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Removal of triglyceride soil from fabrics by a novel lipase fromCryptococcus sp. S-2

K. Thirunavukarasu a, N.G. Edwinoliver a, S. Durai Anbarasan a, M.K. Gowthaman a,H. Iefuji b, N.R. Kamini a,*a Department of Biotechnology, Central Leather Research Institute, Adyar, Chennai 600020, Tamilnadu, Indiab National Research Institute of Brewing, 3-7-1 Kagamiyama, Higashi-Hiroshima 739 0046, Japan

1. Introduction

The detergent industry has remained the largest market forindustrial enzymes and new enzyme products are constantly beingdeveloped for use [1]. A major problem in laundries is the removalof adsorbed lipids from fabrics, which often contain oily, long-chained and water-insoluble triacylglycerols like oils. This could beaccomplished using detergent formulations containing lipolyticenzymes (lipases, formally triacylglycerol acylhydrolase, E.C.3.1.1.3), which degrade triacylglycerols into free fatty acids, di-and mono-acylglycerols, and possibly glycerol [2]. The lipasesimprove the washing capacity of detergents as well as removal offatty food stains and sebum from fabrics, which are difficult toremove under normal washing conditions [3]. Although microbiallipases from various sources have been widely used in industrialapplications, such as in food, chemical, pharmaceutical, anddetergent industries [4,5], there are still substantial currentinterests in developing new lipases, specifically with high activities

and commercially useful properties [6]. However, their majorapplication in terms of quantity is as an additive for laundrydetergents [7].

The lipase from Thermomyces lanuginosus, formerly Humicola

lanuginosa, was the first major commercial lipase used indetergents. The T. lanuginosus lipase (TLL) is produced and soldas Lipolase by Novozymes, Denmark since 1989 [8]. ThoughLipolase is stable in detergent formulations, at high pH and hightemperatures, two to three wash cycles (which includes drying ofthe textile) are needed to obtain a significant removal of lipidicstains [9]. Later, Kamini et al. [10] showed the application ofAspergillus niger lipase as an additive in detergent formulation byconventional method with a single wash cycle. Snabe et al. [2]demonstrated the removal of a triacylglycerol film from a surface,which could be dramatically enhanced in a sequential system,when pH is shifted to alkaline conditions after an initial lipolyticreaction period at or below neutral pH. They obtained significantlipid removal by carrying out the washing procedure at twodifferent pH values with the initial hydrolysis at pH 6.0 and thesubsequent rinse at pH 10.0, using triolein as model substrate.Recently, Saisubramanian et al. [11] have shown the use of A. niger

Process Biochemistry 43 (2008) 701–706

A R T I C L E I N F O

Article history:

Received 5 September 2007

Received in revised form 14 December 2007

Accepted 14 February 2008

Keywords:

Triglyceride soil

Cryptococcus sp.

Lipase

Fabrics

Oil removal

A B S T R A C T

Removal of triglyceride molecules from fabrics could be accomplished in laundries using detergents

containing lipases. The yeast, Cryptococcus sp. S-2 (CS2) produces a novel lipase, a cutinase-like enzyme

that is compatible with various ionic, cationic and non-ionic surfactants as well as commercial detergents

and oxidizing agents. In this study, the efficacy of CS2 lipase was assessed for the removal of triglyceride

soil, like olive oil from fabrics in a single wash cycle using a tool of response surface methodology (RSM),

so as to use the enzyme as an additive in laundry detergent formulations. A five-level four-factorial

central composite rotatable design (CCRD) was used to evaluate the interactive effects of detergent

concentration, lipase concentration, buffer pH and washing temperature on the percentage removal of

triglyceride soil from cotton fabric. The model suggested that all the factors chosen had a significant

impact on oil removal and the optimum conditions derived via RSM were 0.5% detergent, 1000 U of lipase,

buffer pH of 8.0 and washing temperature of 37 8C. Under optimal conditions, the removal of olive oil

from cotton fabric was 19.6 and 43.1% higher at 20 and 37 8C, respectively, in the presence of lipase, when

compared to treatment with detergent wash alone. Moreover, the present work will also serve as a

baseline of initial studies on cutinases and other related enzymes for its exploitation in laundry

detergents.

� 2008 Elsevier Ltd. All rights reserved.

* Corresponding author. Tel.: +91 44 24430273; fax: +91 44 24911589.

E-mail address: nrkamini@rediffmail.com (N.R. Kamini).

Contents l is ts ava i lab le at ScienceDirec t

Process Biochemistry

journa l homepage: www.e lsev ier .com/ locate /procbio

1359-5113/$ – see front matter � 2008 Elsevier Ltd. All rights reserved.

doi:10.1016/j.procbio.2008.02.011

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lipase in detergent formulation and optimization of washingconditions using response surface methodology (RSM), which is aneffective statistical technique, faster and less expensive method forthe investigation of complex processes [12].

The yeast, Cryptococcus sp. S-2 (CS2) produces a novel lipase[13], a cutinase-like enzyme [14] that could be effectively usedin the production of methyl esters, which are excellentsubstitutes for diesel fuel [15]. The deduced amino acidsequence of the purified protein showed remote homology toproteins of the cutinase family with the consensus sequenceGYSQG along with the catalytic triad Ser, Asp and His as that ofthe catalytic triad of lipases [14]. However, the level ofhomology of the CS2 lipase was too low to categorize it as acutinase and hence referred to as a cutinase-like enzyme (CLE),which effectively degraded the high-molecular-weight com-pound polylactic acid (PLA), a biodegradable plastic [14].Cutinases also hydrolyse fatty acid esters and emulsifiedtriglycerides as efficiently as lipases without showing enhance-ment of activity in the presence of a lipid–water interface [16–18]. The surface hydrophobicity characteristic and lipolyticactivity of cutinases have been exploited in laundry detergentformulations [19,20]. Moreover, it was reported earlier thatcutinases exhibit an ‘‘in-the-wash’’ effect, making the proteinsuitable for use in laundry detergents [21]. In 1986, Fujii et al.[22] initially showed the removal of olive oil from cotton fabricusing yeast lipase, Candida cylindracea and there is no report onthe use of Cryptococcus sp. lipase in detergent formulations.Hence, an attempt was made to determine the efficacy of CS2lipase for removal of oil from the soiled fabric using centralcomposite rotatable design (CCRD), a tool of RSM, in order to usethe enzyme as an additive in laundry detergent formulations.

2. Materials and methods

2.1. Materials

Lipase used in this study was produced from Cryptococcus sp. S-2 (CS2) and the

organism was maintained on potato dextrose agar (PDA) slants at 4 8C [13]. Triton

X-100, cetyl trimethyl ammonium bromide (CTAB) and p-nitrophenyl laurate ( p-

NPL) were purchased from Sigma Chemical Co. (St Louis, USA). Detergents used

were selected from the laboratory, viz. anionic detergent – sodium dodecyl sulphate

(SDS), cationic detergent – CTAB, nonionic detergent – Tween 80 and commercial

detergents of Indian market namely Ariel, Tide (Procter and Gamble Home Products

Ltd.), Henko (Henkel Spic India Ltd.), Rin Advanced and Surf Excel (Hindustan Lever

Ltd.). All other chemicals used were of analytical reagent grade and commercially

available.

2.2. Preparation of enzyme solution

For production of lipase, the yeast, CS2 was grown in a production medium

containing soybean oil (1%, v/v) as an inducer for 120 h on a rotary shaker at

100 rpm and at 25 8C [13]. The culture was centrifuged at 8000 rpm for 20 min and

the supernatant was concentrated by ultrafiltration using a 10 kDa cut-off

Pellicon12 Mini filter membrane (Millipore, USA).

2.3. Enzyme assay

The lipase activity was estimated using a spectrophotometric method with p-

NPL as a substrate [23]. One unit of lipase activity was defined as the amount of

enzyme that liberates 1 mmol of p-nitrophenol per minute under the standard assay

conditions.

2.4. Compatibility of lipase with surfactants and commercial detergents

To investigate the compatibility of lipase in various surfactants and commercial

detergents, respective surfactants and detergents were added to the reaction

mixture at a concentration of 7 mg/ml and assayed under standard assay conditions

and expressed as percent relative activity. To determine the stability, an aliquot of

enzyme sample was incubated with equal volume of detergent solution (7 mg/ml of

respective detergent) in 0.1 M sodium phosphate buffer, pH 7.0, for 1 h at 30 8C. The

residual activity (%) of each sample was determined and compared with the control

without detergent. The enzyme activity of control sample was taken as 100%.

2.5. Effect of oxidizing agents on lipase stability

Lipase stability in the presence of oxidizing agents was determined in 0.1 M

sodium phosphate buffer, pH 7.0, containing 0.5–2.0% (v/v or w/v) of hydrogen

peroxide, sodium perborate and sodium hypochlorite for 1 h at 30 8C and residual

activity was estimated and compared with the control without oxidizing agent.

2.6. Preparation of soiled fabric and washing solution

For preparation of soiled fabrics with triglycerides, olive oil was selected as a

model substrate as reported in earlier studies [22,24,11]. The cotton fabric

(5 cm � 10 cm) was defatted in boiling chloroform for 4 h and soiled by spotting

with 0.5 ml of olive oil in benzene (100 mg/ml concentration) twice with a

micropipette. The washing solutions (B/BL/BD/BDL) were prepared as shown in

Table 1. Solution BDL contained buffer and the detergent solution, pre-incubated at

37 8C for 10 min to which lipase solution (1000 U) was then added. The volume of

the final solution was adjusted to 100 ml by adding distilled water. Ten pieces of the

soiled fabric were put into the flask containing the washing solutions.

2.7. Washing procedure and determination of olive oil

The soiled fabrics were washed at 37 8C by shaking at 100 rpm for 20 min. At the

end of 20 min, the fabrics were removed and rinsed thrice with 100 ml of water,

each for a period of 2 min and then air-dried. Olive oil from the soiled fabrics was

extracted using petroleum ether (bp 40–60 8C) for 6 h in a soxhlet extractor. The

petroleum ether was completely evaporated and the weight of olive oil was

determined. The removal of olive oil was calculated by the following equation based

on the weight of olive oil before and after washing.

Oil removal ð%Þ ¼Wb �Wa

Wb� 100

where Wb is the weight of olive oil before washing and Wa is the weight of olive oil

after washing.

2.8. Factorial design and data analysis

Design-Expert Version 7.0.3 (Stat-Ease Inc., Minneapolis, USA) was used for

experimental design (Central Composite Rotatable Design, CCRD), regression and

graphical analysis for the results obtained. A five-level four-factorial CCRD was

employed in this study that consisted of 16 factorial points, 8 axial points and 6

centre points. Four independent variables, including detergent concentration (A,

0.25–0.75%), lipase concentration (B, 500–1500 U), washing temperature (C, 25–

49 8C), and buffer pH (D, pH 6.5–9.5) were studied at five different levels, based on

preliminary results on removal of olive oil from cotton fabric with various

detergents. Oil removal was considered as the dependent output variable and the

data on percent oil removal obtained from RSM were subjected to the analysis of

variance (ANOVA). The results obtained were fitted to a second-order polynomial

equation:

Y ¼ bo þX4

i¼1

bixi þX4

i¼1

biix2i þ

X3

i¼ j

X4

i¼ jþ1

bi jxi j

where Y is the percentage of oil removal, bo, bi, bii, bij were constant coefficients, and

xi, xij were the uncoded independent variables. Statistical significance of the above

model equation was determined by Fisher’s test value, and the proportion of

variance explained by the model was given by the multiple coefficient of

determination, R squared (R2) value. The values presented in each experiment of

this study were the averages of triplicates, unless otherwise specified.

3. Results and discussion

Lipase is used in detergent formulations to remove fat-containing stains such as those resulting from frying fats, salad

Table 1Composition of the washing solutions

Constituents Volume (ml)

Ba BLb BDc BDLd

0.1 M sodium phosphate buffer (pH 8.0) 40 40 40 40

Detergent solution (0.5%) – – 50 50

Lipase solution (1000 U) – 10 – 10

Distilled water 60 50 10 –

a B: buffer.b BL: buffer + lipase.c BD: buffer + detergent.d BDL: buffer + detergent + lipase.

K. Thirunavukarasu et al. / Process Biochemistry 43 (2008) 701–706702

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oils, fat-based sauces, soups, human sebum or certain cosmetics[25]. In the present work, the lipase was obtained from CS2using soybean oil, which is an effective and alternative inducerto triolein as reported by Kamini et al. [13]. Besides there was nosignificant difference in enzyme activity, when the lipase wasproduced from CS2 using soybean oil as an inducer and theactivity was 60.0 � 2.0 U/ml of the optimized culture medium in120 h at 25 8C. Furthermore, soybean oil, a vegetable oil, whichcould be effectively used in the production of lipase [6], is muchcheaper than the synthetic and model substrate, triolein. The crudeenzyme was concentrated by ultra filtration and the concentratedenzyme with an activity of 1000 � 40 U/ml was used for furtherstudies. The suitability of an enzyme preparation for use indetergent formulation depends on its compatibility with thedetergents over a wide alkaline pH and temperature range.Accordingly, the lipase prepared from CS2 was assessed for itscompatibility and stability in the presence of various laundrydetergents. The concentrated crude enzyme exhibited optimumactivity at pH 7.0–8.0 and at 37 8C and showed broad pH stabilitybetween pH 5.0 and 10.0 and temperature stability between 4 and60 8C. Hence, an attempt was made to use this yeast lipase as anadditive in detergent formulations.

3.1. Effect of surfactants and commercial detergents on lipase activity

and stability

The CS2 lipase was evaluated for its potential as an additive indetergents. The enzyme showed excellent compatibility andstability in the presence of ionic, cationic and non-ionic surfactantsas well as in commercial detergents (Fig. 1). CS2 lipase showed anincreased activity in all commercial detergents tested and itshowed 94 and 98% of activity in SDS and Tween 80, respectively.However, the lipase activity of Ralstonia pickettii [24] andAspergillus carneus were inhibited in the presence of SDS [26],while the activity was increased in H. lanuginosa [27]. The enzymewas stable in the presence of all commercial detergents and similarresults were reported for lipases from Pseudomonas glumae [28]and A. niger [10,11].

3.2. Effect of oxidizing agents

The lipase was highly stable towards oxidizing agents at 1.0%concentration for 1 h at 30 8C and it retained 90% of activity even at2.0% concentration of hydrogen peroxide, while the activity wasgradually decreased with increase in concentrations of sodiumperborate and sodium hypochlorite from 1.0 to 2.0% (Fig. 2).

Remarkably, the CS2 lipase exhibited better resistance towardsstrong oxidizing agents especially hypochlorite (90% activity at1.0% concentration) compared to the residual activity of Lipolase(Novozymes, Denmark), which exhibited 43% activity after 1 htreatment as reported by Rathi et al. [29]. Thus, the current thrustfor novel enzymes that tolerate oxidative stress enhances themarketable value of the present lipase.

3.3. Effect of lipase on removal of olive oil from cotton fabric

Various surfactants and detergents were first evaluated for theirefficiency in removal of olive oil from cotton fabric with andwithout lipase. The percentage of oil removed from the fabric washigher (6.6–21.1%) in the presence of lipase for all detergents(Table 2). Similar results have been reported for lipases from A.

niger [11], C. cylindracea [22] and R. pickettii [24]. Among thedetergents, Rin Advanced was the most effective detergent inremoving the triglycerides from the fabric and the removal of oliveoil with the addition of 1000 U lipase was 21.1% higher thanwithout lipase. However, only 14% of the oil was removed whenwashing was carried out in buffer solution and addition of lipase tothe buffer solution increased the percent oil removal to 32.5%under the same conditions. Since the percent oil removal was highwith Rin Advanced, a popular commercial detergent, it wasselected for further optimization studies using response surfacedesign.

3.4. Model fitting and ANOVA

Based on the results on removal of olive oil from cotton fabric(Table 2) experiments were performed to obtain a quadratic model

Fig. 1. Effect of various surfactants and detergents on lipase activity and stability.

For the control, enzyme was incubated with buffer devoid of surfactants and

detergents.

Fig. 2. Effect of different oxidizing agents on lipase stability. For the control, enzyme

was incubated with buffer alone.

Table 2Effect of lipase on removal of olive oil from cotton fabric with various detergents

Detergents Oil removal (%)

BDa BDLb

SDS 35.2 43.2

CTAB 38.4 45.0

Tween80 38.9 48.0

Tide 48.3 64.3

Rin Advanced 46.0 67.1

Surf 47.6 59.0

Henko 44.5 55.0

Ariel 46.4 63.3

a BD: buffer + detergent.b BDL: buffer + detergent + lipase.

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with four independent variables as shown in Table 3 and the dataon the percentage of oil removal by CS2 lipase are given in Table 4.Since the predicted values obtained from model fitting techniqueusing the software Design-Expert Version 7.0.3 were found tocorrelate to the observed values, the quadratic polynomial modelwas seen to be highly significant to represent the actual relation-ship between the response (oil removal%) and the significantvariables. The results of the CCRD indicated that the percentincrease in oil removal was from 7.6 to 43.1% when compared to BDwash alone (46%; Table 2). The varied nature of the resultsindicated that the interactions among the factors played a moresignificant role than the effect of individual factors alone.

The ANOVA for quadratic regression model (Table 5) showedthat the model was highly significant with an F value of 59.7 as isevident from Fisher’s F-test along with a very low probability value(Pmodel > F = 0.0001). At the same time, relatively lower value ofcoefficient of variation (CV = 2.949%) indicated a better precisionand reliability of the experiments carried out [30]. The determina-tion coefficient (R2) of the model was 0.9847 explaining 98.4% ofthe variability in the response could be accounted by the modeland indicated that the model was suitable to represent the realrelationship among the selected factors. The insignificant lack of fit

test also indicated that the model was suitable to represent theexperimental data and the final predictive equation was as follows:

Percentage oil removal ¼ 88:13þ 1:34ðAÞ þ 5:42ðBÞ � 0:68ðCÞ

þ 2:63ðDÞ � 1:62ðA2Þ � 6:02ðB2Þ

� 12:20ðC2Þ � 6:05ðD2Þ þ 0:40ðAÞðBÞ

� 0:31ðAÞðCÞ � 0:27ðAÞðDÞ � 0:49ðBÞðCÞ

� 0:97ðBÞðDÞ � 0:062ðCÞðDÞ:

It was clear that all the linear coefficients and three quadraticcoefficients were highly significant (P < 0.05) from the model andamong the four variables, lipase, pH and detergent were the mostsignificant in the process, while temperature had a lessersignificant effect on oil removal. A significant quadratic regression,insignificant lack of fit and a small total variation (1.6%), which wasnot explained by the model, suggested that the model preciselyrepresented the data in the experimental region. Moreover, thepredicted values for percent oil removal were in good agreementwith RSM plots.

Three dimensional response surface plots (Figs. 3 and 4) wereconstructed according to the above-mentioned equation. Thecumulative effect of detergent concentration and enzyme con-centration on removal of olive oil from cotton fabric at 37 8C andpH 8.0 was shown in the response surface plot of Fig. 3. Theremoval of oil was 88%, when the washing was carried out with anenzyme concentration of 1000 U and a detergent concentration of0.5%. Further increase in enzyme concentration and detergentconcentration did not show any significant effect on oil removal.An ideal detergent enzyme should be effective in removing thefatty food stains at low concentrations of detergent along withenzyme. Accordingly, our results also showed that the lipase fromCS2 was effective in removing the olive oil from the fabric usinglow concentrations of detergent. However, A. niger lipase [11] and

Table 3Experimental range of variables for the central composite design in terms of actual

and coded factors

Variable Symbol coded Range of variables

Low (�1) Mid (0) High (+1)

Detergent concentration (%, w/v) A 0.25 0.5 0.75

Lipase (U) B 500 1000 1500

Temperature (8C) C 25 37 49

pH D 6.5a 8.0a 9.5b

a 0.1 M phosphate buffer.b 0.1 M glycine–NaOH buffer.

Table 4Composition of the various runs of the central composite design, actual and predicted values of the percentage oil removal using CS2 lipase

Run Detergent (%) Lipase (U) Temperature (8C) pH Actual oil removal (%) Predicted oil removal (%)

1 0.25 500 25 9.50 58.4 59.7

2 0.25 1500 25 6.50 64.4 64.8

3 0.75 1500 49 6.50 67.9 66.6

4 0.75 500 49 9.50 60.9 60.5

5 0.75 500 25 6.50 55.7 55.4

6 0.25 1500 49 9.50 67.3 67.5

7 0.50 1000 37 8.00 88.0 88.4

8 0.25 500 49 6.50 53.6 52.8

9 0.75 1500 25 9.50 72.1 72.9

10 0.50 1000 37 8.00 88.7 88.4

11 0.50 1000 37 8.00 87.2 89.1

12 0.25 1500 25 9.50 69.8 70.0

13 0.25 500 25 6.50 54.3 53.0

14 0.50 1000 37 8.00 89.0 89.1

15 0.25 500 49 9.50 62.3 60.4

16 0.75 500 49 6.50 55.9 55.2

17 0.75 1500 49 9.50 69.7 70.5

18 0.25 1500 49 6.50 64.5 63.8

19 0.75 500 25 9.50 62.1 62.3

20 0.75 1500 25 6.50 68.7 70.1

21 0.50 1000 20 8.00 65.6 63.5

22 0.15 1000 37 8.00 80.2 81.8

23 0.50 1000 37 10.12 79.6 78.5

24 0.50 293 37 8.00 64.7 67.2

25 0.50 1000 37 5.88 69.0 71.1

26 0.50 1000 37 8.00 88.8 86.9

27 0.50 1707 37 8.00 84.0 82.5

28 0.50 1000 37 8.00 89.1 86.9

29 0.85 1000 37 8.00 86.1 85.6

30 0.50 1000 54 8.00 58.4 61.6

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R. pickettii [24] lipase were effective in removing the triglyceridesoils at 1.0 and 0.6% detergent concentrations and the increase inpercent oil removal was found to be 33 and 27%, respectively, overtreatment with detergent alone, while CS2 lipase showed anincrease in oil removal of 42.13% (Fig. 3).

The cumulative effect of temperature and lipase on removal ofolive oil at 0.5% detergent concentration and pH 8.0 was depictedin Fig. 4. The percent oil removal was optimum, when the washingwas performed at 37 8C with 1000 U of lipase concentration.However, decrease in temperature from 54 to 37 8C caused asignificant increase in percent oil removal (30.7%) at a buffer pH of8.0 and the increase in percent oil removal was 19.6 and 43.1% at20 and 37 8C, respectively, in the presence of lipase over treatmentwith detergent alone. The effectiveness of CS2 lipase to remove oilfrom soiled fabric at low temperature is one of the most desirabletraits for its use in detergent formulations because of theincreasing use of synthetic fabrics, which do not toleratetemperatures above 50–60 8C and under these conditions CS2lipase might be used for removal of oil.

The interactive effect of pH and lipase on removal of olive oil at0.5% detergent concentration and 37 8C was shown in Fig. 5. The

dome shaped plot indicated that the range and levels of variablesselected in this study were optimal and similar types of plots wereobtained with lipase catalyzed esterification systems [31] andwith synthesis of palm-based wax esters [12]. The percent oilremoval was high at pH 8.0 and 1000 U of lipase concentration.Further increase or decrease in concentration led to decrease inpercent oil removal. Though the amount of lipase used for oilremoval was slightly high (1000 U), the activity was estimatedusing p-NPL as substrate [23], which was equivalent to 75 U oflipase when it was assayed by the titrimetric method of Yamadaet al. [32]. Similar results were reported for lipase from A. niger

[11], while Hemachander and Puvanakrishnan [24] used 100 U ofR. pickettii lipase for removal of oil from fabrics and the lipaseactivities were estimated by the titrimetric method, respectively[32]. Moreover, because of the broad substrate specificity and thediversity of reactions catalyzed by lipases, it is difficult to define auniversal test for lipase activity as reported recently by Sandovaland Marty [33].

Table 5ANOVA for quadratic model for percent oil removal

Source Sum of squares Degrees of freedom Mean square F-value Prob (P) > F

Block 486.6 2.0 243.3

Model 3654.6 14.0 261.0 59.7 <0.0001a

Detergent (A) 35.8 1.0 35.8 8.2 0.0134

Lipase (B) 588.6 1.0 588.6 134.7 <0.0001a

Temperature (C) 9.2 1.0 9.2 2.1 0.17

pH (D) 138.3 1.0 138.3 31.6 <0.0001a

A^2 23.7 1.0 23.7 5.4 0.0367

B^2 326.4 1.0 326.4 74.7 <0.0001a

C^2 1339.0 1.0 1339.0 306.4 <0.0001a

D^2 329.1 1.0 329.1 75.3 <0.0001a

AB 2.6 1.0 2.6 0.6 0.4577

AC 1.6 1.0 1.6 0.4 0.5601

AD 1.2 1.0 1.2 0.3 0.6076

BC 3.8 1.0 3.8 0.9 0.3679

BD 7.3 1.0 7.3 1.7 0.219

CD 0.1 1.0 0.1 0.0 0.9066

Residual 56.8 13.0 4.4

Lack of fit 54.9 10.0 5.5 8.6 0.0513

Pure error 1.9 3.0 0.6

Corrected total 4198.0 29.0

R2 = 0.9847; CV = 2.949%.a Significant at ‘‘Prob > F’’ less than 0.05.

Fig. 3. Response surface plot showing the interactive effect of lipase and detergent

concentration on the removal of olive oil from cotton fabric using CS2 lipase. Other

variables were constant: temperature, 37 8C and pH, 8.0.

Fig. 4. Response surface plot showing the effect of temperature and lipase

concentration and their mutual effect on the removal of olive oil using CS2 lipase at

detergent concentration, 0.5% and pH, 8.0.

K. Thirunavukarasu et al. / Process Biochemistry 43 (2008) 701–706 705

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The design illustrated that the best conditions for the removalof olive oil from cotton fabric were 0.5% detergent, 1000 U of lipase,a buffer pH of 8.0 and washing temperature of 37 8C, which wasalso depicted in all the response surface plots. The present modelcould be considered suitable, since the predicted value under theoptimized conditions (88.13%) was almost the same as that of theobserved value (89.1%). The lipase from CS2 was an ideal candidatefor use in laundry detergent formulations, since it possessedconsiderable activity and stability in various surfactants, deter-gents, and oxidizing agents tested. By the use of statistical design,the enzyme concentration required for maximum oil removal wasfound to be almost the same as that of A. niger lipase [11] and thedetergent concentration was reduced by 50% along with anincrease in oil removal of 10.1% with CS2 lipase. Moreover, thepercent oil removal was 19.6 and 43.1% higher at 20 and 37 8C,respectively, in the presence of lipase over treatment withdetergent wash alone under the optimal conditions. Hence, thelipase from CS2 could be effectively used as an additive in laundrydetergent formulations for removal of triglyceride soil from fabricsin both cold and warm wash conditions.

Acknowledgements

The authors thank Dr. A.B. Mandal, Director, CLRI, Chennai,India, for his kind permission to publish this work. The financialassistance extended by the Council of Scientific and IndustrialResearch, New Delhi, India, to Thirunavakarasu K. is gratefullyacknowledged.

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Fig. 5. Response surface plot showing the interactive effect of pH and lipase on

removal of olive oil from cotton fabric at detergent concentration, 0.5% and

temperature, 37 8C.

K. Thirunavukarasu et al. / Process Biochemistry 43 (2008) 701–706706