Abstract: We aimed to optimize esterase production of Candida rugosa lipase (CRL). Active culture of C. rugosa (DSM 2031) was revived and the culture medium containing the most frequently used ingredients was optimized using a fraction of factorial design method, Taguchi. Temperature and pH of the culture was also optimized using one factor at a time method. The optimum combination of the major medium ingredients, in order of their magnitude, was (g L-1): Corn Steep Liquor (CSL) powder, (40), triolein (glyceril trioleate) (10), glucose (0) and oleic acid (2). The optimum temperature and pH were 30°C and 7, appropriately. Using this combination and conditions, esterase activity of the enzyme preparation was increased up to 9 U mL-1, which was equivalent to 20611 U mL-1 of Sigma® lipase lipolytic activity.
INTRODUCTION
Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) catalyze both hydrolysis and esterification of esters made up of glycerol and long chain fatty acids (Sharma et al., 2001). Lipases can be found in many organisms, but those originated from microorganisms are cheaper and more stable.
The yeast Candida rugosa is an important producer of lipase and C. rugosa lipase (CRL) has been used in many biotransformations, organic acids and alcohols, detergent making, food and flavour industry, production of ice cream and single cell protein, production of carbohydrate esters, amino acid derivatives, biocide making, biosensor modulations, bioremediation, cosmetics and perfumery (Benjamin and Pandey, 1998; Bornscheuer and Kazalauskas, 1999; Ferrer et al., 2001; Dominguez de Maria et al., 2006). It has been claimed that CRL poses versatile catalytic reactions, broad specificities and more applications than any other biocatalyst. It has also been introduced as the best lipase for detergent industries (Ratledge and Tan, 1990).
The molecular mass of CRL is 60 kDa, containing 534 amino acids and is moderately glycosylated (Bornscheuer and Kazlauskas, 1999; Lotti and Alberghina, 2000). C. rugosa secretes multiple lipase isoenzymes (CRLs), which are strongly related in their amino acid sequence with the same molecular weight, but partially differ in their catalytic properties (Alberghina and Lotti, 1997; Lotti and Monticelli et al., 1998; Ferrer et al., 2001; Dominguez de Maria et al., 2006). Heterologous expression of CRL isoenzymes has been reported as well (Brocca et al., 1998). Commercial CRLs contain 2-11 percent proteins and the remaining compositions are sugars or inert vehicles.
Different carbon sources (Valero et al., 1991; Obradors et al., 1993; Benjamin and Pandey, 1996; Benjamin and Pandey, 1997; Lakshmi et al., 1999; Sanchez et al., 1999; Dalmau et al., 2000; Song et al., 2001; Tan et al., 2003; Wei et al., 2004; Dominguez de Maria et al., 2006), nitrogen sources (Benjamin and Pandey, 1996; Dalmau et al., 1998; Lakshmi et al., 1999; Sanchez et al., 1999; Dalmau et al., 2000; Tan et al., 2003; Wei et al., 2004), culture temperatures (Benjamin and Pandey, 1997; Dalmau et al., 1998; Sanchez et al., 1999; Dalmau et al., 2000; Song et al., 2001; Wei et al., 2004) and pH values (Dalmau et al., 1998; Sanchez et al., 1999; Dalmau et al., 2000; Song et al., 2001; Tan et al., 2003; Wei et al., 2004) have been reported for CRL production. The composition of culture medium influences on the ratio of CRL isoforms and therefore on the catalytic activity (Ferrer et al., 2001; Dominguez de Maria et al., 2006). However, there is a lack of comprehensive optimization procedure for CRL esterase activity. There is also an inconsistency on repressive/inductive effect of glucose on CRL production (Obradors et al., 1993; Song et al., 2001). Nowadays, CRL is frequently used in esterification biotransformations for production of chiral pharmaceuticals, such as S-ibuprofen (Contesini and de Oliverira Carvalho, 2006; Won et al., 2006). Therefore, we decided to optimize esterase production of Candida rugosa lipase (CRL) and clarify the effect of glucose on this issue, to use the enzyme preparation for esterification biotransformations in future study.
MATERIALS AND METHODS
This study was conducted in 2006-2007 at Isfahan University of Medical Sciences, Iran.
Microorganism, medium and culture conditions: Active culture of C. rugosa (DSM 2031) was revived (30°C) on liquid YM medium and then maintained at 4°C (up to maximum 1 month) on YM-agar containing (g L-1): peptone from soybeans, 5; malt extract, 3; yeast extract, 3; glucose, 10; agar, 15. Long-term stocks were preserved in 20% (v/v) glycerol at -80°C. The medium was sterilized using autoclave (121°C, 15 PSI, 15 min), but glucose (stock sol.) was autoclaved separately and added to the other ingredients aseptically, considering the final desired concentration. Basal main medium contained (g L-1): KH2PO4, 12.3; K2HPO4, 5.25; MgSO4, 1; FeCl3, 0.01; urea, 1; CaCl2, 0.0001; yeast extract, 3. The other ingredients were added in different compositions based on Table 1. The yeast was grown in 1 L flasks containing 200 mL of medium. Forty eight hours old cultures of C. rugosa on YM-agar were resuspended in 0.9% NaCl solution and each flask was inoculated by 5 mL of this suspension. The flasks were incubated in a shaker-incubator at 30°C, pH 6.3 and 180 rpm.
Design of experiments (DOE): The culture medium was optimized using a fraction of factorial design method, Taguchi. Considering the best previous results of the researchers studied lipase production, 9 combinations (L9 orthogonal array) of the major medium ingredients were studied. The factors to be optimized were the following 4 ones in 3 levels: oils, fatty acids, nitrogen sources and glucose concentration (Table 1).
Maximum total (internal + external) esterase activity was considered as the response factor. Using Minitab® 14 (PA, USA) software, the levels of each factor posing the most influence on the esterase activity and also the rank of each factor were identified. Temperature and pH of the culture were also optimized by one factor at a time method, using the best combination of Taguchi`s method (Shojaosadati and Asadollahi, 2002).
Viable count: Dilutions of the samples were counted by spread plate method after incubation.
Esterase activity assay: Esterase activity was determined by the enzymatic method described by Dalmau et al. (2000) except that culture samples were centrifuged (5840 x g, 15 min, 4°C), the pellets were collected for internal activity assay and the supernatants were used for extracellular activity assay.
Extracellular esterase activity assay: The method of Dalmau et al. (2000) was used, except that the formation of p-nitrophenol after 1 min was measured at 348 nm.
Internal esterase activity assay: The method of Dalmau et al. (2000) was used, except that the disrupted cells were passed through the filter (0.2 μm) and after this stage, the procedure was the same as earlier.
Lipase lipolytic assay: A convenient method (Baillargoeon et al., 1989) was used to measure lipolytic activity, except that the lipolytic activity unit was defined as the amount of enzyme preparation, which hydrolyzed 1 μeq h-1 of the free fatty acid (Sigma® definition).
Protein assay: Protein concentration was determined by a well known method (Bradford, 1976) and bovine serum albumin was used as the standard protein in the calibration curve.
RESULTS
Optimization of medium for lipase production using Taguchi design:
Considering the best previous results of the researchers studied lipase
production (Table 1), combinations of the major medium ingredients were
studied using Taguchi fraction of factorial design method.
| Table 1: | Taguchi`s L9 orthogonal array for optimization of medium composition |
| *- CSL: Corn Steep Liquor | |
| Fig. 1: | The mean effects of each factor on total esterase activity. The mean effects of each factor on total esterase activity (the response factor) was plotted by Minitab®14 software |
| Fig. 2: | Effect of different temperatures on esterase activity. Total esterase activity was determined in frequently used temperatures, grown on the optimum combination of the major medium ingredients determined by Taguchi method at pH 6.3, 25 h after the start of culture growth |
Optimization of conditions for lipase production using one factor
at a time method : Using the best combination of medium ingredients
determined above and changing only one condition at a time, such as temperature
or pH, these conditions were optimized as well. Total esterase activity
was determined in frequently used temperatures. The esterase activities
between 25-30°C were increased (Fig. 2) and reached to the maximum
at 30°C. After this temperature, the activity declined.
| Fig. 3: | Effect of different pH values on esterase activity. Total esterase activity was determined in frequently used pH values, grown on the optimum combination of the major medium ingredients determined by Taguchi method, 25 h after the start of culture growth. Data points (duplicate) are averages and error bars are standard deviations |
Using the best combination of medium ingredients determined previously and changing only pH values, the optimum pH was determined at 30°C. Total esterase activity was determined in frequently used pH values. The optimum pH was 7 (Fig. 3).
Time course of the enzyme activity: In order to investigate the
best time for harvesting CRL from the culture growing in the optimized
medium at optimized conditions, total esterase activity was studied during
the growth. Two steps were seen in the growth curve as well as esterase
activity and the latter one seemed to be growth dependent (Fig. 4). The
maximum growth and activity was observed at 24 h.
| Fig. 4: | Time course of growth and esterase activity. Microbial count (Ο) and total esterase activity (∼) of the culture, were determined during the growth. Data points (duplicate) are averages and error bars are standard deviations |
| Fig. 5: | Comparison of internal and external esterase activity. Internal (Ο) and external (∼) activities were determined during the time. Data points (duplicate) are averages and error bars are standard deviations |
DISCUSSION
CSL as the nitrogen source had the most impact between the factors tested by Taguchi method and showed much better results than ammonium sulfate and soybean flour. Ammonium sulfate (Dalmau et al., 1998; Sanchez et al., 1999; Dalmau et al., 2000; Tan et al., 2003; Wei et al., 2004) urea (Triantafyllou et al., 1993; Benjamin and Pandey, 1996; Lakshmi et al., 1999; Sanchez et al., 2000) and soybean meal (Tan et al., 2003) have been used as nitrogen sources. Previously, Dalmau et al. (1998, 2000) and Sanchez et al. (1999) had suggested (NH4)2SO4 and Benjamin and Pandey (1996) and Lakshmi et al. (1999) had announced urea as nitrogen sources for lipase production. This is the first time, which CSL is introduced as N source for this purpose and due to its low price, this finding seems to be interesting for industries, financially.
It was shown that the combination of glyceryl trioleate (an ester) and oleic acid (a fatty acid) increased CRL activity better than each of them solely, or better than the other studied growth substrates as carbon sources and lipase inducers. Researchers have reported various major carbon sources and inducers for production of CRL, including oleic acid (Lakshmi et al., 1999; Sanchez et al., 1999; Tan et al., 2003; Wei et al., 2004), palmittic acid (Dalmau et al., 2000), olive oil (Valero et al., 1991; Benjamin and Pandey, 1996; Song et al., 2001), sesame oil (Lakshmi et al., 1999) and glycerol trioleate (Wei et al., 2004). This is the first report, which a combination of an ester and a fatty acid is introduced as the carbon sources and inducers for lipase production.
The negative effect of glucose on lipase production mentioned by Obradors et al. (1993) was also verified in the current study, but was in contrast with the results of Song et al. (2001).
It was determined that the optimum temperature for lipase activity was 30°C, which was consistent with some results (Benjamin and Pandey, 1997; Dalmau et al., 1998; Sanchez et al., 1999; Dalmau et al., 2000; Song et al., 2001; Wei et al., 2004). The optimum pH was 7, but Wei et al. (2004), Song et al. (2001), Dalmau et al. (2000), Sanchez et al. (1999) and Lakshmi et al. (1999) have not used this pH. Tan et al. (2003) was the only researcher who previously optimized pH for lipase activity and reported pH 7 as the optimum value, which is consistent with the current study. Tan et al. (2003) only studied pH values with 1 unit difference, but in this work pH was optimized changing the values by 0.5 unit steps, which seems to be more accurate.
Two steps which was seen in the growth curve as well as esterase activity, might be due to switch from low amounts of the carbon sources in yeast extract at early stages to oleic acid and glyceril trioleate, which are metabolized more slowly.
It was shown that 24 h after the start of C. rugosa growth is the optimum time to extract esterase from the culture. Lakshmi et al. (1999) had reported that the maximum productivity of lipase production was obtained within 48 h. But, the results of current study and Lotti et al. (1998) showed that the maximum yield was obtained earlier due to difference in the media composition. It might be concluded that the latter media are better considering productivity.
The ratio of internal to external activity was increased between hours 0-24 about 2 times, which supports hypothesis of Lotti et al. (1998), saying during batch growth in media producing high amounts of lipase, the synthesis of lipase becomes faster than its transport, causing intracellular accumulation.
CONCLUSION
The culture medium was optimized using a fraction of factorial design method, Taguchi. Temperature and pH of the culture was also optimized using one factor at a time method. Using this combination and conditions, the activity of enzyme preparation was increased to 9 U mL-1, which was equivalent to 20611 U mL-1 of Sigma® lipase lipolytic activity, with a productivity of 0.362 U mL-1 h-1. After a semi-purification, in case of using appropriate substrates, this enzyme preparation can be considered as a potent biocatalyst for production of enantiopure pharmaceutical products. It might be concluded that 24 h after growth in the optimized medium at the optimized conditions, will be the optimum time to extract and purify the enzyme as a biocatalyst for esterase activity.
ACKNOWLEDGMENT
We wish to express our gratitude to Isfahan University of Medical Sciences for financial support of this study.