately, as nitrogen sources. The results (Table 5) indicated that a mixture of alkali-extracted soybean (5%) with ammonium phosphate (1%) was the best nitrogen source. On the other hand, the enzyme activity decreased dramatically when using wheat bran, corn steep liquor or urea, whereas untreated soybean was ineffective as a nitrogen source. These results were in accord with those reported for an alkaline protease from Bacillus sp. (Fujiwara and Yamamoto, 1987).

Different concentrations of ammonium phosphate and alkali-extracted soybean were also tested (Table 6). Six per cent alkali-extracted soybean and 1.2% ammonium phosphate caused 40% increase of the activity, which reached 14920Um1-1, whereas the

Table 6 Effects of different concentrations of the nitrogen source on the production of alkaline protease by Bacillus licheniformis ATCC 21415

Combined N source concentrations (% w/v)

Alk. extracted soybean Amm. phosphate

Alkaline protease activity (U/ml ~)

4 0.8 8756 (Control) 5 1 10655 6 1.2 14922 7 1.4 7912 8 1.6 5786

3.1.5. Effects of some supplements The effects of addition of some metal ions to the

culture medium on alkaline protease productivity are shown in Table 7. ZnCI2 and SPP had adverse effects on enzyme production at all concentrations used. CaCI2 at 0.07% concentration markedly affected protease activity and caused 26.6% increase in the activity over the control, and this might be attributed to the stabilizing effect of Ca 2+ on the alkaline protease (Kelly and Fogarty, 1976; Strongin et al., 1979; Ward, 1983). Also Manachini et al. (1988) reported that Ca 2+ had a stimulating effect on enzyme action.

In addition, B. licheniformis 21415 could grow very well in the medium to which sodium lauryl sulphate (SLS) had been added to a final concentration of 0.15% (Table 7), but it produced the same amount of protease whether SLS was added to the medium or not.

Adding 1% corn oil to the medium as suffactant led to a good increase in activity, to 20380 U ml 1. Also, elevation of agitation speed from 250 to 400rpm increased activity to 29550 U ml 1. Bae and Lee (1988) found that the highest protease productivity was obtained when the bacterial strain was incubated in an alkaline medium with shaking at 450 rpm.

The optimization of the culture medium caused a 9.9-fold increase in enzyme production compared with the original basal medium. B. licheniformis 21415 protease activity was higher than those reported for other Bacillus strains (Fujiwara and Yamamoto, 1987; Manachini et al., 1988). The optimized medium in a 14-1itre laboratory fermentor could produce 16500 U ml ~ after 5 days incubation.

S.S. Mabrouk et aL/Bioresource Technology 69 (1999) 155-159 159

3.2. Some properties of the crude alkaline protease preparation from B. licheniformis 21415

After heating the crude enzyme solution in the absence of its substrate, at 50, 55 and 60C for 15 min, it still retained about 83.6, 60.2 and 51.2%, respec- tively, of its original activity and also retained more than 80% of its activity after 60 min at 50C. On the other hand, the activity dropped to only 42.7 and 17.6% after 60 min at 55 and 60C.

The enzyme solution was mixed with EDTA (0.02 M) or SPP (0.5%) and preincubated for 15 min at 25C, then protease activity was determined as described earlier. The results were recorded as the percentage residual activity calculated with reference to activity controls incubated in the absence of the aforementioned substances. The SPP caused negligible inhibition of the enzyme (3%), but the enzyme was inhibited by EDTA and lost about 62% of its original activity and this may be attributed to chelation of calcium ions, which are necessary for enzyme activation or participate in the enzyme molecule. On the contrary, Manachini et al. (1988) found that the enzyme was not activated by 1 m M EDTA.

Collectively, these results may justify the suitability of the bacterial strain B. licheniformis 21415 for commercial production of alkaline protease, using inexpensive materials.

References

Bae, M., Lee, Y.H., 1988. Identification of alkalophilic bacteria from compost and properties of its alkaline protease. Nonchong- Han'guk Saenghwal Kwahak Yonguwon 41, 19-31.

Bergkvist, R., 1963. The proteolytic enzymes of Aspergillus oryzae. 1. Methods for the estimation and isolation of the proteolytic enzymes. Acta Chem. Scand. 17, 1521-1540.

Boer, A.S. de, Priest, F., Diderichsen, B., 1994. On industrial use of Bacillus licheniformis: a review. Applied Microbiology and Biotech- nology 40, 595-598.

Dawson, P.S.S., Kurz, W.G.W., 1969. Continuous phased culture - - a technique for growing, analyzing and using microbial cell. Biotech- nology and Bioengineering 11, 843-851.

Fujiwara, N., Yamamoto, K., 1987. Production of alkaline protease in a low-cost medium by alkalophilic Bacillus sp. and properties of the enzyme. Journal of Fermentation and Technology 65, 345-348.

Fukumoto, J., Yamamoto, T., Tsuru, D., 1974. Process for producing detergent resisting alkaline protease. Patented No. 3, 838, 009.

Kelly, C.T., Fogarty, W.M., 1976. Microbial alkaline enzymes. Process Biochemistry 11, 3-9.

Kole, M.M., Draper, I., Gerson, D.F., 1987. Production of protease by Bacillus subtilis using simultaneous control of glucose and ammonium concentration. J. Chem. Tech. Biotech. 41, 197-206.

Levisohn, S., Aronson, A.I., 1967. Regulation of extracellular protease production in Bacillus cereus. Journal of Bacteriology 93, 1023-1030.

Malathi, S., Chakraborty, R., 1991. Production of alkaline protease by a new Aspergillus flavus isolate under solid-substrate fermenta- tion conditions for use as a depilation agent. Applied and Environ- mental Microbiology 57, 712-716.

Manachini, P.L., Fortina, M.G., Parini, C., 1988. Thermostable alkaline protease produced by Bacillus thermoruber - - a new species of Bacillus. Applied Microbiology and Biotechnology 28, 409-413.

May, B.K., Elliott, W.H., 1968. Characteristics of extracellular protease formation by Bacillus subtilis and its control by amino acid repression. Biochim. Biophys. Acta 157, 607-615.

Ming Chu, I., Lee, C., Shun Li, T., 1992. Production of degradation of alkaline protease in batch cultures of Bacillus subtilis ATCC 14416. Journal of Enzyme and Microbial Technology 14, 755-761.

Schaeffer, P., 1969. Sporulation and the production of antibiotics, exoenzymes and exotoxins. Bacteriology Review 33, 48-71.

Strongin, A.Y.A., Abramov, Z.T., Yaroslavtseva, N.G., Baratova, L.A., Shaginyan, K.A., Belyanova, L.P., Stepanov, V.M., 1979. Direct comparison of subtilisin-like intracellular protease of Bacillus licheniformis with the homologous enzymes of Bacillus subtilis. Journal of Bacteriology 137, 1017-1019.

Van-Kessel, K.P., Van-Strijip, J.A., Verhoef, J., 1991. Inactivation of recombinant human tumer necrosis factor-alpha by proteolytic enzymes released from stimulated human neutrophils. Journal of Immunology 147(11), 3862-3868.

Ward, O.P., 1983. Proteinases. In Microbial Enzymes and Biotech- nology, W.M. Fogarty (ed.). Applied Science, New York, pp. 251-317.

Xiaohang, Ma, Guanchui, Xu, 1990. Isolation of a bacterial strain (BsO8) producing alkaline protease and properties of the enzyme production. Weishengwuxue Zazhi 10(4), 20-24.