Lipase production by Bacillus stearothermophilus S-203 in shake flasks

Zbl. Mikr o biol. 139 (1984), 61-68

l Waksrnan Institute of Microbiology, Rutgers U.S.A.]

The State University, Piscataway, New Jersey,

Lipase Production by Bacillus stearothermophilus S-203 in Shake Flasks M. S. AMMAR and L. E. McDANIEL With 3 Figures

Summary Fermentation conditions for the production of lipase by Bacillus stearothermophilus S-203 have been established in 300 ml baffled shake flasks. The optimum medium consists of yeast extract 0.3 %, ammonium acetate 0.5 %' cotton-seed soil 0.4 %, sucrose 0.5 %, Tween 40 0.4 %, phosphate buffer 0.04 M pH 6.9, and other inorganic salts. The optimum temperature is 45-55 "C, volume per flask 50 ml, shaking speed 300 rpm, and incubation time 24 hours. Under these conditions the yield of lipase was 59.5 units per ml.

Zusammenfassung Es wurden die Fermentationsbedingungen zur Erzeugung von Lipase durch Bacillus stearothermophilus ermittelt. Das am besten geeignete Medium bastoht aus Hefeextrakt (0.3 %), Ammoniumazetat (0,5 %), Baumwollsamenol (0,4 %), Rohrzucker (0,5 %), Tween 40 (0,4 %), Phosphatpuffer (0,04 M, pH 6,9) und weiteren anorganischen Salzen. Die optimale Temperatur ist 45-55 "C, del' Flascheninhalt 50 ml, die Schut.telgeschwindigkeit 300 U/Min. und die Irikubat.ionszeit 24 Stunden. Unter d iesen Bedingungen wurden 59,5 Einheiten Lipase pro ml gebildet.

Lipases are widely distributed in animals, plants, and microorganisms (DESNUELLE 1961, LAWRENCE 1967, WILLS 1965). Most of the microorganisms, which have been reported to produce lipase, are mesophilic (KOSARIC et al. 1979) and the lipases they produce are not thermostable (ARIMA et al. 1972a). The latter authors pointed out the potential value of thermostable lipase and reported the production of a thermostable lipase by a thermophilic fungus, Humicola lanuqinosa 8-38. There were later reports of lipase production by thermophilic bacteria, Bacillus stearothermophiius 8-203 (ELWAN et al. 1977) and Thermoactinomyces vulgaris (ELWAN et al. 1978a, 1978b). The optimum temperature range for activity of the B. stearothermophilus lipase was 55-60 "C, and it was stable for 30 weeks at 60 "C (ELWAN et al. 1977). KOSUGI and KAMIBAYASHI (1972) reported the production of a thermostable lipase by a mesophilic bacterium, Pseudomonas mephitica var. ll:polytica. Their attempts to obtain the production of thermostable lipases by thermophilic bacteria were not successful. In the original tests, B. stearothermophilus 8-203 produced relatively low levels of lipase (0.6 unitsjml) in stationary culture in Dux-yeast extract-tributyrin medium (ELWAN et al. 1977). We continued the work with this organism, growing it in shake flasks in order to develop a process, which could be scaled up to large fermentor size, and substituting natural oils for tributyrin since the hydrolysis of simple triglycerides does not insure the capacity to hydrolyze more complex natural triglycerides (ALFORD

62

1\1.

S. A:\ll1IAR and L.

E. McD AKIEL

and STEINLE 1966, HUGO a nd BEVERIDGE 1962) . W e rep ort her e the development of an improved practical nutri ent medium and of fermentati on cond it ions to giv e maximum yi elds of thermostable lipase.

Materials and Methods Organism The b acter ium used t h ro ughout t h is wo r k wa s isolated fr om soi l and id ent ifi ed as B. st ea rotherm oph il us by ELWAN e t a l. (197 2) a nd d esi gn a ted 8 -203. It wa s selected as t he mo st lip ol y ti c of 21 3 thermophilic b a cterial isol at es,

Chemi cals R ea gent g ra des of tributyrin, a m mo ni um acetate, and so d ium phospha te (mono- and diba sic) were o b ta ine d from .J. T. Baker Che mical Co. , Phillipsburg, New J ersey. Co t t on -seed oil (Proflo) came f ro m Traders Oil Mill Co., F o rt Wo rth, Texas. Yeast extract (A rd amine Z) was obtained fr om Y ea st Products, Inc., Clifton, N ew Jersey . Tweens were from Sig ma Che m ica l Co., St. Louis, Missouri . All other chemicals and oils were obtained from commerci al so u rces.

Growth m edium Nutr ien t Agar (Difco L aborator ies, D etroit, Michigan), su p ple m ented wi th 0.2 % s u crose , wa s used for s u bc ult ur in g the organism at 55 °C for 18 hours in P etri di sh es o r on s la n ts . The s to ck cu lt u res we re s t ored at 5 °C_

Inoculum medium D o x. y ea s t e xtract medium co nt a ine d (%, w {v) : suc rose 0. 2, N aN 0 3 0. 2, K 2HP0 4 0.1, Mg S0 4 , 7 H ~O 0.05, K Cl 0.0 5, F eS0 4 7 H 20 0.000 1, and ye ast extra ct 0 .1 in di st ille d wa t er. The p H wa s 7. 2 before auto cla ving. A loopful of ba ct eria l gro wt h fr om t he su rface of a growth medium slant wa s transferre d into 100 ml of inoculum m edium in a 30 0 ml baffled fl a sk (Bellc o Gl a ss Co. , Vin el and, N ew J ersey ) and sha ken at 300 rpm for 18-24 h ou rs at 55 °C.

Production m edium The co m posit ion o f the production m edium, u sed a t t he beg inning of th is work, was t hat of ELW AN e t a l. (1977), e xcep t th at olive oil wa s used in pl a ce o f t rib u tyrin . Its co m pos it ion wa s th e sa m e as in oculu m medium, e xcept t hat it co n taine d 0.2 % o liv e oi l a n d n o s uc rose. Preparati on o f the oil em u ls ion wa s done b y the m ethod of S TARR(1941), except tha t t he ra t io of acacia p owde r t o oil wa s 1: 1 (w{v). Th e pH wa s a dj us t ed t o 7.2. Fifty ml volumes were u sed in 300 ml baffled fl a sk s, which were capped with cot.ton -ga u ze pads, sterilized for 20 minutes at 121°C, and eac h wa s in oculated with 2 ml of inoculum c u lt u re. The flasks were sha ken for 24 hours at 55 °C and 30 0 rpm in a Psycrotherm incuba t or-s ha ker (New Brunswick Scientifi c Co ., Edison, New Jersey) .

Lipase assay Th e t ri b uty ri n clearing-zon e assay (L AWREN CE e t al. 1967), mo di fi ed for t he determination of Iipa ses f rom t he r mo p h ilic b a cteri a (Er.w AN et a l. 1977) and fr om t he rmophi lic a ctinomy cetes (EL . WAN e t a l. 197 8 a , 197 8 b), wa s used for t he assay of lipase in broth fil tra tes. Prep arati on of a ssa y pl a tes wa s ca rrie d out. as follo ws : t r ib u ty rin e m uls ion was di sp ersed into t.ris- H'Cl buffer ( 1JH 8.8 ) at 0 .2 % co n cen t rat io n , s u pp lem ented wi th 1.5 % agar , a n d ste rilized for 15 min. Aliquots of e q ual volume (20 ml ) were p oured into pla t es. H oles were made, using a s te ri le co r k borer. One -t enth m l q ua ntities of en zy me so l ution were t rans fer re d to eac h hole. P lates were incu bated at 55 °C for 6 h. D iameters of t he clear zon es we re m ea sured b y Vernier ca l ipo r . Curves were contr ucted , which sho we d a linear relation ship b etween d iameter of clea ri ng zon e a n d logarithm of lip a se co n centration , using a p an crea t ic lip a se o f 11,900 units{g (N utritio nal Bioch emi cal s Corp., Clev e land , Ohio) a s a stan d a rd.

63

Lipase Production by Bacillus st eorothermophiius

Results T he st art ing medium for t hi s work was Medium 1 (Table 1), which gave a lip ase yield of 2 .4 units/rnl . Table I. Com p osit ion of m ed ia u sed")

Medium

Sodium n itrate Y ea st ex t ra ct Olive oil K 2H P 0 4 In organic sa lt s-) Cot ton -see d oil (ed ib le) Cotton -seed oil (Profl o) Phosphate buffers) Ammonium acetat e Tween 80 Tween 40 Su crose (R eagent) Su cr ose (edible) Lip a se yi eld (units jrnl]

0. 2 0. 1 0.2 0.1

2

3

4

5

6

i

8

0. 2 0.1

0" 0.1

0.3

0 .3

0 .3

0.3

O.:J

+

+

+

+

+

+

OA

0.02

0.02 0. 5

0.1 0.02 0 .5 0.4

0.4 0 .02 0.5 0.4

0.4 0.04 0.5 0.4

0.4 0.04 0.5

0.5

0.5

29.8

no

0. 1

+

0.4

0.4

0..1-

2.4

i

13.2

.J

20.0

15.2

0.:; 59 .5

I) Co nce n tra t ions are mo lari ty for ph osphate buffer, per cen t w ]» fo r other solids, and per ce n t v [v for oils and Tween s. 2) K CI 0.05 %, l\IgS04' i H 20 0.05 %, a nd F eS0 4· i H 20 0.0001 % . 3) Ph osphate buffer pH 6.9 , 0.04l\f - NaH 2P04 . H 2 0 2.5 gfl it er an d N a 2HP0 4 • i H 2 O 5.9 g/lite r.

Typ e of oil a n d o il con ce ntr ation Th e subst it ut ion of soy bea n oil or cotton- seed oil for oli ve oil in Medi um 1 gave substantial inc reases in yi eld (F ig . 1), the highest bein g 7.2 unitsjrnl with cotton-seed oil (Medium 2). 8

i 7 ~ 6

l

l\

II \.COTTO.; OIL . ~ SEED (EDIBLE)

II

~ ~ J~r _~/'-..~ o~~~

5I 4, w I Cfl

OIL

Z

2

~: t~~_~ I ~

F ig . l. Effect of natural oils on lipa se producti on (Medium I ).

o

L i

o

_

_

- ' - -_

0 .2

---'-

0.4

--"-_

06

---'L -

0.8

OIL CONCENTRATION (%, v/v)

1.0

I

64

M. S. AMMAR and L. E. McDANIEL

14

BUFFER

o NONE PHOSPHATE • CITRATE-PHOSPHATE 6. TRIS-MALEATE o

12

2

---0

a IL-__-----'-----__------'----__-----'--5

7

6

8

pH Fig. 2. pH optimum for lipase production, using different buffers (Medium 2).

Buffering at different pH levels Medium 2 was adjusted to different initial pH levels with: 1) no added buffer (pH range 4.0-9.0), 2) phosphate buffer (pH 5.7-8.0), 3) citrate-phosphate buffer (pH 5.8-7.0), and 4) tris-maleate buffer (pH 5.2-8.6). The buffer solutions (all

0.02 M) were prepared according to the specifications of GOMORI (1955). Phosphate buffer pH 6.9 (Medium 3) gave the highest yield, 13.2 units/ml (Fig. 2). Table 2. Effect of different inorganic and organic nitrogen sources on thermophilic lipase production (Medium 3) Nitrogen sources (0.2

%)

Lipase (unitsjrnl]

%)

Lipase (unitsjml)

Organic sources

Inorganic sources Calcium nitrate Ammonium sulfate Sodium nitrate Ammonium nitrate Ammonium citrate Ammonium chloride Potassium nitrate Ammonium phosphate Ammonium acetate

Nitrogen sources (0.2

3.1 6.0 7.6 7.6 7.6 10.6 11.0 13.2 14.9

Corn steep liquor Urea Soy peptone Soybean meal

4.1 7.6 10.6 13.2

Nitrogen sources A number of inorganic and organic nitrogen sources were substituted for NaNOs. Ammonium acetate gave the highest yield (Table 2). In subsequent experiments the optimum combinations of nitrogen sources was found to be 0.5 % ammonium acetate plus 0.3 % yeast extract (Medium 4). Tests of other natural oils, a simple triglyceride (tributyrin) and a surface active agent (Tween 80).

Lipase Production by Bacillus stearothermoplcilus

65

Several natural oils and tributyrin were tested at 0.4 % (v/v) in comparison with cotton-seed oil (Table 3). A commercial grade cotton-seed oil (Proflo) was adopted as the best practical oil source. Tween 80 (0.4% v/v), added to the Proflo medium, gave a further increase in lipase yield. This became Medium 5. Table 3. Effect of natural oils, a triglyceride, and Tween 80 on lipase production (Medium 4) Substrates (0.4

% vJv)

Plant oils

Lipase units J ml

Olive oil Corn oil Peanut oil Cotton-seed oil (edible) Soybean oil Cotton-seed oil (Proflo)

10.2 12.0 ]3.2 ]4.9 ]5.1

Animal oil Neat's foot oil Marine oil Atlantic menhaden oil Cod liver oil Triglyceride Tributyrin Tween 80 (0.4%) PLUS: Cotton-seed oil (Proflo)

]5.]

9.1 14.9 16.2 9.1 20.0

Other additives In earlier experiments soybean meal, soy peptone and sucrose (Reagent grade) each gave some increase in yield in the medium used at that time. Each one individually and in all combinations of the three were used as additives to Medium 5. Sucrose (0.5 %) gave the greatest increase, to 29.8 units/ml (Medium 6). Concentration of phosphate buffer An increase in the concentration of phosphate buffer from 0.2 M to 0.4 M gave a further yield increase to 37.0 unitsjrnl (Medium 7). Other Tweens and grades of sucrose Further increases to 59.5 unitsjrnl were achieved when Tween 40 was used in place of Tween 80 and edible grade sucrose in place of Reagent grade. This became our final nutrient medium (Medium 8). Optimum physical conditions Tests run in Medium 6 confirmed that the optimum incubation temperature was 45-55 °C or higher (Fig. 3). Other tests in Medium 6 and Medium 7, respectively, confirmed that the shaking conditions, used throughout this work (50 ml volumes in 300 ml baffled flasks and a shaking speed of 300 rpm), were optimum. Incubation time Operating with the optimum nutrient medium and physical conditions, the highest lipase yields were reached within 20.5-24 hours (Table 4). [; ZbJ. }Iikrobiol., Rd. 139

66

;U. S.

24

20

A:I1MAR

and L . E .

1 i

/

/

:J 16 ~

<,

(f)

f-

z

12

~

w

(f)


8

:::::i

l\fCDAKJEL

j

)

D

D

/

I

4

o2 L5

-'-- ---'----- ---'--------'-- L----LJ 30 35 40 45 50 55

Fi g. 3 . T ernperat.ure o pt i m u m for lipase production (Medium 6).

TEM PERATU RE (DC) Table 4. L ip a se production in Medium 8 as a function of time Time

Lipase

H ours

U n itsjm l

Units jml cell dry wt .

6.5 17.5 20.5 24 30 4 1.5

6.5 57. 1 58 .3 59.5 6.5 6.3

0.7 2 4.76 4. 86 5.95 0.6 5 0 .63

o

o

o

Optimum pro cess The opt imum pr ocess is summarized in Table 5. Table 5. Optimum lipase production process

M e d ium N o. 8 (Table 1) Op erating co n d i t i o ns Volume per 300 ml baffled flask Shaker sp eed T emperature Incubation period

50ml 300 rpm 55 °C 24 hours

Yi e ld s Maximum yield obtained

59 .5 unitsjml

L ip a se Production b y Bacillue stearo thermophilue

67

Discussion Lipase producti on by microorganism s is known t o be a ffected by t he nutrients used and by cultur al conditions . Wide variations may occur wit h the same organi sm in d ifferen t media . A single medium is not adequate for lipase production by different organisms (A L F OR D and PIE R CE 1963). Th e varied res po nses of different mi croorgani sm s has not permit t ed a unified conce pt of the effect of t he medium compone nts on lipase pr oduction (NELSON 1953). This is particularly true wh en t he producing organi sm is a t her mo philic bacteri um, which has spec ific nu tri ti onal requirem ents in order t o gro w well a t high tem peratures . The best combination of n utrients and opera t ing conditions, found for lip ase product ion with B. siearothermop hilus 8 -203 in this st udy, ga ve a yie ld of 59.5 unitsjrnl in 24 ho urs . The us e of cot to n -seed oil as a su bst rate solve d t he probl em of producing a lipase, capable of atta ck ing complex natural triglycerides. Production and assa yin g of the lip ase at high temperatures confi rmed its previ ously r eported thermost abl e nature ( E L W AN et al. 1977 ). It was reported by Lru et al. (1972) that the addition of a sur face-act ive ag ent (polyoxy- ethylene nonyl ph enol ether ) gave a pronounced in crease in lipase production by N . lanuginosa 8-38 . Similar results were obtained in this st udy by adding Tween s t o th e culture medium of B . stearothermop hilus 8-203 . The effect of sur fa ce-act ive age nts is, no d oubt, due to emulsification of oil, giving it greater sur face ar ea sub jec t to enzymatic a ttack. Tween s are suitable for this purpose, sinee t hey are miscible wit h wat er and do not ge nera lly inhibit ba ct erial growth ( NIEMAN 1954). In compa r ison wit h B . stearothermop hilus 8-2 03, ot her t hermophilic organisms have been r ep ort ed to give lower yields of lipase and ha ve required ionger fermentati on times. A maximum of 30 un it s/ml were obt ained with H .lanuginosa 8-38 in 72 hours (ARD1A et al. 1972b) and 7.5 unl t s/ml with T. vulgaris in 48-72 hours ( E LWAN et al. 1978 a , E LWAN et al. 1978 b). Ac k now l edge m en t R esearch and F ello wship su pport was provided b y t he Charles and Johanna Busch Memor ial Fund.

References ALFORD, J . A., and PIERCE, D . A.: Produ cti on of lip ase b y P seud omon as fragi in a synthetic m ediu m . J. Bact. 86 (1963) , 24. - and STEINLE, E. E.: Improved pl a t in g m ethod for the det ermination of microbial lipolysis. B a ct. Proc, 80 (1966), 14. ARIIotA, K., Lru, W. H., and BE PPU, '1'.: Studies on the lipase of t hermophilic fungus Humicola la nu g in osa. Agr. BioI. Ch ern. :16 (197 2 a), 893. -- - - I sols .eion and id en ti fica t ion of the lipolytic and thermop hilic fungus. Agr. BioI. Chern. 36 (19 72 b ), 19 13. D ESNUELLE, P.: P ancr eatic li pa se, p . 129-161. In : N ORD, F. F . (ed .), Advan. Enzymo!. Vol. X X III (196 1), Interscience P ublishers, Inc., Ne w Y ork, L on d on . ELWAN, S. H ., R ADWAN , S . S., a nd A)lMAR, M. S .: Studi es on t he thermop h ilic bacteria of so me E gy p t ia n soi ls. 1. G row t h a nd nutritional requiremen t s in rela ti on to temperat ure . ZbJ. B uk k II 127 (1972), 25 1. - E r,.NAGGA R, M. R ., and AMMAR, M. S.: Characteristics of lipases in the growth f il trate d ia lysa te of Bacillus stea ro t he r mo p hi lus g row n at 55 °C us ing a t r ibutyrin-cu p plate assay. Bull. Fac. Sc i., Riyad Univ. (Sa udi A rabia) 8 (19 77), 105 . - MOSTAFA, S. A., KH ODAIR, A . A ., and ALI , 0.: L ip a se p roductivity of T he rmoactino rnyces v ulgaris . ZbL B akt. II 133 ( 1978 a ), 706. 5*

68

M. S. AMMAR and L. E. McDANIEL, Lipase Production by Bacillus stearothermophilue

F.LWAN, S. H., MOSTAFA, S. A., KHODAIR, A. A., and ALI, 0.: Identity and lipase activity of an isolate of Thermoactinomyces vulgaris. Zbl. Bakt. II 133 (1978b), 713. GOMORI, G.: Preparation of buffers for use in enzyme studies. In: COLOWICK, S. P., and KAPLAN, N. O. (eds.), Methods in Enzymol. Vol. 1 (1955). Academic Press, New York. HUGO, 'V. B., and BEVERIDGE, E. G.: A quantitative and qualitative study of the lipolytic activity of single strains of seven bacterial species. J. App. Bact. 25 (1962), 72. KOSARIC, N., ZAJIC, J. E., ABOUE, G., JACK, T., and DERSAN, D.: Lipase synthesis in hydrocarbon fermentation. Biotechnol. Bioeng. 21 (1979), 1133. KOSUGI, Y., and KAMIBAYASHI, A.: Thermostable lipase from Pseudomonas sp. Cultural conditions and properties of crude enzyme. Rep. Ferment. Res. lnst. (Chiba) 41 (1972), 31. LAWRENCE, R. C.: Microbiallipases and related esterases. 1. Detection, distribution, and production of microbial lipases. Dairy Sci. Abstr. 29 (1967), 1. - FRYER, T. F., and REITER, B.: Rapid method for the quantitative estimation of microbial lipases. Nature (London) 213 (1967), 1264. LIU, W. H., BEPPU, T., and ARIMA, K.: Cultural conditions and some properties of the lipase of Humicola lanuginosa S-38. Agr. BioI. Chem. 36 (1972), 1919. NELSON, W. 0.: Nutritional factors influencing growth and lipase production of Geotrichum candidus. J. Dairy Sci. 36 (1953), 143. NIEMAN, C.: Influence of trace amounts of fatty acids on the growth of microorganisms. Bact. Rev. 18 (1954), 147. STARR, M. P.: Spirit blue agar. A medium for the detection of lipolytic microorganisms. Science 93 (1941), 333. WILLS, E. D.: Lipases. Advan. Lipid Res. 3 (1965), 197. Eingegangen am 1. 10. 1982. Authors addresses: Dr. M. S. AMMAR, (present address) Dept. of Botany, Faculty of Science, AI-Azhar University, Medinet Nasr, Cairo, Egypt; Dr. L. E. McDANIEL, Waksman Institute of Microbiology, RutgersThe State University of New Jersey, P. O. Box 759, Piscataway, New .Jersey, U.S.A. 08854.