Process Biochemistry 37 (2002) 637–641

Production of lipases by Rhizopus oligosporous by solid-statefermentation

Ikram ul-Haq a, Shumaila Idrees a, M. Ibrahim Rajoka b,*a Biotechnology Research Laboratories, Department of Botany, Go�ernment College Lahore, Lahore, Pakistan

b National Institute for Biotechnology and Genetic Engineering (NIBGE), P.O. Box 577, Jhang Road, Faisalabad, Pakistan

Received 26 January 2001; received in revised form 23 May 2001; accepted 24 June 2001

Abstract

The present investigation describes the production of lipase by solid-state fermentation. Ten mould cultures were screened forthe production of lipases. Rhizopus oligosporous GCBR-3 supported maximum production of lipases (48.0 U/g substrate) underoptimum environmental and cultural conditions such as selection of efficient organism from the available microorganisms,selection of almond meal as the best substrate among different substrates, inoculum size, extractants/diluents and incubationperiod. The organism secreted the enzyme extracellularly. To study the kinetics of enzyme production, the organism was grownunder optimum growth conditions for enzyme production. The lipase value supported by the organism and calculated as units pergram substrate per hour was greater than the values reported by other workers. This organism can be exploited for large-scaleproduction of enzyme for commercial purposes. © 2002 Published by Elsevier Science Ltd.

Keywords: Lipases; Rhizopus ; Meals; Oils; Sugarcane; Baggase

www.elsevier.com/locate/procbio

1. Introduction

Lipases (triacyl glycerol acyl hydrolases) are hy-drolytic enzymes, which catalyze the hydrolysis of theester linkage of long chain acylglycerols at an oil waterinterface. Lipases also bring about bioconversion reac-tions viz., interinterifaction, esterifaction, alcoholysis,acidolysis and aminolysis [1]. They are implicated in theprocesses and products of food and flavoring industries.They are also used in the manufacture of fine chemicalswith special emphasis on pesticides, detergents, cosmet-ics, biosensors, and pharmaceuticals. They are also usedin the waste management and improving tanning tech-niques [2]. Many bacteria, fungi and yeasts have beenused for the production of lipases [3–5]. Mould pro-duces lipases of high catalytic potential and stability [6].Kundu and Pal [7] described a new method for theisolation of lipolytic fungi from soil on oilmineralmedium, which was spread on silica gel plates.

Lipases are synthesized in the presence of inducers.The inducers of lipases are wheat bran, rice bran,dextrins, sugar cane bagasse, coconut cake, olive oilcake, and gingley oil cake [2,4,8–10]. Lipases can beproduced by solid-substrate or submerged fermenta-tion. Because of simplicity and being economicallymore attractive, solid-state fermentation has been usedby many workers for the production of various en-zymes [11–13]. Bhushan et al. [3] reported the produc-tion of lipases from an alkalophlic yeast species bysolid-state fermentation using rice bran and wheat branas alternative cheap substrates. The substrate was en-riched with urea (1%), peptone (3%), maltose (5%) andolive oil (10%) as carbon and nitrogen sources. Christenet al. [14] studied Rhizopus delemar using polymericresin (amberlite), dextrin, maltose or glucose as sub-strate. Destain et al. [15] reported production of extra-cellular lipases by Yarrowia lipolitca at optimalincubation temperature of 37 °C. Essamri et al. [16]studied Rhizopus oryzae at optimum temperature 30 °Cand pH 8.5. Lipase activity at 48 h was 12-fold greaterthan that obtained with the initial medium, used forlipase production. Among different fungi, Rhizopusoligosporous showed higher productivity of lipases [17].

* Corresponding author. Tel.: +92-411-651-475; fax: +92-411-651-472.

E-mail address: ibrahimrajoka@yahoo.com (M.I. Rajoka).

0032-9592/02/$ - see front matter © 2002 Published by Elsevier Science Ltd.

PII: S 0 0 3 2 -9592 (01 )00252 -7

I. ul-Haq et al. / Process Biochemistry 37 (2002) 637–641638

The present study deals with the production of li-pases by R. oligosporous by solid-state fermentation.Different environmental variables were employed tomaximize the lipase production.

2. Materials and methods

2.1. Microorganism

Cultures of R. oryzae, Mucor lipolytica, R.oligosporous, Aspergillus niger, Penicillium sp., Mucorsp., R. nigrican, R. arrihizum and A. wentii were col-lected from the culture collection of this institute. Theywere selected on the basis of availability and satisfac-tory activity of lipase.

2.2. Culture maintenance

The isolated cultures were maintained on potatodextrose agar medium [13].

2.3. Inoculum preparation

The spore inoculum was used in the present study.Spores from 3 to 5-days-old slant culture were used forinoculation. The spore suspension was prepared in ster-ilized 0.005% Monoxal O.T. (dioctyle ester of sodiumsulfosuccinic acid). An inoculum needle was used tobreak clumps of conidia.

2.4. Fermentation technique

Ten grams of wheat bran were transferred in 250 mlof cotton plugged Erlenmeyer flask and moistened withphosphate buffer. After sterilization, the flasks wereinoculated with 1.0 ml of fungal inoculum and incu-bated at 30 °C for 72 h. After 72 h, 100 ml of theextractant was added to each flask and rotated at theincubator shaker (200 rpm) for 1 h; the contents of theflasks were then centrifuged (4000×g) and substratefree enzyme extract was used for the estimation oflipase.

2.5. Sterilization

All the media, unless otherwise stated, was sterilizedat 15 lb pressure (121 °C) for 15 min.

2.6. Lipase assay

Lipase activity was determined after Kundu and Pal[7]. One unit of activity is defined as the amount ofenzyme, which release 1 �mole fatty acid per min perml under specified assay conditions.

Lipase activity=�V×N×1000

Vsample

60

where �V=V2−V1 is the volume of NaOH usedagainst control flasks, V2 the volume of NaOH usedagainst experimental flasks, N the normality of NaOHand Vsample is the volume of enzyme extract.

2.7. Kinetic studies

The kinetics of the rate of lipase production wasstudied after Pirt [20].

3. Results and discussion

3.1. Screening of stock cultures

Ten moulds available in the culture collection (Table1) were screened for lipase activity, following growth on10 g wheat bran/flask and incubated at 30 °C for 48 h(found optimum). Of these cultures, R. oligosporousproduced maximum lipase activity (30�2.1 U/g sub-strate). Three cultures gave 7.0�1.5–11.6�0.5 U/gsubstrate, four cultures produced 13.3�0.6–20.0�2.4U/g substrate. R. oligosporous provoked the maximumlipase activity and substantiated the work of Toshiko etal. [17] and Korn and Fujio [18]. The enzyme activitieswere greater than the nine cultures studied by Rivera-munoz et al. [8] and Cordova et al. [5].

Table 1Chemical analysis of substrates used for lipase productiona

Carbon source Moisture (%) Crude protein (%) Oil (%) Carbohydrates (%) Ash (%)

Almond meal 11.5�0.4a 22.0�0.6c 4.0�0.1a 40.0�1.1c 7.0�0.2bc9.0�0.3b 21.3�0.6cCoconut meal 2.0�0.1b 6.0�0.1c47.3�1.3b9.0�0.3b 7.5�0.2bMustard meal 30.0�1.0d1.8�0.1b34.0�1.0b9.0�0.3b 46.0�1.2aSoybean meal 1.0�0.0c 30.6�1.1d 6.4�0.2bc8.0�0.2c 11.1�0.3eRice husk 1.0�0.0c 10.2�0.3e 20.6�0.6a

11.5�0.3a 14.0�0.4dWheat bran 4.0�0.1a 54.0�1.5a 6.2�0.1cHS HS HSSignificance at P�0.05 HS HS

a Each value is average of three replicates. � , Stands for S.D. among replicates. Values followed by different letters differ significantly atP�0.05. HS stands for highly significant.

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Table 2Comparative growth and substrate utilization kinetic parameters of R. oligosporous following growth on different substrates in solid substratefermentation under optimum fermentation conditionsa

Yx/s (g cells per g substrate utilized) Qs (g substrate utilized per l/h)Carbon source Qx (g cells per l/h)

0.65�0.02aAlmond meal 0.35�0.01a0.35�0.01a0.55�0.02bcCoconut meal 0.33�0.01ab0.30�0.01b0.51�0.02c0.27�0.01b 0.31�0.01abcMustard meal0.63�0.02aSoybean meal 0.28�0.01bc0.24�0.01b0.60�0.02ab0.26�0.01b 0.25�0.00Rice husk

0.29�0.01bWheat bran 0.63�0.02a 0.31�0.01abcSignificance at P�0.05 HS HS HS

a Each value is an average of three replicates. � , Stands for S.D. among replicates. Values followed by different letters differ significantly atP�0.05. HS stands for highly significant. Qx is the volumetric rate of cell mass production, Yx/s the cell yield coefficient, and Qs is the volumetricrate of substrate consumption.

Table 3Comparative product formation kinetic parameters of R. oligosporous following growth on different substrates in solid substrate fermentationunder optimum fermentation conditionsa

Yp/s (IU/g substrate utilized) Yp/x (IU/g cells)Carbon source Qp (IU/1 h)

48.0�2.1aAlmond meal 100.0�3.0a2.23�0.08a35.0�2.5c 90.0�2.7cCoconut meal 1.87�0.07b23.0�1.0f1.75�0.06b 60.0�1.8eMustard meal

1.07�0.06cSoybean meal 28.0�0.84d 80.0�2.4d0.87�0.03cRice husk 26.0�0.1e 60.0�1.8e

46.0�1.2b2.20�0.4a 95.0�2.1bWheat branSignificance at P�0.05 HS HS HS

a Each value is an average of three replicates. � , Stands for S.D. among replicates. Values followed by different letters differ significantly atP�0.05. HS stands for highly significant. Qp is the product formation rate, Yp/x the specific product formation, and Yp/s is the product yieldcoefficient.

3.2. Selection of substrate

Different agricultural byproducts including almondmeal, coconut meal, soybean meal, wheat bran, ricehusk, sunflower meal, cotton seed meal and mustardmeal were evaluated to select the best supporter oflipase production by R. oligosporous GCBR-3 (Table2). Maximum activity occurred on the almond meal(48.0�2.1 U/g substrate). Almond meal provides allrequired carbon, nitrogen and contained sucrose, gum,asparagines and proteins. Cordova et al. [5] reportedlow value of lipases (2.67–20.24 U/g substrate). Butwhen the medium was supplemented with mixed carbonsources, the product yield increased to 79.6 U/g sub-strate. This work is not in agreement with work re-ported by Rao et al. [19] and Kamini et al. [10] whoused other substrates and obtained higher activities oflipase.

3.3. Effect of temperature

The fermentation medium along with the inoculumwas incubated at 25, 30, 35, 40 and 45 °C (Fig. 2).Maximum enzyme was supported at 30 °C. At lowerand higher temperature, the maintenance energy re-

quirement increase and less product formation occurs[20]. Different workers have used different cultivationtemperatures and obtained higher values calculated asunits per liter [1,3,4,12]. When values were divided bythe time of fermentation, all values were lower thanthat for the isolate used in these studies.

3.4. Effect of inoculum size

The data of Table 3 show the effect of inoculum sizeon enzyme production. Maximum production (48�2.1U/g substrate) occurred with 1.0% inoculum size.Lower or higher inoculum size did not support higherenzyme activity. This may be due to the fact that theoptimum level of mycelium produces an optimumamount of enzyme for which 1.0 ml of inoculum issufficient. This is in agreement with the finding ofToshiko et al. [17]. With the increase in mycelial mass,the production of enzyme declined due to exhaustion ofnutrients in the fermentation mash.

3.5. Selection of diluents/extractant

The data obtained revealed that enzyme extractionwas maximum with phosphate buffer. This may be due

I. ul-Haq et al. / Process Biochemistry 37 (2002) 637–641640

to the fact that the phosphate buffer extracted theenzyme from all fermented meal by increasing thepermeability of cell membrane.

3.6. Rate of enzyme synthesis

The kinetics of lipase production was studied usingR. oligosporous following growth on almond meal un-der optimized conditions and results have been shownin Fig. 1. After 48 h, maximum synthesis of enzyme wasachieved. At lower or higher time of cultivation,product formation declined. Initially the organism wasadapting to the experimental conditions, but after 48 hthe enzyme lost some of its activity. A similar deactiva-tion during liquid production of lipase has been ob-

Fig. 3. Effect of fermentation temperature on relative lipase produc-tion parameters. Qp, open circle, Yp/s, open triangle and Yp/x, closedtriangle following solid substrate fermentation of wheat bran (controlpH 6.0, inoculum 1 ml/10 g wheat bran, temperature 30 °C and time48 h; control values of Qp, Yp/s and Yp/x were 2.2�0.035 IU/l per h,48�2.1 IU/g substrate and 95�2.1 IU/g cells, respectively).

Fig. 1. Kinetics of lipase production in solid substrate fermentation.(1) Almond meal, (2) wheat bran, (3) coconut meal, (4) soybean meal,(5) rice husk and (6) mustard meal. The initial pH of the medium was6, inoculum size 1 ml/10 g wheat bran, and temperature 30 °C.

served by Riveramunoz et al. [8]. In the present case,this might have occurred due to exhaustion of nutrientsor loss of moisture. The enzyme activity measured asunits per gram substrate per hour from the slope of Fig.1 was 2.16 U/g substrate per hour. These values arehigher than the values (0.024–0.65 U/g substrate perhour) reported by Rivermunoz et al. [8]; Rao et al. [19]1.95 U/g substrate per hour, Benjumin and Pandey [2]0.91 U/g substrate per hour and Cordova et al. [5]. Thevalues reported here are higher than the values reportedby other workers except the value reported by Kaminiet al. [10], which is 5.05 U/g substrate per hour).

3.7. Enzyme properties

The enzyme shows maximum activity at 45 °C. Froman Arrhenius plot (Fig. 3), it is clear that at lower andhigher temperature, the enzyme required 114.25 and116 kJ energy, respectively, for conversion of 1 molesubstrate. This indicates that the enzyme can be safelyused in detergents for washing clothes. Similarly en-zyme was stable at pH 7.0–8.5. This is the normal pHof detergent solution used in laundry (Figs. 4 and 5).

4. Conclusion

R. oligosporous was selected as the best mould tosupport maximum enzyme production without supple-ments. Mutational work may further increase the yieldof enzyme and will be used as well to further enhancethe product yield. Other workers have added additionalnitrogen and carbon sources to enhance enzyme pro-

Fig. 2. Effect of initial pH of the fermentation medium on relativelipase production. Qp, open circle; Yp/s, open triangle; and Yp/x closedtriangle following solid substrate fermentation of wheat bran (controlpH 6.0, inoculum 1 ml/10 g wheat bran, temperature 30 °C and time48 h; control values of Qp, Yp/s and Yp/x were 2.2�0.035 IU/l per h,48�2.1 IU/g substrate and 95�2.1 IU/g cells, respectively).

I. ul-Haq et al. / Process Biochemistry 37 (2002) 637–641 641

Fig. 4. Relative tolerance of temperature by lipase produced by R.oligosporous. Control temperature 30 °C, pH 6.0, inoculum 1 ml/10 gwheat bran, and time 48 h; control values of Qp, Yp/s and Yp/x were2.2�0.035 IU/l per h, 48�2.1 IU/g substrate and 95�2.1 IU/gcells, respectively).

S. Idrees. The chairman, Department of Botany isthanked for providing the research facilities.

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Fig. 5. Relative tolerance of pH by lipase produced by R.oligosporous. Control temperature 30 °C, pH 6.0, inoculum 1 ml/10 gwheat bran, and time 48 h; control values of Qp, Yp/s and Yp/x were2.2�0.035 IU/l per h, 48�2.1 IU/g substrate and 95�2.1 IU/gcells, respectively).

duction. Similar practice may further enhance enzymeproduction and the organism may be used for industrialproduction of lipases.

Acknowledgements

This work was supported in part by Pakistan ScienceFoundation. This work formed a part of M.Sc. work of