Indian Journal of Experimental Biology Vol. 37, May 1999, pp. 481-486

Bioreactor operated production of lipase: Castor oil hydrolysis using partially-purified lipase

Chetna Sharon, Midori Nakazato, Hiroaki I. Ogawa & Yasuhiko Kato·

Dept. of Applied Chemistry, Kyushu Institute of Technology, I-I Sensui-cho, Tobata-ku, Kitakyushu-shi - 804-8550, Japan

Received 21 September 1998; revised 21 January 1999

A highly stable lipase from Pseudomonas aeruginosa KKA-5 was produced by batch cultivation technique employing shake flask and 5 L - bioreactor. The bioreactor was run at different airflow rates. Low airflow rates (I and 3 Umin), did not lead to effective growth and lipase production. Growth increased by about one order and lipase production increased by about 6 times , at an airflow rate of 5 Umin. Lipase production occurred during decelerated cell growth. A highly stable lipase was produced which ~etained its activity in the running bioreactor, even after a period of one month . This stable lipase was partially-purified using ammonium sulphate precipitation technique. Castor, oil was hydrolyzed using 300U crude and partially-purified lipase, each . Approximately 21-fold, partially-purified lipase could hydrolyze 81 % castor oil within a period of 96 hr, where as only 63% hydrolysis was obtained, in 216 hour, when ,rude lipase was used.

In recent years, much attention has been focused on enzyme stability owing to its significance in various industrial applications . Microbial lipases have numerous applications in detergent, oleochemical, pharmaceutical and food industries l

.

Recent studies have shown that an extracellular lipase gets inactivated within minutes in stirred gas/water, trioleoylglycerol/water or oleic acid/water mixtures2

. In a typical aeration system, oxygen from the air bubbles is transferred through the gas-liquid interface followed by a liquid phase diffusionlbulk transport to the cells. Production of a stable lipase that can retain its activity even in the presence of continuous air bubbles would prove of great industrial importance. Conditions suitable for lipase production in bioreactors vary from microorganism to microorgani sm:l.4. To date, lipases have been purified to execute hydrolysis of various oils5

.6

. Alkaline lipase from Pseudomonas pseudoaLcaLigenes F-III (ref. 7), lipase from Penicillium cyclopiumB, stable in pH range of 4.5 to 6.0, and a thermostable pllOspholipase D from Streptomyces Sp.9 have been purified.

Lipases, catalyze hydrolysis of oils and fats at the oil-water interface yielding free fatty acids and glycerol. By-products like salts of fatty acids are not produced. Using lipase, hydrolysis can be effectively and economically conducted under mild conditions.

* Corresponding author

Few reports have been published on castor oil hydrolysis using lipase from Humicola lanuginosa No.3 (ref. 10), Pseudomonas sp. f-B-24 (ref. 11) and Oat seeds (Avena sativa L.)12.

Castor oil contains about 90% 12-hydroxy-cis-9-octadecenoic acid, ricinoleic acid. Different derivatives of castor oil are formed owing to the presence of two reactive functional groups, a hydroxyl group and a double bond . Enzymatic hydrolysis of castor oil produces an odourless and light-colored product called Ricin'oleic acid, which is applicable in a number of cosmetic and food industries. Detailed study on castor oil hydrolysis by lipase can clarify the relationship between lipase and its substrate.

To facilitate production of a variety of products, possession of a large amount of lipase is crucial. In an attempt to accomplish this, present work deals with production of lipase from Pseudomonas aeruginosa KKA-5 by batch culture technique in shake flask and bioreactor. We also execute castor oil hydrolysis using crude and partially-purified lipase.

Materials and Methods Organism and medium-Pseudomonas aeruginosa

KKA-5 was isolated from industrial wastes of Kitakyushu city, Japan. Cells were grown on Luria Bertani plates (I % Tryptone, 0.5% Yeast extract, 0 .5% NaCI and 1.5% Agar, pH7.2) for 24 hr at 37°C. Growth media (Media A) consisting of 0.1 %

482 INDIAN J EXP BIOL, MAY 1999

KH2P04, O. I % NaCI, 4% polypeptone, 0.05% yeast ex tract and 0 .02 l}l 1M MgS04.7H20, pH 7.2 and

lipase production media (Media B) consisting of 0.1 % KH 2P04, 0.1 % NaCI, 4% polypeptone, 0.05%

yeast ex tract , 0.4% polyoxyethelene lauryl ether, 0.02% 1M MgS04.7H20 and 1% Antifoam (Silicone

KM-72F, ShinEtsu Chemicals Ltd, Japan) were used.

Analytical methods Assay of lipase-Lipase activity was measured

using 2,3-dimercaptopropan-l-ol tributyrale as the substrate (Lipase Kit S, Oainippon Pharmaceutical Co .. Ltd ., Osaka, Japan). One Unit lipase was defined as the number of Ilmol acid liberated per minute at 30°C.

Pm/ein Assay-Protein concentration was detel*rnined by Bradford's method" using Bio-Rad protein assay, Dye reag~nt concentrate (Bio·Rad Laboratories, Inc., USA). Bovine serum albumin (Bio- Rad laboratories, Inc ., USA) was used as the standard protein .

Viahle cell ('ount-Cell s were serially diluted in lOX Davis Minimal Medium {1.05% K2HP04, 0.45%

KH 2P04, 0.1 % (NH4hS04, 0.05% sodium citrate and 0.2ml 5% MgS04.7H20 , per 100 ml distilled walter},

and plated on Luria Bertani plates, followed by incubation at 37°C for 24 hr.

Culti vatiofl conditions in Shake flask-Single colony of Pseudomonas aeruginosa KKA-5 was grown in 5 ml Media A at 30°C for 24 hr, on a hot water bath . 2 ml Media A pre-culture broth was inoculated in 100 ml Media B in a 500 ml capacity shake fl ask at 121 rpm at 30°C.

COfls/ruc/ion of bioreactor-A double-jacketed 5

d e

L- reactor having 3.5 L working volume (Methane Fermentation Bioreactor, Sibata Hario Glass Co., Ltd., Japan), was filled with 3.43 L Media Band sterilized at I i I °C for 20 min (Fig. I ) . Temperature of the culture media in the reactor was controlled at 30°C by a water bath (Low Temp Thermostatic Water Bath, TRL-112, THOMAS Kagaku Co. Ltd., Japan) . Continuous oxygen was supplied by an Air pump (APN-2ISNV-I, Iwaki Air Pump, Iwaki Co., Ltd ., Japan), via a cylindrical glass tube carrying Silica gel­Blue (5-10 mesh, Nacalai Tesque, Japan). Silica gel was changed every 48 hour. Temperature, pH and airflow rate were continuously monitored and data was collected using a specified Bioreactor-Instrument Box (Model 2P-IO, Sibata Hario Glass Co., Ltd ., Japan). To evaluate lipase activity, protein content and viable cell count, samples were collected using a sampling-outlet located on top of the reactor lid, via a Peristaltic Pump (Micro Tube Pump MP-3, EYELA, Tokyo Rikakikai Co., Ltd., Japan) .

Cultivation conditions in bioreactor-A single colony of Pseudomonas aeruginosa KKA-5 was inoculated in 80 ml Media A in a conical flask, at 121 rpm for 24 hr at 30°C, on a rotary shaker (Biorotary Shaker l3R-200L, T AIYO, Japan). 70 m1 of this pre­culture was re-inoculated in the reactor containing Media B. Samples were analyzed for li.pase activity, protein content, 00 at 600nm and viable cell count, every 24 hr.

Lipase purification-Ammonium sulfate was added to Media B culture supernatant (hereby called 'Crude lipase') to 45% saturation, at 4°C. The precipitate obtained was collected, centrifuged at 10,000 rpm for 20 min at 4°C and redissolved in a.OIM potassium

Fig. I-Constructi on of the bioreactor: a-air-pump, b-Silica gel, c-Airflow rate detector, d-pH meter, e-Temperature detector, f­Double-jacketed reactor, g- Autoclaved disposal tank containing distilled w'ater in which exhaust air from the reactor flows out, h­Peri staltic pump. i-Sample collector, j-Hot water bath.

SHARON et al. : STABILIZED LIPASE PRODUcnON & CASTOR OlL HYDROLYSIS 483

phosphate buffer (PH 8.07), followed by overnight dialysis at 4°C, against the same buffer. This preparation is hereby called 'Partially-purified lipase'.

Hydrolysis of Castor oil~astor oil (Nacalai Tesque Inc., Kyoto, Japan) was hydrolysed using crude and partially-purified lipase preparations. The reaction mixture {0.25g castor oil, Irnl O.OIM CaCh, lOrnl lOmM potassium phosphate buffer (PH 8.07) and the lipase preparation (300U)}, was placed in IOOrnl air-tight Erlenmeyer bottles and. incubated at 30°C on a reciprocal shaker and titrated every 24 hr. 20rnl acetone/ethanol (1: I, v/v ) was added to stop the reaction. Liberated fatty acids were titrated with O.IM alcoholic KOH, usmg phenolphthalein as an

10000.-------------~-----B-la-n-k~

Ii 2% , 8000 :J

.0 6000 'S;

.~ 4000 Q) V) a 2000 ....

.....l

.. 5%

00 48 96 144 192 216 240

Incubation time (l1ours) Fig. 2-Effect of various concentrations of anti foam on lipase production.

indicator. Percentage of castor oil hydrolysis was calculated using the following relation:

Hydrolysis (%) = (Acid value/Saponification value)xIOO

The saponification value of castor oil was 181 (value being measured by the supplier).

Results Effect of Antifoam on lipase production-It is

known that frothing can promote air-oxidation of protein (e.g., its sulfhydryl group) and surface denaturation l4

• To avoid these possible occurrences, an antifoam Silicone KM-72F was added in the lipase production media. In case of regulated batch culture in shake flask, within the ~ange of 0-5% antifoam, lipase production increased linearly with increase in antifoam concentration. Due to economical reasons, 1 % anti foam was used in all the experiments. As seen from Fig.2, anti foam possibly acted as a lipase inducer when supplemented in Media B. A similar phenomenon was reported in Pseudomonas nitroreducens nov. var. thermotelerans l5

•

Lipase production by shake flask technique-On the second day of cultivation, an increase in pH to 9.1 was followed by steady production of lipase (Fig. 3). Maximum growth and lipase activity were recorded on the second and sixth day of cultivation, respectively . Lipase was stable for 5 days, propounding that an alkaline condition was necessary to induce lipase production. It was also seen that lipase was produced during decelerated cell growth .

8oo0~----------------~ 3 10

-• :::: 6000 c::::: ::>

f 4000 ·u CO Q)

rJ 2000 a. ::J

2

1

o~~.-~,+~,-~~~~

2SJ o 5 10 15 20

Days

--a -T"" -~

... T""

C 0 :J 0r-

O X u

0000 .~ Q) --0 .... a.

Q5 E 0) u -.

>. E Q) c -§ .Q

0

000 co --0 :> u ....... I-

0

Fig.3-Lipase production in Shake fl ask culture.

9

~ 8

I Cl.

7

6

484 INDIAN J EXP BIOL, MAY 1999

On the eleventh day, the cells perished followed by sudden decrease in protein concentration.

Lipase production in bioreactor-Batch culture technique performed in shake flask was scaled-up in 5 L- bioreactor. The reactor was run at three different airflow rates. At low air flow rates, 1 and 3 Umin, maximum growth was observed on the fourth day, while lipase activity increased to about 800 and 1000 UIL pn the third day of cultivation, respectively (Fig. 4).

.-E' :::>

~ .:; n re 0) en re a. ::J

remained stable for a period of over a month, in the running reactor (Fig. 5) . In the present experiment, due to limitation of the reactor capacity, we could not try this experiment at further higher airflow rates. This has to be investigated by an experimental study.

Purification of lipase-On the day of maximum lipase activity, I L culture was collected from the bioreactor and centrifuged at 6000 rpm for 15 min at

1200 8 8.5

1000 8.0 6 :::::::

~ 800 _S!

c:o :::l .... 7.5 8x

600 4 Q)E rJ~

7.0 0) c: 400 J3.Q

reo >~ 2

200 6.5

a a 6.0 a 48 96 144 192

Hours

Finally, we ran the reactor at an airflow rate of 5 limin. On the second day of cultivation, maximum growth was observed along with an increase in pH (similar to results of shake flask cultivation). Compared to the results of low airflow rates, growth increased by about one order (data not shown) while lipase production increased by about 6 times. When the reactor was run at low air flow rates ( I and 3 limin) , initial pH (6.0) did not increase to the alkaline range on cultivation of the culture (data not shown) . Lipase activity constantly increased till 14th day of cultivation. Fifteenth day onwards, a slight loss in lipase activity was observed but the enzyme Fig.5-Effect of an airflow rate of 5 Umin on lipase production

Table I-Summary of partial-purification of Pseudomonas aerugillosa KKA-5 lipase

Steps Total protein Lipase activity Specific activity Yield Purification (mg) (U) (U/mg protein) (% lipase activity) fold

Crude lipase 1299.98 6526.80 5.02 100.0

Dialyzed ammonium sulphate 1125.00 4451 .28 41.62 68.2 8.29 precipitate dissolved in buffer A

1200 8 8.5

1000 8.0 .- 6 ~ ~ c::: 800 0

=> Co 7.5 ........ :::JT"""

~ 8x S-.;:; 600 4 ~E ~ I u u--ca >- 7.0 a.

Q,l Q,le -0 en 400 .0_

~ cao .- u :.::J 2 >-

6.5 200

0 6.0 48 96 144 192

Hours Fig.4. Effect of an airflow rate of I and 3Umin on lipase production open and closed symbols determine readings fo r I and 3Umin, respectively [square-lipase activity (Ufl); circle· viable cell count (colony / mix 1010

) and triangle-pH)

~ I a.

SHARON et al.: ST ABIUZED LIPASE PRODUcnON & CASTOR OIL HYDROLYSIS 485

100.---------------------------. --0---- Crude lipase

til 80 ·til >. "0 .a 60 >. :r:: Q)

~ 40 .... t:: Q) U I-<

~ 20

o

---.-.- Partlally-putified lipase

o 48 96 144 192 240

Reaction time (hours) Fig. 6-Hydrolysis of castor oil using 300U of crude and partially-purified lipase. For reaction mixture and conditions see materials and methods.

4°C. Precipitate fonned by addition of ammonium sulfate was dialyzed overnight against Buffer A in order to remove the ammonium ions. Approximately 21-fold purification of lipase was achieved (Table 1).

Cas/or oil hydrolysis-Castor oil was hydrolysed using crude and partially-purified lipase, 300U each . Using crude lipase as the lipolytic source, only 63% hydrolysi s was obtained in a period of 216 hr, where as about 81 % hydrolysis was attained within 96 hr, whel] partially-purified lipase was used (Fig. 6). It must be viewed that in 96 hr period barely 50% of hydrolysis was percieved, when crude lipase was used.

Discussions In case of Pseudomonas aeruginosa KKA-5,

similar culture conditions were suitable for consistent growth and lipase production, while conditions that supported high lipase production were different from those supporting high biomass production, as noticed in Pseudomonas aeruginosa MB 5001 (ref. 16). The cause for increase in lipase activity on addition of an anti foam is not yet clearly understood. It is conceivable that its addition improved the oxygen­transfer coefficient, which influences the cell membrane and stimulates lipase excretion l5

.

Possibility that operation of the reactor at higher aeration and agitation rates could result in cell lysis, leading to loss in lipase production could not be ruled out. It has been reported that purified lipase loses its

actIvity drastically on addition of an air bubble2.

Although presence of antifoam can destroy air bubbles in the reactor, some amount perpetuates throughout the cultivation period. It should be noted here that present lipase from Pseudomonas aeruginosa KKA-5 retained its activity in-spite of continuous addition of oxygen in the fonn of air bubbles, throughout the cultivation period.

All the above observations suggest that apart from prevalent parameters like pH an~ temperature, growth and lipase production depended on the following four limitations: (i) Lipase production was induced when maximum growth was observed on the second day of cultivation; (ii) Furthennore, the amount of air supplied to the cells played an important criterion in production of lipase. High aeration, which also caused better agitation, was essential to produce lipase; (iii) An increase in pH to the alkaline range within 48 hr of growth was mandatory to obtain maximum lipase activity and (iv) Low air flow rates leads to oxygen starvation, which gives a negative impact on lipase production and growth. Such a condition must be avoided.

The cells excreted lipase in late or post exponential growth phase. It should be pointed that lipase retained its activity over a period of one month and bacterial colonies were also surviving. This apparently suggested that surviving cells were living on bacterial debris as their major source of carbon.

Partially-purification of Pseudomonas aeruginosa KKA-5 lipase by ammonium sulfate precipitation lead to an increase in specific activity of lipase. Many accompanying proteins and impurities present in the crude lipase were also removed by purification. The lipase produced was in an appreciably active stage and hence could react the oil molecules more specifically, as compared to the crude lipase preparation. Thus the lipase in partially-purified state could effect castor oil hydrolysis. In the present article we have shown that 300U partially-purified lipase could hydrolyse about 81 % castor oil within a period of 96 hr. We have earlier demonstrated 90% hydrolysis of castor oil using lOOU of lipase (purified to homogeneity)l7. This difference in percentage of castor oil degradation could be due to lower specific activity of partially-purified lipase. It must be noted that inspite of the presence of impurities and lower purification fold of Pseudomonas aeruginosa KKA-5 lipase, this partially-purified lipase could hydrolyse about 81 % of castor oil. It has been reported that

486 INDIAN J EXP BIOL, MAY 1999

Pseudomonas sp. f-B-24 lipase, puritied to homogeneity, could hydrolyse merely about 80% of castor oil 1 1 •

Conclusions In conclusion, we have presented construc[ion and

operation of a bioreator. Using batch culture technique we obtain production of a highly stable lipase from Pseudomonas aeruginosa KKA-5 . In case of a reactor having 5 L capacity, an airflow rate of 5 Umin gave appreciable growth and stabl{: lipase production. The lipase was stable in the running bioreactor for a period of over one month. Owing to its high stability this lipase can be used in many biotechnological industries. Further studies on prodtJction of lipase by continuous culture tet::hnique are under progress.

The present lipase from Pseudomonas aeruginosa KKA-5, in crude and partially-purified state could efficiently hydrolyze castor oil. This lipase has a future not only in biotechnology industries but is also applicative in chemical industries. With this illl mind, we therefore propose that in order to procure. higher amount of hydrolysis, the concentration of lipase can be modified to further enhance the rate of castor oil hydrolysis.

Acknowledgement Thanks are due to Mr. Tsuyoshi Yarnakido,

Kyushu Institute of Technology, for his valuable

discussions. This research .is partly supported by a Grant-in-Aid for Scientific Research from The Ministry of Education, Science, Sports and Culture.

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