Lipase production by immobilised Rhizopus arrhizus

Process Biochemistry 36 (2000) 219 – 223 www.elsevier.com/locate/procbio

Lipase production by immobilised Rhizopus arrhizus Murat Elibol *, Dursun O8 zer Chemical Engineering Department, Fırat Uni6ersity, 23279, Elazig, Turkey Received 25 October 1999; received in revised form 10 April 2000; accepted 3 May 2000

Abstract Immobilisation of Rhizopus arrhizus cells on a solid support (polyurethane foam) for lipase production has been explored. Lipase biosynthesis was repressed at a high glucose level. Maximum productivity was recorded at 1 g l − 1 glucose concentration. The inclusion of 0.5 g l − 1 corn oil (as an inducer) in the fermentation medium resulted in 2.5-fold higher lipase production compared to the control where no oil was added. Repeated-batch experiments revealed that immobilised R. arrhizus cells demonstrated reproducible behaviour, producing the same amount of enzyme over a 120 h period. The storage stability of the enzyme was investigated and enzyme activity reduction was 26% within 160 h. © 2000 Published by Elsevier Science Ltd. All rights reserved. Keywords: Lipase; Immobilised Rhizopus arrhizus; Lipolytic activity; Inducer

1. Introduction Lipases are of considerable commercial and industrial potential. Moreover, there is an increasing interest in the development of new applications for these enzymes on products and processes [1]. Studies of the fermentation conditions for production of lipase by freely suspended cells have been previously carried out [2 – 5]. In most technical applications of immobilised cells the objective is to increase the extent of reaction and to facilitate downstream processing. If the cells can, in some way, be retained in the system, prolonged use of the cells may be achieved by keeping them viable and consequently inducing production of the enzyme. The application of immobilised biocatalysts for the production of lipase is less well-documented than their application for bioconversions and for the production of useful low molecular mass components [6]. There is little literature published about immobilisation of microbial cells for lipase production [7 – 9]. Immobilisation of * Corresponding author. Tel.: + 90-424-2370000, ext. 3687; fax:+ 90-424-2122717. E-mail address: [email protected] (M. Elibol).

Rhizopus arrhizus cells on a solid support for industrial production of lipase could offer several advantages. In contrast to ordinary suspension culture systems, immobilised whole cells have the merits of: (1) avoiding wash-out of cells at a high dilution rate, (2) higher cell concentration in the reactor and (3) easy separation of cells from the system or the product containing solution [10]. Owing to their importance engineering research of immobilised cells has also been realized and has covered a wide range of aspects, such as immobilising supports, mass transport effects, physical and chemical environment, kinetics and process modelling. One of the most used immobilisation methods is the entrapment of cells in gel matrices. Due to the mass transfer limitation in this matrix the lipase productivity is often limited. To prevent this problem a natural immobilisation techniqe was employed in this study. The cells were immobilised by physical entrapment in the open pore network of reticulated polyurethane foam. This natural immobilisation technique has several advantages over the other methods. Having given the important advantages that any immobilised biocatalyst may offer a priori, the present work has focused its interest in fermentation conditions for lipase production.

0032-9592/00/$ - see front matter © 2000 Published by Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 0 0 ) 0 0 1 9 1 - 6

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2. Materials and methods

2.1. Microorganisms and medium R. arrhizus NRRL 2286 was maintained on agar plates. It was grown at 30°C for 2 days and then stored at 4°C. After growth and sporulation, 10 ml distilled water was aseptically added to each agar plate which was then scraped to release the spores. This spore suspension was centrifuged at 4000 rpm for 10 min, the spores were washed and resuspended in 1 ml distilled water. 250 ml spore suspension was used to provide a spore inoculum for each 250 ml shake-flask containing 50 ml medium. The flasks were then placed on a rotary shaker at 30°C and 150 rpm. The growth liquid medium contained per litre of distilled water: glucose variable (1–10 g), 0.2 g MgSO4.7H2O, 0.5 g K2HPO4, 0.5 g KH2PO4 and 2 g yeast extract. The pH of the medium was initially adjusted to 6 and then allowed to follow its natural course.

2.2. Analytical procedure The biomass concentration within the polyurethane foam matrices were determined as follows: a ten flasks set containing medium and support material of known dry weights was inoculated with spore suspension. One flask was taken every sampling time. Polyurethane matrices were placed on a piece of aluminium foil and dried in an oven at 80°C for 24 h and then reweighed. The biomass concentration was calculated from the difference in weight. Residual glucose was measured with a Beckman glucose analyser. Lipolytic activities in filtrates were determined as previously reported where the method was based on titration of free fatty acids released by lipases for 1% (v/v) tributyrin [11]. All experiments reported here were carried out in duplicate.

Fig. 1. Time course of lipase fermentation (glucose, g l − 1: , 1; , 2.5; , 5; and ", 10).

distilled water were autoclaved three times for 15 min at 121°C, the distilled water being replaced each time to remove any chemical that might have otherwise leached out into the culture medium. One foam slab (55×20× 8 mm) was placed in each flask and held stationary by fixing onto a stiff L-shaped stainless steel wire. After sterilization 50 ml nutrient medium was placed in each flask.

3. Results and discussion

2.3. Polyurethane foam preparation 3.1. Effect of glucose Foam matrices (15 ppi) were used throughout the work. Prior to use, the support materials submerged in Table 1 Some kinetic parameters in lipase productiona S0 1 2.5 5 10

Xm 1.23 1.53 2.85 3.80

Pm 132 145 62 60

mmax

YX/S

YP/X

YP/S

0.131 0.132 0.134 0.137

1.230 0.612 0.570 0.380

101 95 22 16

132 58 12 6

a S0, initial glucose concentration (g l−1); Xm, maximum biomass concentration (g DW l−1); Pm, maximum lipolytic activity (mmol l−1 min−1); mmax, maximum specific growth rate (h−1); YX/S, yield coefficient for cells on glucose (g cells per g glucose); YP/X, yield coefficient for product on biomass (mmol per g cells per min); and YP/S, yield coefficient for product on glucose (mmol per g glucose per min).

One of the most important parameters in fermentation is the level of substrate used. In this study four different glucose concentration varying from 1 to 10 g l − 1 were used. The maximum lipolytic activity was recorded at 1 and 2.5 g l − 1 glucose concentrations (Fig. 1). Any further increase in glucose level resulted in a decrease in lipase production. This can be attributed to the repressive effect of glucose on lipase fermentation. However, yield coefficients at 1 g l − 1 glucose concentration were higher than those at the other concentrations (Table 1). This concentration was used in the rest of the study. In the presence of rapidly utilised carbon sources, such as glucose, many fermentation processes are repressed [8]. For example, Ates et al. [12] reported that

M. Elibol, D. O8 zer / Process Biochemistry 36 (2000) 219–223

Fig. 2. Time course of biomass concentration (glucose, g l − 1: , 1;

, 2.5; , 5; and ", 10).

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Fig. 4. Variation of maximum activity with various spore concentration.

3.3. Effect of inoculum size actinorhodin fermentation was repressed at high concentrations of glucose. In that study the repressive effect of glucose was eliminated by using a fed-batch technique. Marek and Bednarski [13] also reported that catabolite repression influenced lipase synthesis in Rhizopus miehei. Lipase production was also affected by pH changes during the fermentation (Fig. 1). Unlike lipolytic activity the biomass concentration increased with increasing glucose concentration (Fig. 2).

3.2. Effect of initial pH Microbial growth and metabolism inevitably lead to a change in the hydrogen ion balance and, hence, the pH of the culture medium. Preliminary experiments showed that lipase production was highly sensitive to pH alteration during the fermentation. In all cases maximum activity was recorded at pH 5.5 9 0.5 of the medium. The effect of initial pH of the medium was conducted at five different pH values varying from 4 to 8. The result is depicted in Fig. 3. The maximum lipolytic activity was recorded at pH(initial)= 6.

Fig. 3. Effect of initial pH on enzyme production.

The nature of the inoculum as well as its size may effect the microbial process [14]. In order to find out the effects of inoculum level on lipase production a series of experiments were performed using four different spore concentrations. Spore counts were made with a haemocyometer. There was no significant effect of size of spore inoculum on lipolytic activity (Fig. 4). Visual observation also showed that no morphological differences occurred, apparently, once the spores had germinated and grown on or within the support matrices, enzyme production was not significantly affected.

3.4. Effect of agitation speed Enzymes are susceptible to mechanical force, which may disturb the elaborate shape of a complex molecule to such a degree that denaturation occurs [15]. Lipase activity was investigated at four different agitation speeds, i.e. 75, 100, 150 and 200 rpm. The maximum lipolytic activity was measured at 150 rpm (Fig. 5). The

Fig. 5. Effect of agitation speed on lipase production.

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M. Elibol, D. O8 zer / Process Biochemistry 36 (2000) 219–223

3.6. Repeated-batch experiments One of the most important advantages of using immobilised cells is to increase the extent of growth and production period and repeated batch experiments were, therefore, conducted. When maximal lipolytic activity was recorded the used medium was replaced with fresh medium, aseptically. While, initially, the maximum activity was measured after 18 h of fermentation, after 120 h of fermentation this activity was obtained within 2 h (Fig. 7). This result showed that immobilised R. arrhizus cells might be kept viable and could produce the enzyme for a 120 h period. Fig. 6. Effect of corn oil as an inducer on lipase production (", control; and , 0.5 g l − 1 corn oil).

negative effect of agitation speed at 200 rpm may be due to the perturbation of protein structure during the biosynthesis of lipase. Charm and Wong [16] have reported that the enzymes, catalase, rennet, and carboxypeptidase were inactivated by shear effects.

3.5. Effect of inducer In many fermentation processes some materials may induce the synthesis of product. In this study 1g l − 1 corn oil was used as an inducer. The inclusion of corn oil in the fermentation medium resulted in a significant increase in lipase production (Fig. 6). This can be attributed to the presence of an inducer (oil) which is also a substrate for the enzyme. In the presence of corn oil the maximum lipolytic activity was 325 mmol l − 1 min − 1 which was approximately 2.5 times higher than that in the absence of inducer. Valero et al. [2] similarly reported that the presence of olive oil promoted yeast growth as well as lipolytic activity [2]. Ferrer and Sola also found that the addition of gum arabic (as an inducer) to the culture medium of Candida rugosa enhanced lipase activity [9].

3.7. Storage stability In biotechnological recovery processes instability of the product can lead to large losses in the sequence of recovery processes needed to purify the product. As the cost of the final active product is strongly dependent on the recovery yield this will lead to an increase in product cost [17]. Therefore, knowledge of storage stability of the enzyme produced is important. In order to determine storage stability a filtered sample was kept in a refrigerator at 4°C and lipolytic activity was measured at timed intervals. The enzyme lost 26% of its original activity after 160 h (Fig. 8).

4. Conclusions R. arrhizus cells may be easily immobilised on or within polyurethane foam matrices. The maximum lipolytic activity was obtained at 1 g l − 1 glucose concentration. The addition of 0.5 g l − 1 corn oil to the fermentation medium resulted in 2.5-fold higher lipase production compared to the control. This preliminary work revealed that lipase production by R. arrhizus could easily be affected by fermenta-

Fig. 7. Time course of enzyme activity in the repeated-batch system.

M. Elibol, D. O8 zer / Process Biochemistry 36 (2000) 219–223

[4]

[5]

[6]

[7]

[8] Fig. 8. Storage stability of the enzyme. [9]

tion conditions. Considering all these results this study will be followed up using solid state fermentation.

[10]

[11]

Acknowledgements [12]

The authors wish to thank TUBITAK, the Scientific and Technical Research Council of Turkey, for financial support of this study.

[13]

[14]

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