Indian Journal of Experimental Biology Vol. 37, August 1999, pp. 818-824

Isolation and identification of alkaline thermostable lipase producing microorganism, cultural conditions, nutritional requirements and

some properties of crude enzyme

Nityananda Ray, Lalitagauri Ray, B N Srimani & Pari mal Chattopadhyay*

Department of Food Technology and Biochemical Engineering, Jadavpur University, Calcutta 700032, India

Received 18 January 1999; revised 30 March 1999

A bacteri al strain isolated from spoil ed coconut and identified as Corynebacterium species was found capable of producing alkaline thermostable extracellular lipase. Li pase activi ty of the strain was increased 2.3-fold by mutagenic technique using a chemical mutagen NTG (N- methyl-N'-nitro-N-nitrosoguanidine). Optimum temperature, time and pH for enzyme substrate reaction were found to be 60° C, 10 min and 8.0 respectively. Co mmon surfactants have no or very little inhibitory effects on the acti vity of enzyme, whereas bile salts are inhibitory. The maximum lipase production was achieved at 33°C, pH 8.0 on 72 hr of fermentation. Optimum age and volume of inoculum were 24 hr and 2% respecti vely. Superior carbon and nitrogen sources fo r lipase production were starch (2%), ammonium chloride (nitrogen level 0.1 OOgi l OOml) and peptone (nitrogen level 0.320g I 100 m1 ) in combination respectively. The optimum C/N ratio was found to be 4.76 : I. Glycerol and oil when used as sole sources of carbon low levels of enzyme was produced. The maximum enzyme activity obtained was 28.0 lUI mi .

Lipases have been used industrially for decades, but the range of application and the volume of manufacture have been limited . Recently, however, many new potenti al applications have been proposed, the modification of fat s and oils to produce higher value products and the use of lipase in detergents being of particular significance i

. Demand for microbial lipases have increased due to their specificity of reaction , mild conditions required for reaction , stereospecificity and less energy consumption than conventional chemical methods. The importance of heat stable lipase in biotechnology is growing rapidly, and at present a large number of lipases are commercially available2.4. Microbial lipase has wide application in the processing of food, leather, domestic and industrial wastes and in cosmetics, detergent and pharmaceutical preparations.

This paper deals with the isolation, identification, and mutation of an alkaline thermostable lipase producing microbial strain and some properties of the crude extracellular enzyme. Studies have also been made to measure the optimum environmental conditions, nutritional requirements and CI N ratio for the maximum enzyme production using the mutant strain .

*Correspondent au thor

Materials and Methods Isolation, media and culture conditions-

Corynebacterium species (C7) was isolated from spoiled coconut using plate and dilution technique5

.

Plate and maintenance media were composed of olive oil 2%, (NH4) 2 S04 0.5%, (NH2) 2 CO 0.2%, MgS04, 7H20 0.1 %, yeast extract 0 .05% and agar 2%. The mixture was heated and emulsified and the pH was adjusted to 8.0. Fermentation medium and inoculation medium were composed of soluble starch 2% , peptone 2% , KH2P04 0.5 %, (NH4)2S04 0.1 %, (NH2h CO 0.1 %, MgS04, 7H20 0.1 %6. Identification of the selected strain was performed according to Bergy 's Manual of Determinative Bacteriology7.

Preparation of crude enzyme--Inoculum was prepared by transferring one loop - full of cells from slant culture to the inoculum medium (50 mlJ 250 m] Erlenmeyer flask) and incubating the flask at 33° ± 2°C in a rotary shaker at 120 r.p.m. for 24 hr. Fermentation medium (total volume 50 ml in 250 ml Erlenmeyer flask ) was inoculated with 2% (vi v) inoculum and incubated for 72 hr under the same conditions. After 72 hr of fermentation culture fluid was centrifuged at 4000 r.p .m for 15 min. Lipase activity of the supernatant was measured.

Enzyme assay -Unless otherwise stated, all experiments were run in triplicate. The reaction

RAY et. at.: ALKALINE THERMOSTABLE LIPASE FROM CORYNEBACTERIUM 819

mixture containing 5 ml olive oil emulsion , composed of 25 ml o li ve oil and 75 rnl 2.0% polyvinyl alcohol solution , 4 ml 0 .2 M Tris-buffer (PH 8.0 ), I rnl 110 mM CaCh and I ml enzyme solution8 was incubated at 600 C for 10 min . Controls containing inactivated enzyme were treated similarly . Immediately, after incubation, the emulsion was destroyed by the additi on of 20 mt of acetone-ethanol (l : I) mixture and the liberated free fatty acid was titrated with 0 .02 N sodium hydroxide . One unit of lipase was defined as the amount of enzy me which liberated I Ilmol of fatt y ac id I min . I rnl of 0 .02 N sodium hydroxide is equi valent to 100 Ilmol of free fatty ac id .

Induced mutation and selection of high yielding strain- The induction of mutants was carried out by using N-methyl-N' -nitro-N-nitiosoguanidine at final concentrati on ranging from 10 - 100 Ilg/ rnl in 15 rnl of 24 hr culture broth and incubated at 33°C at 120 r.p .m. Samples were collected at different time interval s, viz. 1,2,3 ,4,5 and 24 hr. Isolates were collected using plate and dilution technique using the same pl ate medium. Isolates (82) were tested for lipase acti vity.

pH stability and thermostability of crude enzyme -The volume of crude enzyme solution was reduced to one third using a vacuum evaporator (400 C) . I rnl concentrated enzyme was mixed with 2 rnl buffer (at different pH ) to restore the initial concentration of the enzyme at eac h pH . The enzyme solution was kept at 4°C for I hr and then enzyme activity of each sample was determined. The thermostability of the enzyme was determined by incubating the enzyme solutions at optimum pH va lues and a range of temperatures for 1 hr. Acti vity of each sample was then determined as usual.

Results Isolation and identification of a lipase producing

strain -46 bac terial isolates were collected from soil , spoiled coconut , o il residue and soil samples mixed with coconut oil and kept for one month, using plate and dilution technique. Of the II isolates capable of producing lipase, one strain (C7) having enzyme acti vit y 7. 3 lUI ml was selected.

Identificati on of the selected strain was carried out according to Bergy's Manual of Determinative Bac teri olog/. The media for different biochemical tests were prepared according to Harrigan et at Results are shown in Table I . On the basi s of these taxonomical studies, the strain C7 was identified as Corynebacterium spec ies (Fig. 1 A).

Induced mutation - During this expriment 82 isolates were collected, of which 12 strains were low lipase yielding, 4 strains were similar in activities to the parent strain and 66 strains showed high lipase yielding activity . Isolate mlJ showed 2.3-fold higher activity (16 .65 IUI ml) compared with the parent strain C7 (7 .3 IUI rnl ). The selected mutant strain of Corynebacterium species m n was used for further studies (Fig. B).

Some properties of crude enzyme Effect of temperature, incubation time and pH -

Optimum temperature , pH and reaction time were determined by varying on! y one parameter and keeping the others constant. Optimum pH, temperature and reaction time were found to be 8.0 (Fig. 2), 60°C (Fig. 3) and 10 min (results not shown)

Fig. I-Photomicrograph of isolated strain (A) and mutant strai: (ml3) (8 ) of Corynebacterium species Magnification x 1000.

820 INDIAN J EXP BIOL, AUGUST 1999

Table I -Taxonomical characteristics of strain C7

Parameters (a) Cellular characlerislics Morphology

Stain ing characteri sti c

(b) Cllltllwi characteristil 's ,

utrient agar co lonies

Nutri ent broth (s tationary condit ion )

Fermen tati o n mcdi um

Characteristics

: Straight or slightly slender rods 1,5 x 0,6 ~ , occurring singly, Non motile, : Gram positive, some cells stained unevenly givi ng a beaded appearance, Not acid fast. Non spore former.

:. Round (2 mm), opaque, smooth, off white with entire edge, : Poor growth , ring formation, pellicle formation. o ff fl avour, yell owi sh pigmentation,

(sta tion ary co nd iti on) : Fair growth , turbid , no sedimentati on, ring formation , no pellicle formation, brown pi gmentati on off fl avour,

(c) PhY.I' iolol: iCII I characteristics

Growth facto rs : Optimum growth at 33°C. Range

28°-37°C Toleranct; Amillonia fro m

arginine Arginine used as sole

source o f energy Prot ein liq ue fac ti on

(gelatin ) Catalase reac ti on Indo le producti on Litmus milk test Hydrolys is o f Urea Starch Casein Nitrate reduced to

nitrite Methyl red test

: No growth at 6,5 % NaCI at pH 9,6 : Positi ve

: Positive

: Growth, but no liquefaction

: Positi ve : Positive : No acid : Negati ve : Positive : Negative : Positive

: Negative

respecti ve ly , Three buffers, phosphate (PH range 5.7 to 7.0), tri s-HCI (PH range 7.5 to 9.0) and glycine -NaOH (PH range 8.5 to \0.0) were used for this purpose.

pH stability and thermostability -The crude lipase preparation was most stable at pH 8.0 (Table 2) at 4°C. But it is more or less stable up to 60°C (Fig, 3). So the enzyme may be referred to as thermostable.

Effects of suljactants - Effects of different kinds of surfactants on enzyme activity are shown in Table 3. Surfactants are used in the reaction mixture at a 0.4% concentration during enzyme assay. Table 3 shows most of the surfactants have no or minimum inhibition on lipase activity .

Effect of bile salts -Effect of bile salts were studied using cholic acid, sodium deoxycholate, sodium taurocholate, dehydrocholate (concentration used 0.25% and 0.5%) in the enzyme substrate reaction mixture. Bile salts showed inhibitory effects on lipase activity (Table 4) .

Effect of environmental conditions on production of lipase - Lipase production was carried out using different volumes of medium, viz. 40, 50, 60 and 70 ml ' using 250 ml Erlenmeyer flask, other conditions remaining the same. Samples were collected at 24 hr interval and tested for enzyme activity. Volume of medium and fermentation time had a significant effect on maximum production of lipase (Fig. 4). Maximum lipase production was occurred using 50 ml medium at 72 hr of fermentation.

Effect of initial pH of the culture medium was studied using a wide range of pH .and the maximum lipase production was obtained at pH 8.0 (Fig 5) lipase production was carried out at different

.->

0-0 Phosphate buHer

I:r-Il Tris - HCt bulle r

0-0 Glyc ine NoOH buller 30

v

~20~ o a.

10L-~ ____ ~ ______ L-____ ~ ____ -L~

5.5 60 7 ·0 8 ·0 9 · 0 10 ,0

Reaction pH

Fig .2-Effect of pH on enzyme -substrate reaction .

30 E --:::> ~

~ 20 ,-> , -

o

.. 10 .. a a. ,--'

OL-~ ____ ~ ____ ~ ____ ~ ____ ~

Storage temperature ("C I ~ Reaction temperature I'C) 0---0

Fig. 3-Effect of reaction temperature on enzyme substrate reaction and thermostability of crude lipase.

RAY el . al.: ALKALINE THERMOSTABLE LIPASE FROM CORYNEBACTERIUM 821

temperature, viz. 28°, 33°, and 35°C. Maximum lipase activity 24 IUI m! was achieved by growing Corynebacterium species at 33°C (Fig. 5) after 72 hr of fermentation. Effect of age of inoculum (range used 16 to 48- hr), volume of inoculum (range used 0.5% to 4%) on lipase production were also studied most suitable conditions were 24 hr and 2% respectively (Fig. 6) .

Nutritional fac tors affecting lipase production -Carbohydrates, oli ve oil and some salts were tested as carbon source for lipase production, other ingredients of the medium remaining the same. The results are shown in Fig 7. From the experiment, inulin was

Tablc 2-Determination of pH stability

Storage

pH

5.7

6.0

6.5

7.0

8.0

8.6

9.0

10.0

Buffer used

Phosphate buffer

(0.2 M )

Tis-buffer (0.2 M )

Gl yci ne

NaOH buffer

<02 M)

Enzyme activity ( lUI ml)

8.0

8.0

4.0

5.0

30.0

10.0

4.0

0.0

Table 3 - Effect of surfactants on enzyme-substrate reaction

Surfactant used Relative activity (0.4%) (%)

I. Control 100 2. Triton x 100 100 OJ. Sp:m 40 110 ·1. Tween 40 100 ) . Trut.:izin.l 70 6. Tween 60 140 7. Tween 20 80 8. Tween 80 100 9. Brij 35 80 10. Benzal konium chloride 10

Table 4--Effect of bile salts on enzyme activity

Bile salt used Concentration Enzyme activity (% ) (lUI ml )

Control 24.0 Cholic acid 0.5 8.0

0.25 9.0 Sodium deoxycholate 0.5 3.0

0.25 6.0 Sodium taurocholate 0.5 3.0

0.25 :.0 Dehydrocholate 0.5 20.0

0.25 20.0

found to be the supenor and starch as the second superior carbon sources respectively. As inulin is very costly for large-scale fermentation, starch was selected as carbon source for further study. Then different levels of starch were used to determine the optimum concentration (2.0%) required for maximum enzyme production (Fig. 8). Simple nitrogen sources like (N~h S04 , NH4CI , (~hHP04 , (N~)H2P04

, N~03 , urea, sodium nitrate (N2 level 21.2 mg/lOO rnl for A and 70 mg/lOOrnl for B) etc . were used along with peptone (nitrogen level 320 mg/1OOmI) in presence of urea (nitrogen level 68.0mg/100rnl) (A) and in absence of urea (B) (:able 5). NH4CI (nitrogen level 70 mg/lOOrnl) along with peptone (nitrogen level 320 mg/lOOrnl) having total nitrogen level 390mg/100mI in absence of urea gave the m3ximum production of lipase. Different complex nitrogen sources like peptone, tryptone, beef extract, yeast extract, com steep liquor, soybean meal, malt extract were used with (NH4)2S04 and urea in the fermentation medium for lipase production. Then the' experiment was carried out without adding (N~)2S04 and urea in the fermentation medium. The results are shown in Table 5. Lipase production was totally stopped when complex nitrogen sources were used as sole source of nitrogen, in case of peptone it is only 5 IUI ml (Table 6) . Then complex nitrogen source (having nitrogen level 320 mg/ 100 rnl) were used with (NH4hS04 (nitrogen level 21.2 mg/lOO rnl) and urea (nitrogen level 68 mg/ IOOrnl). Lipase activity was obtained only when tryptone (14.0 IUI rnl), soybean meal (20.0 IU/rnl) and peptone (22.0 IUI rnl) were used (Table 6).

30 E --~ ,. 20 >

v o

: 10 o Q.

~

40 ml *---1(

50 ml 0--.,..0

60 ml lJr--t.

70 ml ~

O~~==~ __ -L __ ~U 12 48 96

Time of f.rmentotion (hI

Fig. 4 -Effect of volume of medium and fermentation time on lipase production.

822 INDIAN J EXP BIOL. AUGUST 1999

Since the combination of NH4Cl and peptone gave the maximum production of en2!.yme, this combination was chosen as the superior nitrogen source for enzyme production . The nitrogen level was 390 mg! 100 ml where contribution of NH4CI was 70 mg / 100 ml. Now keeping the nitrogen level in peptone constant , nitrogen level in NH4C1 was varied together with the variation in starch concentration (Table 7) . From the ex periment, nitrogen level of NH4CI was fixed to 100 mg II OOml. The same experiment was

E 30 :3

:> ·20 ~

v a .. .. a a. ~

10

6·0

25

pH Ii-Il

Temperature 0-0

In i tia I pH

I I

30 35

T e m per a t u r e (·C )

9·0

r ig .5 -Effect of initi al pH and fe rmentation temperature enzyme production.

Age 01 the inoculum 0----0

Inoculum concentration 6-----6

-! 30 :::> ....

.-:>

.. ... o a. .-oJ

20

10

5L-____ ~ ____ ~ ____ ~ ____ _L ____ ~

o 10 20 30 40 50 Age 01 inoculum (h)

o 2 3 4

Inoculum concentration (% )

Fig. 6 -Effect of age of inoculum and inoculum concentration on lipase production

carried out keeping nitrogen level of NH4CI (100 mg / looml) constant, that of peptone was varied from 270 mg / 100 ml to 370 mg! 100ml together with variation of starch concentration. The results are shown in Table 7 . From the above experiments, optimum CIN ratio and nitrogen level for the production of lipase were found to be 4.76 : 1 and 420 mg / 100ml respectively.

Discussion Lipase production by the strain Corynebacterium

species was found to be active at alkaline pH and high temperature. Moreover the enzyme IS

Table 5- Effect of simple nitrogen source on production of enzyme

Simple nitrogen source

nitrogen level for A 21.2 mg/IOO ml B 70 mg I 100 ml

Ammonium nitrate Ammonium chloride Sodium nitrate Di-ammonium hydrogen phosphate Ammonium di­hydrogen phosphate Ammonium sulphate Urea

Lipase activity (IU / ml ) in presence of

Peptone 2% (N2 level 320 mg 1100 ml )

+ Urea 0.1 % (N2 level 68 mg 1100 ml)

A 4.0 14.0

22.0

2.0

Peptone 2% (N2 level 320 mg 1100 ml )

B

26.0

22.0

Table 6-Effect of complex nitrogen source on enzyme production

Complex nitrogen source used with nitrogen level

A 320 mg 1100 ml B 390 mg 1100 ml

Yeast extract Malt extract Com steep liquor Beef extract Tryptor.c

Peptone Soybean meal

Enzyme activity (lUI ::11) in presence of ammonium sulphate 0.1 % (N2 level 21.2 I 100 ml)

+ UreaO. I% (N2 level 68 mg I ml)

A

14.0

22.0 20.0

Enzyme activity (IU I ml) in absence of (NH4hS04

+ urea

B

5.0

RAY et. at.: ALKALINE TIlERMOSTABLE LIPASE FROM CORYNEBACTERIUM 823

thermostable. These characteristics are important in biotechnology. Again common surfactants have little or no effect on the enzyme. Due to above mentioned characteristics this enzyme may be used in detergent formulations. Mutation of the strain was successfully carried out using chemical mutagen NTG (N-methyl­N'-nitro-N-nitrosoguanidine) as the activity of the enzyme was increased 2.3-fold. Bile salts have inhibitory effect on enzyme activity. These studies were performed because bile salts were found

. I f 10 stlmu atory or some enzyme system .

Regarding nutritional requirements, inuline was selected as superior carbon source but it is tou costly for commercial use and so starch (2 %) was used for further studies . Maximum lipase production occurred at 72 hr of fermentation . Lipase production was very little when olive oil and glycerol were used as sole source of carbon . Lipase furmation IS often suppressed by the presence of monosaccharides or glycerol 10. Appreciable lipase production occurs after glucose has been exhausted from the medium and growth has almost ceased II. Regarding nitrogen source, combination of ammonium chloride (nitrogen level 100 mg I 100 ml) and peptone (ni trogen level

35

30 f-.Ii:

" ~ c -0 2 - ~

E -=> f- '" - ."

" 0 - 25 ~ • c -0 .- 0 0 ->-

- .. .-> • oS)

~ ~

0 ~ VI u 20

0 - -• 2 .-.. c 0 c c 0

Q)

'" 15 0 .-~ .... 0. ....

-' 0 -Q<

10 r- ~

• .. • 0 .. ~

" 0 , c 5 '" .-

oS)

n 0 ~

« 0

• .. 0 -v 0 .....

r-

320 I 100 ml) was used for maximum production of the enzyme. The maximum enzyme activity was 28.0 IUI ml at the optimum C/N ratio 4.76 : I. Complex nitrogen sources cannot be used as sole source of

Table 7-Effect of C / N ratio on the production of lipase.

Starch Peptone Ammonium CI N Enzyme cone. cone. chloride ratio acti vity (%) (mg / 100 ml ) cone. (IU / ml )

(mg / 100 ml ) 320 30 4.28 : 1 8.5

1.5 320 70 3.84: I 6.0 320 100 3.57 : I 5.0 320 30 5.71 : 1 12.0

2.0 320 70 5.12: 1 25.0 320 100 4.76: I 28 .0 320 30 7.14: I 6.5

2.5 320 70 6.41 : I 12.5 320 100 5.95 : I 6.5 270 100 4.05: I 7.0

1.5 320 100 3.57 : I 5.0 370 100 3.19 : I 4.0 270 100 5.40 : 1 22.0

2.0 320 100 4.76: I 28.0 370 100 4.25 : I 19.0 270 100 6.75 : I 18.0

2.5 320 100 5.95:1 14.5 370 100 5.31 : I 11.0

c .--:> c

;::.

• .. 0 c E

• 0 .. .r. 0 Cl< -.. v .. .. :> - 0 ... 0 -- " • 2 II >- ! ~ >

v >< r- 0 .--.. 0 ~ - 0 0 .. - r- - • ~

0 0 . - v - • - " v 0 v - 0 . >-0 n 0 0 -

~ z z " Cor bon source (2%)

Fig. 7 -Different carbon sources used for lipase production.

824 INDIAN 1 EXP BIOL, AUGUST 1999

Soluble storch concentrotion ("!o)

Fig. 8-0ptimum soluble starch concentration for lipase production.

nitrogen due to presence of some unknown factors detrimental to lipase production .

Acknowledgement The authors gratefully acknowledge the financial

support of Department of Biotechnology,

Government of India m carrymg out this research work.

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