Production of an alkaline lipase by Acinetobacter radioresistens

JOURNAL OFFERMENTATION ANDBIOENGINEERING Vol. 86, No. 3, 308-312. 1998

Production

of an Alkaline Lipase by Achetobacter SHU-JEN

CHEN,

CHU-YUAN

CHENG, * AND TEH-LIANG

radioresistens CHEN

Department of Chemical Engineering, National Cheng Kung University, Tainan 70101, Taiwan, R.O.C. Received 13 March 19981Accepted 15 June 1998 Alkaline lipase production by Acinetobacter radioresistens was investigated and the role of culture conditions was examined. The enzyme had an optimum pH of 10 and was stable over a pH range of 6-10; it could have great potential for application in the detergent industry. The optimal temperature and pH for lipase fermentation were 30°C and 7, which were related to cell growth, protease formation, and stability and reactivity of lipase. A high lipase yield could be obtained during prolonged cultivation in the presence of n-hexadecane. With additional olive oil supplementation, the volumetric productivity of lipase could be improved; however, the lipase yield decreased with increasing concentrations of olive oil. The increase in the rate of lipase formation by olive oil in the presence of n-hexadecane was attributed to its enhancement of the uptake of n-hexadecane; the hydrolytic products of olive oil promoted the emulsification of n-hexadecane. Because olive oil repressed the synthesis of lipase, optimization in formulating the medium composition is considered necessary. [Key words:

alkaline lipase, Acinetobacter radioresistens, batch fermentation, n-hexadecane uptake] lular and had an optimal temperature and pH of 37°C and 10.0, respectively, for olive oil hydrolysis. Prior to the preparation of precultures, the cells were transferred to a LB (Luria-Bertani) agar slant and incubated overnight at 30°C. The LB slant contained (per liter): 10 g tryptone (Difco, Detroit, MI, USA), 5 g yeast extract (Difco), log NaCl and 15 g agar. Precultures were prepared by inoculating one loopful of cells from an agar slant into a 500-ml Erlenmeyer flask containing 100 ml of LB broth, and incubating for 10 h in a rotary shaker. The operating conditions for all shake-flask cultivations were set at 120rpm and 30°C. The basal medium for lipase production contained (per liter): log tryptone, 5 g yeast extract, 10 g NaCl and 1 g NH&I. During cultivation, the basal medium was supplemented with varying concentrations of olive oil and n-hexadecane, as indicated. Tank fermentations were carried out in a 2.5-l fermentor (M-100, Tokyo Rikakikai, Tokyo) with a working volume of 1.2 1. The inoculum size was 2%. Temperature and pH were controlled at 30°C and 7.0, respectively, unless otherwise stated. The pH was controlled using 3 N NaOH/l N HCl. The agitation speed was 400 rpm, and the aeration rate was 1 vvm. Cell growth was monitored by turbidimetry (600nm, UV-2000, Hitachi, Tokyo) and correlated with the drycell weight. The interference by the remaining organic phase with the turbidity measurement was eliminated by centrifuging the sample twice in a Du Pont Sorvall RC-5B centrifuge at 10,000 x g and 4°C for 5 min, and re-suspending the cells in 0.1 M phosphate buffer (pH 7.0). Lipase activity was determined by the pH-stat method. The substrate was prepared by stirring 20 ml olive oil (Sigma, St. Louis, MO, USA) and log gum arabic (Sigma) in 200ml deionized water. While performing the lipase assay, 1 ml of enzyme solution was stirred into 10ml of the substrate solution. Titration was performed in a Mettler DL-21 titrator with appropriate concentrations of NaOH (0.01-0.15 N). One unit of lipase activity was defined as the amount of enzyme required to release 1 pmol fatty acid per min at 37°C and pH 10.0. Protease activity was measured by proteolysis of azocasein (Sigma) according to the method of Ginther (8). The substrate solution contained 5 mg/ml azocasein. Samples

A large number of surface active agents are used in the detergent industry. However, as environmental regulations become stricter, there is increasing concern about reducing the surfactant concentration in effluent streams. One method to reduce the concentrations of surfactant in effluent streams is to include lipase in the detergent formulation. To this end, lipases capable of functioning at alkaline pHs require to be developed. Lipases can be obtained from many species of plants, animals and microorganisms; however, most attention has been paid to microbial sources. Lipases from microbial sources are usually extracellular. In general, fungal lipases have a pH optimum in the neural or slightly acid range, while the optimal pH of bacterial lipases is in the neutral or slightly alkaline range (1). Unfortunately, relatively rare species capable of producing alkaline lipases have been reported in the literature. Some examples of microbes that possess this capability are Bacillus subtilis (2), Pseudomonas nitroreducens (3), Pseudomonas fragi (3, 4), Pseudomonas fruorescens (5) and Pseudomonas pseudoalcaligenes (6, 7). In this article, we report on the production of an alkaline lipase by a newly isolated strain, Acinetobacter radioresistens. The aim of this work was to determine the optimum culture conditions and, most importantly, to investigate the role of culture conditions for optimum lipase production. The factors examined were temperature, pH and medium composition. MATERIALS

AND METHODS

A. radioresistens, originally isolated from the sludge of wastewater, was provided by Professor Ming-Chung Chang, Department of Biochemistry, National Cheng Kung University, Tainan, Taiwan. The strain was maintained in bacterial preservation beads (Protect, Technical Service, UK) and stored at -30°C. Preliminary experiments revealed that A. radioresistens showed poor growth and little lipase production if the cells were cultivated in the absence of oils or at acidic pHs; in addition, A. radioresistens lipase was determined to be extracel* Corresponding

author. 308

LIPASE FERMENTATION BY A. RADIORESISTENS

VOL. 86, 1998

309

were incubated at 37°C for 20min, and the reaction was terminated by the addition of 5% trichloroacetic acid. One unit of protease was defined as the amount of enzyme required for an increase per min in absorbance of 0.1 at 440 nm. RESULTS

AND DISCUSSION

The inclusion of NH&l in the fermentation medium was according to Cordenons et al. (9); they suggested that NH&l could repress protease formation during lipase fermentation by Acinetobacter calcoaceticus. The formation of proteases, which cause proteolytic degradation of the produced lipase, has been observed during the production of microbial lipases (9-11). The effect of NH&l on A. radioresistens lipase fermentation in shakeflasks using LB broth supplemented with 0.1% (w/v) olive oil is shown in Fig. 1. It seems that there is no correlation between protease formation and the concentration of NH&I; nevertheless, 1 g/l NH&l was considered optimal for lipase production by the strain reported in this study. Figure 2 shows the effect of temperature on A. radioresistens lipase fermentation, when the cells were cultivated in basal medium supplemented with 0.1% (w/v) olive oil. In the temperature range of 20 to 37°C cell growth increased with increasing temperature; however, the highest lipase yield was obtained at 30°C. Lipase formation was noted from the middle of the exponential phase to the early stationary phase; in other words, A. radioresistens lipase can be regarded as a growth-associated product. Therefore, the increase in lipase production noted between 20 and 30°C can be attributed to an increase in the growth rate of the organism. The trend of increasing lipase production with increasing growth rate, however, was not observed at 37°C. The existence of a temperature optimum for lipase production indicates that there must be two opposing effects of temperature, favorable and adverse. The favorable effect, as mentioned above, is via an increase in cell growth. The adverse effect, on the other hand, may be due to the thermal instability of the enzyme. In the examination of the thermal stability of the lipase (Fig. 3), it was found that the lipase was stable below 3.0 2.5

1 .o

=

2.0 5 s 1.5 :z P

1.0

g $

0.5 2 0.0

0

3

6

NW

9

12

0.0 2

0

4

6

0

10 Time

12

14

16

18

20

(h)

FIG. 2. Effect of temperature on A. rudioresisfens lipase fermentation. Solid symbols, cell concentration; open symbols, lipase activity. Symbols: 0 (o), 20°C;

n

(o), 25°C; A (A), 30°C; + (0), 37°C.

3O”C, but lost 30% of activity at 37°C within 2 h. At higher temperatures, the lipase lost its activity even more rapidly. Thermal instability could thus account for the decrease in detectable lipase activity at 37°C. The effect of pH on A. radioresistens lipase fermentation is shown in Fig. 4, when the cells were cultivated in basal medium supplemented with 0.1% (w/v) olive oil and 1.5% (w/v) n-hexadecane. The inclusion of nhexadecane as the carbon source for lipase production was according to the work of Kok et al. (12) for A. calcoaceticus; they found that the use of n-hexadecane resulted in a high lipase yield. Compared with the data in Fig. 2, the addition of n-hexadecane to the medium could result in a two-fold increase in cell concentration and a ‘I-fold increase in lipase yield. As can be seen in Fig. 4, lipase productivity was in the order pH 7 > pH B>pH 9, which is consistent with the sequence of cell growth. In addition to cell growth, the lipase production would also depend on three other factors: (i) protease formation, (ii) pH stability of the lipase, and (iii) reactivity of the lipase at different pH values. Figure 4 shows that protease formation begins at the end of the exponential phase, with protease activity in the order of pH 9 >pH 8>pH 7. This order indicates that the condition of pH 7 favors the production of A. radioresistens lipase. The effect of the second factor that could possibly affect lipase yield,

0.8 z 3 0.6 s ..> 0.4

2 $ m al

0.2 f

0.0

15

Ml)

FIG. 1. Effect of NH,CI on protease formation and lipase production by A. rudioresisfens. The cultivations were performed in shake-flasks and the data were taken at the maximum lipase yields. Symbols: 0, cell concentration; w , lipase activity; A.,protease activity.

0 0

20

40

60

80 Time

100

120

140

160

(min)

PIG. 3. Thermal stability of A. radioresbtens lipase. Symbols: A, 30°C; 0, 37°C; A, 50-Z; 0, 6O’C; n , 70°C; 0, 80°C.

(),25”C;

3 10

J. FERMENT. BIOENG.,

CHEN ET AL.

od k

30

I

6

r

6

9

10

11

12

PH FIG. 6. Effect of pH on the reactivity of A. radioresistens lipase. The relative activity (%) was calculated with reference to the maximum value.

o.oh 0

4

6 12 16 20 24 26 32 36 40 44 46 Time (h)

FIG. 4. Effect of pH on A. rudioresistens lipase fermentation. Symbols: 0, pH 7; A, pH 8; = , pH 9.

namely pH stability of the enzyme, is shown in Fig. 5. It indicates that the lipase is stable over a pH range of 6-10. Therefore, the influence of pH on lipase yield through enzyme inactivation is excluded. The effect of reactivity of the lipase at different pH values on lipase production is given in Fig. 6. The optimum pH was determined to be 10, and the lipase activities at pH 7, 8, and 9 were 20, 65, and 85% of the maximum value, respectively. The much lower lipase activity at pH 7 could cause insufficient nutrient uptake, leading in turn to starvation of the cells, and stimulates the cells to secrete more lipase for survival. Thus, a pH of 7 favors

4

5

6

7

6

9

10

11

12

PH FIG. 5. pH stability of A. rudioresistens lipase. The enzyme preparation was incubated at different pH values for 2 h at 30°C before the enzyme assay.

the Iipase production. Because the results showed that a temperature of 30°C and a pH of 7 were optimal for lipase production by A. radioresistens, the following experiments were performed under these two conditions. Since, as shown above, the addition of n-hexadecane to the basal medium resulted in a marked increase in lipase production, the roles of olive oil and n-hexadecane on lipase production are worthy of further examination. Figure 7 shows the cultivation of A. radioresistens on media containing various concentrations of olive oil and/or n-hexadecane. It can be seen that, compared with its growth on n-hexadecane, A. radioresistens grew quite fast on olive oil and attained a relatively high cell concentration; however, the lipase yield was poor. Cell lysis was obvious in a medium containing 1.5% olive oil at 20 h, probably because of saponification of fatty acids and depletion of the nitrogen source. The former, due to the reaction between fatty acids which are hydrolytic products of olive oil, and cations (e.g., sodium ion) originating from the medium itself or the NaOH added to the medium for pH control, leads to solubilization of the cell membrane. The latter, namely depletion of nitrogen, is due to the presence of a high cell concentration. In this case, however, depIetion of the nitrogen source may not be primary as concerning the sharp decrease in cell concentration. In the case of 1.5% n-hexadecane (Fig. 7), A. radioresistens grew quite slowly, but with a relatively high lipase yield. Examination of the growth curve reveals a biphasic growth pattern: the cell concentration increased slightly during the first 8 h and the second growth phase started at 60 h. It can be assumed that during the first 8 h, the cells grow using the constituents of the basal medium until the carbon source is depleted, and between 8 and 60 h, they adapt themselves for growth on n-hexadecane. It has been proposed that the uptake of hydrocarbons as microdroplets is the most common mechanism employed by unicellular microorganisms; very often the degradative microorganisms produce biosurfactant compounds in order to emulsify the freephase hydrocarbon and thereby enhance substrate availability (13). In addition, Navon-Venezia et al. (14) reported that A. radioresistens KA53 produced a high-molecular weight biosurfactant (alasan) that was capable of emulsifying long-chain aliphatics. The adaptation time can therefore be regarded as a lag period during which

LIPASE FERMENTATION

1998

311

BY A. RADIORESISTENS

9

a 7

0

35

6

12

16

24

30

36

42

46

54

60

Time(h) 30

FIG. 8. A. rudioresistens lipase fermentation with both olive oil and n-hexadecane as carbon sources. Filled symbols, cell concentration; clear symbols, lipase activity. Symbols: 1).(A), 0.2,‘4 (w/v) olive oil and 1.3,% n-hexadecane; n (a), 0.5% olive oil and 1.0% nhexadecane.

25 20 15 10 5 0

0

20

40

60

80

100

120

140

160

180

Time (h)

FIG. 7. A. radioresistens lipase fermentation in the presence of olive oil and n-hexadecane either singly or in combination, as the carbon source(s). The basal medium was supplemented with: A, 1.5% (w/v) olive oil; A, O.l,?& olive oil; 0, 0.1% olive oil and 1.5% nhexadecane; 0, 1.5% n-hexadecane.

the cells synthesize emulsifying agents to aid the uptake of n-hexadecane. Once the cells start growing on nhexadecane, lipase synthesis is induced. An interesting observation was made during cultivation of A. rudioresistens in a medium containing 0.1% olive oil and 1.596 n-hexadecane (Fig. 7). Recall that A. radioresistens needs a very long adaptation time to grow on n-hexadecane. In the medium containing olive oil and n-hexadecane, however, cell growth was almost uninterrupted, indicating that the adaptation time was much shorter or even negligible. As mentioned above, n-hexadecane requires to be emulsified before uptake by the cells. In addition, fatty acids, and mono- and di-glycerides have been identified as the biosurfactant compounds for Acinetobacter species (15). Accordingly, we may suggest that the reduction in the adaptation time of A. radioresistens for growth on n-hexadecane is due to incorporation of the hydrolysates of olive oil for emulsification of the hydrocarbon. Examination of the growth- and lipase-curves in the case of a medium containing 0.1% olive oil and 1.5% nhexadecane (Fig. 7) reveals that lipase production occurs almost simultaneously with cell growth. Recalling that olive oil leads to poor lipase production, it can be assumed that, (i) the cells can simultaneously consume olive oil and n-hexadecane, thus leading to a high volumetric productivity of lipase, (ii) the presence of n-hexadecane enhances the stability of lipase by protecting the enzyme against proteolytic degradation (lo), and (iii) the fatty acids (mainly oleic acid) produced as a result of hydrolysis of olive oil are adsorbed by n-hexadecane (forming an emulsion), thus reducing the repression of lipase synthesis by oleic acid (10). In addition to verifying

the emulsification of the sodium oleate-n-hexadecanewater system, we also examined the effect of oleic acid on cell growth and lipase production. The cultivations were performed in shake-flasks, in basal medium supplemented with various concentrations of oleic acid. It was found that although the presence of oleic acid led to good growth, the lipase yield was on the order of 0.1 U/ml (see Table 1). Compared with similar experiments with n-hexadecane, in which the lipase yields were around lOU/ml, the repression of the lipase gene of the present strain by oleic acid is evident. It is worthy of note that cell growth was very poor in the presence of 1.5% oleic acid, owing to saponification of the oleic acid. At this point, some characteristics of A. radioresistens lipase fermentation can be concluded: (i) the basal medium is the main source of nitrogen and salts; (ii) olive oil is a suitable carbon source for cell growth but not for lipase production; (iii) the presence of n-hexadecane is associated with excellent lipase production but with a long fermentation time (or low volumetric productivity); and (iv) the cells can consume olive oil and n-hexadecane simultaneously resulting in a high productivity of lipase. Accordingly, when concerning lipase yield as well as volumetric productivity, a combination of olive oil and n-hexadecane as the carbon source for lipase fermentation by A. radioresistens should be examined. Two more experiments using olive oil and n-hexadecane as the combined carbon source for A. rudioresistens lipase fermentation is shown in Fig. 8. Together with results shown in Fig. 7, a common trend for cell growth and lipase TABLE 1.

Effect of oleic acid on growth and lipase production by A. radioresistens

Oleic acid

p/ (w/v)

Cell concentration (g/l) 12h 16h 24h

Lipase activity (U/ml) 12h

16h

24h

0.1 0.3 0.5 0.8 1.0

0.3 2.0 2.8 3.1 3.9 4.9

0.3 2.1 2.7 3.0 3.8 4.1

0.3 2.0 2.6 2.9 3.1 4.1

0.14 0.20 0.18 0.21 0.38 0.17

0.15 0.22 0.21 0.22 0.27 0.14

0.12 0.19 0.17 0.20 0.25 0.17

1.5

0.1

0.1

0.1

0

0

0

0

312

J. FERMENT.BIOENG.,

CHEN ET AL.

production can be summarized. As the concentration of olive oil increases, higher extents of cell growth can be achieved, but with lower levels of lipase yield. Cell lysis is marked in the presence of high concentrations of olive oil (over 0.5%). Nonetheless, addition of olive oil to a medium containing n-hexadecane could speed the formation of lipase. The results of the aforementioned experiments indicate that the combination of 0.1% (w/v) olive oil and 1.5% (w/v) n-hexadecane is optimal for the production of lipase by A. radioresistens. ACKNOWLEDGMENT

This study was supported by the Research Grant NSC86-2745-E006-OOlR, National Science Council of Taiwan. REFERENCES

1. Crueger, W. and Crueger, A.: Biotechnology: a textbook of industrial microbiology, p. 177-178. Science tech. Madison, WI, USA (1984). 2. Lesuisse, E., Schanck, K., and Colson, C.: Purification and preliminary characterization of the extracellular lipase of Bacillus subtilis 168, an extremely basic pH-tolerant enzyme. Eur. .I. Biochem., 216, 155-160 (1993). N., Ota, Y., Miooda, Y., and Yamada, K.: lsola3. Watanabe, tion and identification of alkaline lipase producing microorganisms, cultural conditions and some properties of crude enzymes. Agric. Biol. Chem., 41, 1353-1358 (1977). 4. Nishio, T., Chikano, T., and Kamimura, M.: Purification and some properties of lipase produced by Pseudomonas fragi 22.39B. Agric. Biol. Chem., 51, 181-186 (1987). 5. Kojima, Y., Yokoe, M., and Mase, T.: Purification and characterization of an alkaline lipase from Pseudomonas fluorescens AK102. Biosci. Biotechnol. Biochem., 58, 1564-1568 (1994). 6. Lin, S. F., Chiou, C. M., Yeb, C. M., and Tsai, Y. C.: Purification and partial characterization of an alkaline lipase from

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LipA, of Acinetobacter calcoaceticus BD413 and sequence analysis of the cloned structure gene. Mol. Microbial., 15, 803-818 (1995). 13. Morgan, P. and Watkinson, R. J.: Biodegradation of components of petroleum, p. 1-31. In Ratledge, C. (ed.), Biochemistry of microbial degradation. Kluwer Academic Publishers, Dordrecht, Netherlands (1994). S., Zosim, Z., Gottlieb, A., Legmann, R., 14. Navon-Venezia, Carmeli, S., Ron, E. Z., and Rosenberg, E.: Alasan, a new bioemulsifier from Acinetobacter radioresistens. Appl. Environ. Microbial., 61, 3240-3244 (1995). 15. Makula, R. A., Lockwood, P. J., and Finnerty, W. R.: Comparative analysis of the lipids of Acinetobacter sp. grown on hexadecane. J. Bacterial., 121, 250-258 (1975).