Indian Journal of Experimental B iology Vol. 40, November 2002, pp. 1280- 1284

Staphylococcus warneri BW 94-A new source of lipase

G S Walavalkar & M M Bapat*

Departmel1l of Biochemistry, The Institu te of Sc ience, Madam Cama Road, Mumbai 400032, India

Received 9 April 2001; rel'iseti 5 Allgllst 2002

StaphylococclIS isolated from a common Ind ian weet viz. basundi was tested for its ab ilit y to produce lipase. The co l­orless zone of hydrolysis around the colony grown on Baird Parker agar con taining egg yo lk produced extrace llular lipase. Colony morphology, coagulase production, haemolysis, ac id production in carbohydrate med ium and enzyme activ ity stud­ies showed that the organism was STaphylococclis 1I'1Imer i. Growth of S. It'omer i was obtained after I I hr at 37"C, pH 7.5, while the maximum producti on of lipase was obtained at 30°C at pH 6.5 after 9hr of incubation. Agitation did 110t increase li pase production. A sudden fa ll in the acti vity of l ipase was noted after II hr. Add ition of sucrose which is a growth stimu ­lant for Staphylococclls, did not stimu late produc ti on of lipase by these organisms. Al so, add ition of oleic acid, Tween 80 or ethanol did not stimulate format ion of l ipase.

A large number of microorgani sms are capable of us­ing natural oi ls and fats as carbon source for their growth and produce extracellular lipase (glycero l es­ter hydrolase 3.1 . 1.3) , that catalyses the hydrolys is of tri glycerides to free fatty acids, parti al glycerides and glycero l. Microbial li pases constitute an important group of enzy mes because of versatility of their appli­cation and ease for mass production. At present the main microbial sources2 of lipase are yeasts Candida cylandraceae and Saccharolllysis /ipolytica. Lipases from fungi3, Aspergillus niger, Aspergillus oryzae, Penicillium cye/opium, Mucor lI1 ehii , Humicula lal/u­ginosa, Geotricul11 candidulIl , are well studied. ft has been reported that a few bacteria e.g . Chromobacte­riu l1I viscosum4, Acinetobacter calcoaceticLls, Bacillus sllbti/is5 can produce lipase, but widely studi ed bacte­ri a fo r lipase producti on is Pseudomonas Spp6.

In the present paper, all attempt has been made to isolate and identi fy a new strain of Staphylococci from common Indian sweet, basundi , and optimi zed culture conditi ons required for max imum yield of en­zy me from thi s organism. Effects of chemical stimu­lants on producti on of lipase by the new strain have also been di scussed.

Materials and Methods Isolation of lipase producing Staphylococci fr0 11l

basundi-Basundi (eatable sweet) sample acquired from a loca l sweet shop was diluted I : 10 with di s­tilled water. 0.5ml of this sample was spread on Staphylococcus agar 110 (HiMedi a, India). lates

COiTespondent author: E-mail : mmbapat @bom7.vsnl.net. in

were incubated at 37°C fo r 24 hr. C lonies were ob­served under mi croscope fo r confirmation. Stappyla­cocci co lonies isolated on Staphylococclls agar 11 0, were spot inocul ated on Baird Parker agar (HiMedia, India) containing (gil ) tryptone, 10; beef extract, 5; yeast ex tract, I ; sodium pyru vate, 10; glycine, 12; lithium chloride, 5; agar, 20; p H 7.0 ± 0.2, supple­mented with egg yolk and checked for the zone of hyd rolys is around.

Characterization of the strain - Sl ide and tube co­agul ase tests7 were carried out fo r the detection of bound and free coagulases. Broth culture of S. al/rellS was used as coagulase positi ve control and culture of S. epidermidis was used as negati ve contro l. Clear­ance zone around the colony after incu bation at 3]DC fo r 24 hr was checked fo r the presence of haemolysins by spot inocul ating the organism on blood agar21. Other tests e.g. colony morphology, phosphates pro­ducti on , urease and nitrate reductase were carried out as per standard procedures to identi fy Staphylococcl/s Sp.7 Acid production from carbohydrates was studi ed by adding appropriate amount of steri le carbohydrate stock solution to a sterili zed purple agar base medi um, containing bromo cresol purple indicator (Hi-Medi a, India) to have fin al carbohydrate concentration ( I %)7. Cultured streaks were prepared by inocul ating 0.5 to 1 cm long streak on the surface of agar plate with loop full of an overnight culture. Cul tu res incubated at 37°C and examined at 12,24 and 72 hI'.

Enzyme assay-Trioline (Flub :) substrate was prepared as descri bed by Peled and Krenz8

. Trioline ( I g) was mi xed with triton X-I 00 (30 ml ) and healed

WALAVALKAR & BAPAT: STAPHYLOCOCCUS WARNER! BW 94-A NEW SOURCE OF LIPASE 1281

at 55°C in a water bath . NaCi so lution (9%; 200 ml) heated upto 55°C was then added gradually with stir­ring. p H was adjusted to 8 by NaOH (0.0 1N). For each assay 6.25 ml of emu lsion was incubated at 3rC fo r 10 min. Broth (0.25 ml) contain ing enzy me was added to emul sion. Change in pH was recorded. Fatty ac ids liberated were titrated against 0.0 1 M, NaOH , p H 8 (Vs). For blank , boiled broth (0.25 ml) was added to emulsion (Vb). Amount of enzy me was cal­

culated as-U/mL= (Vs - Vb) X 103 /0.25.

Preparation oj siandard inoculum and growth curve-For mai ntaining culture on brain heart infu­sion (BHI ) agar slant, a loop fu ll of culture was inocu­lated in sterile BHI broth and incubated at 37°C for 24 hr under non-shaking conditi ons. Cells were har­vested by centri fugation at 3000 rpm for 30 min. The cells were repeatedly washed with no rmal ste rile sa­line and suspended in sterile distilled water just before inoculation, so as to give an inoculum of approx i­

mately 3 x 104 cellslml as determined by opac ity stan­dards') (at 660 nm).

BH I broth (200 ml ) was taken in two Erlenmeyer flasks. Medium was adjusted to pH 7.5 in one and to 6.5 in the other flask . Medium was inocul ated with 2 ml of standard inoculum and incubated at 37°C. Broth (5 ml) was removed from the flask under asept ic con­ditions after every half an hour interval and the opti cal density of the broth was measured at 660 nm.

Effect oJphysical Jaclors Time- BHI broth (20 ml) was inocul ated with

0.25 ml of standard inoculum, incubated at 37°C, pH 7.5. Production of enzyme was monitored fo r every hour. Similarly, enzyme production was also moni ­tored at 30°C, pH 6.5.

p H-rIasks contai ning 20ml of steril e BHI broth with pre-adjusted pH 5.0, 5.5, 6.0, 6 .5, 7 .0 , 7 .5, 8.0 were inoculated with 0.25 ml inoculum and incubated at 37°C. Production of enzyme in each flask was de­termined.

Temperature-Flasks contai ning 20 ml of BHI broth, pH 7.5, were separately inocu lated with 0.25 ml of standard inoculum and incubated at 1 r, 20°, 28°,30°, 37°,42°, 55°C for the period determined to

e optimum for production of lipase.

Inoculum size-Two sets of flasks (5 in each set) contai ning 20 ml of sterile BHI broth were prepared . In l SI set pH was adj usted to 7.5 and in other set pH was adjusted to p H optimum for max imum lipase

production . Different concentrations (0.2, 0.4, 0.6, 0.8, 1.0 ml) of standard inoculum was added to both the sets. The set with pH 7.5 was incubated at 37°C, while other set was incubated at the temperature op­timum for the production of enzyme. Afte r optimum period of time, production of enzyme was determined .

Agilatiol1-Two sets of flasks contain ing 20ml of sterili zed BHI broth one with pH 7.5 and other with p H optimum for max imum lipase production were inoculated with opt imum amount of inoculum as de­termined in previous experiments. The sets were sub­jected to ag itation (75 , 100, 150 and 200 rpm), one set was maintained at 37°C, while the second set was kept fo r ag itati on at optimum temperature determined for enzy me producti on. Production of enzyme was determined after optimum period of time.

Effect oj chemical Jactors-Effect of sucrose, oleic ac id , Tween 80 and ethanol was stud ied on pro­duction of lipase at the conditions opt imum for growth as well as at the conditions optimum for lipase production. Different concentrati ons of sucrose (5, 10, 15 , 20, 25%), and oleic acid, Tween 80 and ethanol (I, 2, 3, 4, and 5%) were studied in flasks containing sterilized BHI broth.

Detection oj protease actil 'ity-Coloni es of S. lVarneri BW 94 were spot inocu lated on milk agar. Zone of hydrolys is arou nd the colony indicated pro­duction of protease 10. S. warneri were grown in BHI broth at 30°C and pH 6.5 and the amount of proteases production was determined after every hr from 7 to 13 h · . b II r, uSll1g case ll1 as su strate .

Results and Discussion Lipase produc ing strain isolated from basundi

when subjected to various characterizat ion test. (Table I) was identi fied as Staphylococcus warneri fo llowed by Bergey's manual 7

. The stra in was named as S. warneri BW 94.

Cellular growth curve of S. warneri BW 94 at 37°C, pH 7.5 and at 30°C, pH 6.5 has been shown in Fig. I. This Figure also indicates the protease produc­tion by the organism at pH 6.5 which was the opt i­mum pH for maximum enzyme activity (Fig. 2a). Though the range for cellular growth of S. warlleri BW 94 was wider (PH 4 to 8) with the optimum pH between 7.4 -7.6 (data not shown), the range of li pas~

production was detected between pH 6.0 to 7.5 (Fig. 2a). It has been shown that initial pH of the growth medium affects the lipase producti on l2

.

Conditions optimum for lipase production by S. warneri BW 94 were 'different from those for cellular

1282 IND IAN J EX P BIOL, NOVEMBER 2002

Table I- Identifica tion of Staphylococclls spp. producing lipase

Character

Colony morphology

Size

Pigment

Growth

Aerob ic

Anaerobic

Coagul ase

Haemolysis

Phosphates

Urease

Nitrate reductase

Acid prod uction (aerobically) from

Maltose

D-Turallose D-Mannose

D-Ribose

RafTi nose

Lactose D-rructose

D-Trehalose

D-Manni tol

D-Xylose

Xy litol

D-Cell obiose

Sucrose

Obse rvat ion

I mm

Ni l

+ +

+

+ +

+ + +

+

growth of Staphylococci (Fig. 2) . Under the optimum conditions (30°C, pH 6.5) fo r lipase production, or­ganism entered exponenti al growth phase after 7 hr.

The pH of the broth decreased as the incubation time increased. Rate of decrease in pH was high when the organism entered later part of exponenti al phase of growth . It was observed that after 13 hr, pH of the medium became 5.5 when initi al pH was 7.5 , and 4.5 when initi al pH was 6.5 (data not shown) . Decrease in pH of the culture medium could be due to produc­tion and secretion of metabolites in the medium which in turn would reduce the acti vity of lipase in the broth .

S. warneri BW 94 was able to produce enzyme be­tween 17°-41°C (Fig. 2b). Standard inoculum (0.2 ml ) was sufficient to produce max imum enzy me (Fig. 2c) . No apparent positive correlation was found between total number of cells and lipase production under op­timum condition fo r cellular growth or under those fo r lipase production. Max imum lipase production was observed at 30°C, at a temperature which was lower than the temperature required (37°C) for the optimum

0.8

O.G

f ~ ~ 0.4 0 0

0.2

0.0

r--------- - -------,

--0-. pH 6.5, 30 'c . 0.10 ---pH 7.5,37 'c - pH 6.5, 30 'c

° ,0 i5

Time (hr)

::; 0.08 E

~ >.

:~ 006 g

:x '" '" e

0.04 Cl.

0.02

Fig. I -Growth cu rve and protease production by of S. lVam eri BW 94. [(- e -) Growt h at pH 6.5, temp. 30 °C; (- . -) Growth at

pH 7.5 , temp. 37°C; and (-0-) Protease product ion at pH 6.51.

growth . It was noticed that higher temperature had adverse effect on lipase acti vity in the broth. Reduc­tion in lipase acti vity at hi gher temperature may be due to inact ivation of enzyme at higher temperature. S. warneri BW 94 produced max imum amount of lipase after 9 hr of incubati on (Fig. 2d). After 10 hr, the decrease in the acti vity of the enzy me in broth could be due to producti on of protease by the organ­ism at this stage (Fig. 1). Proteases can inactivate li ­pases in their nati ve form. Lipolytic acti vity depends on amount and the acti vity of lipase present in cul ture supernatant. Amount of lipase present at a given mo­ment woul d depend on the di ffe rence between amount produced and destroyed. Lipase has been observed in S. aureus after 18 hr I 3

.14

, whil e Saccharomycis lipoli­tica exhibits max imum lipase activity on day 4 and Micrococus caceolyticus on day 2 and 3 of incuba­tion 13. Penicillium. simplicium take 48 hr to produce max imum amount of lipase IS, whereas Bacillus licheriform is and Sfaphylococus spp need 7 and 2 days respecti vely for max imum lipase productionl3

.

S warneri produced 240 U/mL lipase within 9 hr of incubati on. Continuous shaking decreased the activ ity of lipase rapidly indicating that aeration had adverse effect on lipase production from this organism (data not shown).

Production of lipase was found to decrease on addi­tion of sucrose to the medium (Fig. 3a). Similar re­sults have been obtained wi th Pseudomonas aergi­nosa as addition of glucose decreases lipase act ivit/ 6

.

Under the conditions optimum fo r cellular growth as well as lipase production, addition of oleic ac id de­creased lipase ac ti vity in S. warneri (Fig. 3b). When

WALA VALKAR & BAPAT: STAPHYLOCOCCUS WA RNERI BW 94-A NEW SOURCE OF LIPASE 1283

the concentration of oleic acid in the medium was 3%, enzyme production was nearly same as that produced in absence of oleic acid . Sudden decrease was ob­served with further increase in oleic acid concentra­tion. Oleic acid or oli ve oil has no effect on lipase producti on by Acina/abacter Sp1 7. Lipase producti on level markedly increases by several fatty ac id esters (e.g. triglycerides, Tweens) and represents the key mechanisms fo r making a fatty acid from the carbon source available to the cell during growth on such compounds. It is reported that the level of production

~~~------------------------~ (0 ) --Growth

150

:::J' 100 E 2-.~ .2: U co

~ 50

~ c::

u.J

o

5 e pH

~[jJXlSt

250 ,,-:-----________________ . ___ --,

(C )

--*- Growth 200

150

0.1 0.2 0.3 OJ 0.5

Inoculum size (mL)

of enzyme with free fa tty acid is higher than that ob­tained with tri glycerides, inspite of the fac t that thei r presence makes lipolysis unnecessary'6. But in casc of S. warneri lipase production did not increase with additi on of oleic acid .

A few lipases are di stributed in the cell bound and/or cell free form. It has been reported that mem­brane bound lipases are well extracted in presence of

17 detergents . Tweens are also capable of complete disassociation of lipase from cell surface and thus the addition of Tweens should increase the acti vity of the

2~ T7~----------------------------~ (b) L'

------ Ipa se

2W

150

100

~

0

I

20 3G 40 50 0

Temp ( C) 250

(d)

200

150

100

50

0 __

o 2 6 8 10 12 • 14 16

Tlme(hr)

Fig. 2 - Effcc t of (a) pH ; (b) temperature ; (c) inoculum size; and (d) time on lipase production and growth of S. warneri BW 94.

1284 INDIAN J EXP BIOl, NOVEMBER 2002

200 (a) -.- Lipase

-- Growth

150

100

50

0 0 5 10 15 20 25

Cone. of Sucrose (%)

200

--' E

:::J 150

Z. .;; '13 100 ro <1l E >. N C W

0 0 2 3 4 5

Cone. of Oleic acid (%)

250

200

150

100

50

0 0 2 3 4 5

Cone. of Tween 80 (%)

Fig. 3-Effect of (a) sucrose; (b) oleic ac id ; and (c) Twecn 80 on prod uction of lipase and growth of S. IVa l'll ('ri BW 94.

enzy me in the medium by releasing the enzyme into the supernatant of culture broth , However, production of lipase by S. warneri BW 94 showed no significant increase with addition of Tween 80 to the medium (Fig 3c).

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13 Vadehra 0 V & Harman L G, Isolation and purification of Staphylococcal lipase, Microbiology, 15 (1967) 295.

14 Vadehra 0 V & Harmon L G , Characterisation of purified Staphylococcal lipase, Appl Microbiol, 15 (1967) 480.

15 Helena S, Heinrsch L F, Helmut E, Menge U & Schmid R, PenicillulII SilllplicissilllUIII, Biochilll Biophys Acta, 1124 ( 1992) 253.

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