Optimization and characterization of Candida cylindracea ... - AIChE

OPTIMIZATION AND CHARACTERIZATION OF CANDIDA CYLINDRACEA LIPASE PRODUCED FROM PALM KERNEL CAKE BY SOLID-STATE BIOCONVERSION Md. Zahangir Alam, Amal Ahmed Elgharbawy and Hamzah Mohd Salleh Bioenvironmental Engineering Research Centre (BERC), Department of Biotechnology Engineering, Faculty of Engineering, International Islamic University Malaysia (IIUM), Gombak, 50728 Kuala Lumpur, Malaysia

Abstract The study aimed to produce lipase using abundant raw material in Malaysia employing a cost- effective method which is solid-state bioconversion (SSB). The optimization of the production was achieved with 400 U/gds which was more than 4-folds compared to the screening experiments. Characterization of the produced lipase showed promising results in terms of stability as well as in the hydrolysis of lipids. The enzyme was stable at 50-55°, contact time of one hour and hydrolyzed reaction to 50% free fatty acids. Introduction One of the most important industrial enzymes is lipase. Lipases are gaining interests in terms of research due to their potential applications. Despite that, researchers have to overcome the challenge of the high production costs. Hence, the use of different microorganisms, low-cost nutrients and available substrates may reduce the production costs in industrial scales. A practical method is to use agroindustrial residues as substrates to produce the enzyme using solid-state bioconversion to reduce the cost [1]. Management of waste and turning waste to wealth are attracting the researchers during this few years in order to meet the demand of the current market. Malaysia is one of the largest producers of palm oil in the world with plantation area of 11% of the land along with a yearly production of 13 million tons of crude palm oil [2]. Palm kernel cake (PKC) is one of the solid wastes obtained after oil extraction from palm kernel seeds [3] which reached 2.4 million tons by the end of 2012 [4]. PKC contains 50% carbohydrates and 15-20% proteins. It is also rich source of minerals such as Mg (0.27%), P (0.79%), Fe (4.05 mg/kg), Cu (28.5 mg/kg), Zn (77.0 mg/kg) and Mn (225.0 mg/kg) [5-6]. Solid-state bioconversion (SSB) or solid-state fermentation (SSF) is defined as the fermentation process occurring in the absence or near-absence of free water [7]. However, to date only few studies employed SSB for lipase production from different substrates, whereas many applied submerged fermentation for this purpose. Some of the substrates that have been used for lipase production are olive oil cake, cassava, sweet potato, pineapple and carrot waste and wheat bran [8, 9], rice bran, groundnut oil cake, sesame oil cake, coconut oil cake, and wheat bran [10-11]. Among the reported data, optimization of media and process conditions has not received much attention. Nevertheless, not much works have been conducted on lipases, especially employing SSB to ferment solid agro-industrial wastes and particularly, PKC using Candida cylindracea. Furthermore, SSB seemed to be more attractive economically because the cost was reported to be much lower than the market price [12]. This work aimed to explore the potential of using PKC in optimizing a fermentation medium and to determine the optimal process conditions for microbial lipase production, using Candida cylindracea by employing SSB as well as characterizing the produced lipase.

Materials and Methods Materials The main substrate in this study was palm kernel cake (PKC) was collected in clean autoclaved bags, from West Oil Mill, Sime Darby Sdn. Bhd. (Supercritical Fluid Extraction Unit) in Carey Island, Selangor, Malaysia. The raw material was dried and grinded to 1 mm particles. Chemicals and consumables used in this research were of analytical grade and commercially purchased from Oxoid Ltd, Merck Sdn Bhd., Fisher Scientific, R & M Chemicals, MHM Global, Bumi Pharma Sdn Bhd., Nano-life Quest, Malaysia. The microbial strain, Candida cylindracea (ATCC 14830) used in this study was purchased from the American Type Culture Collection, USA. C. cylindracea was grown on (PDA) plates at 28ºC for four days and sub-cultured every two weeks. Each culture plate was washed with sterile distilled water and the suspension was used to prepare the inoculum. 1.0 ml of suspension was cultivated in a medium containing 0.7% (v/v) Tween-80, 0.2% (v/v) olive oil and 0.5% (w/v) peptone at 28ºC in an orbital shaker at 150 rpm for 48 hrs [13]. Fermentation Process The fermentation was carried on in culture flasks where 6.0 g of PKC was moisturized using sterilized distilled water and supplements to reach a total weight of 20 grams. The medium components consisted of the solid substrate supplemented with nutrients as described in the experimental design later. After autoclaving, the contents were inoculated with the prepared inoculum solution and incubated at 30°C. Samples were taken every 24 hrs for duration of 8 days for analysis. After the incubation period, the fermented medium was mixed with sterile distilled water and agitated for 2 hrs at room temperature on a rotary shaker to facilitate the extraction. The solution then was filtered through Whatman No.1 filter paper and the filtrate was used for the lipase assay [3]. Analysis of lipase was carried out according to the method described by Gopinath et al. [14] using p-nitrophenyl palmitate (pNPP) as the substrate. Lipase activity was expressed as unit/ ml of the extracted solution and units/gram of the initial dry substrate (U/gds). Assays were carried out in triplicates and the averages were recorded. Statistical Optimization Three stages were involved: Plackett-Burman design (PB), one-factor-at-a-time (OFAT) and response surface methodology (RSM). Design Expert v.6.0.8 was used for designing the experiments for screening, optimization and validation of the model. IBM SPSS Statistics v.19 was used to analyze the contribution and the significance of the parameters that influenced the lipase production. Eleven components were selected for screening which are media components: glucose, sodium nitrate, magnesium sulphate, peptone, yeast extract, Tween-80 and olive oil for process conditions: pH, temperature, inoculum concentration and moisture content. PB design was followed by classical OFAT method. From PB design results, factors that contributed at negative level or had relatively small values for the main effect were eliminated from the design. The aim of this method was to find the optimal levels for the optimization process using Face Centred Central Composite Design (FCCCD). The FCCCD under RSM was employed to determine the optimum conditions. After obtaining OFAT results, the data was subjected to One-Way ANOVA Post-hoc Tukey’s test (p ≤ 0.01). The factors that showed their optimal levels at certain points were selected for FCCCD while the remaining factors were fixed. Analysis of Variance (ANOVA) was used along with 2D contour plot and 3D response surface curve to evaluate the accuracy of the model. Main effect calculation was used to analyse PB design results in Microsoft Office Excel 2007.

Characterization of the PKC-lipase Effects of several factors that influence the lipase activity and stability were studied. These factors included temperature, pH, metal ions and organic solvents that can enhance or inhibit the activity. Application in the hydrolytic reaction of lipids was also investigated. Effect of pH on stability was tested by incubating the enzyme with different pH buffers (4.0-9.0) for 24 hrs. Thermo-stability was tested by incubating PKC-lipase in different temperatures (25-70°C) followed by lipase assay for activity. Organic solvents (methanol, ethanol, 2-propanol, acetone, nhexane and toluene) were investigated for their effects on the activity as well as metal ions and detergents. The enzyme was pre-incubated in 1:1 ratio of the following: CaCl2, CoCl2, MnCl2, MgCl2, FeCl3, EDTA, Triton X-100, Tween-80 and sodium dodecyl sulfate (SDS) for 60 minutes followed by the enzyme assay at the same conditions [15]. Hydrolysis of lipids was conducted by weighing 5.0 g of the oil sample (canola, palm and olive oil). Then, 10.0% (0.5 ml) of the crude lipase (43.5 U/ml) was added to start the reaction. The control sample was left without enzyme loading. The mixture was incubated at 37°C in an orbital shaker at 200 rpm to ensure a homogenous mixing [16]. The reaction was followed up for 24 hrs to determine the percentage of hydrolysis. The hydrolyzed sample was dissolved into ethanol and phenolphthalein (prepared in ethanol) was added as a detector. The mixture was titrated with 0.1M NaOH until permanent (for 30 seconds) faint pink colour appeared. The percentage of hydrolysis was calculated according to the American Oil Chemist’s Society method [17]. Results and Discussion Optimization of lipase production Factors that influenced the production at positive level were ranked as: temperature > inoculum concentration > Tween-80 > moisture content > yeast extract> pH > peptone. Lipase production based on Plackett-Burman design showed a fluctuated activity which ranged from 29 U/gds to 94.13 U/gds. The highest lipase activity was obtained in Run 10 in the presence of organic and inorganic nitrogen sources, olive oil, Tween-80 and in the absence of glucose and MgSO4. The lowest activity was observed in Run 3 where the medium was supplemented with only olive oil. The most significant factors that resulted in a maximum lipase activity were Tween-80, yeast extract and olive oil while the effective process conditions were the temperature and inoculum concentration. In order to obtain the optimal level of each factor, the factors that contributed the most in lipase production were further investigated using one-factor-at-a-time (OFAT) method. The results were analyzed using One-Way ANOVA Post Hoc Tuckey’s test. Factors selected to be fixed were: olive oil, Tween-80, moisture content, pH and temperature. Two factors were subjected to face-centred central composite design (FCCCD): yeast extract and inoculum concentrations (Table 1). The results showed that the highest lipase production (400 U/gds) was observed at the centre points (runs 3, 6, 9, 10 and 12). The minimum activity was observed in run 4 (241 U/gds), when both factors were at their low concentrations. This actually showed that the FCCCD design improved the lipase production where the difference between the maximum and minimum responses (241 and 400 U/gds) was significant. A second order equation which described the relation between the two factors and the lipase activity was obtained from the software by analyzing the experimental results. The equation obtained was as follow: Lipase Activity = +272.10 – 162.08A – 5.10B – 193.28A2 – 8.89B2 – 20.31AB

(1)

The goodness of fit was evaluated by the coefficients of determination (R2). The high value of R2 (0.9893) implied a high degree of correlation between the experimental and the predicted values by the model. The R2 value of 0.9893 pointed that only about 1.07 % of the total variations could not be

explained by the model. The adjusted R2 of 0.9816 in this study was close to R2 value which indicates the better prediction of the model. The contour plot (Figure 1A) and 3D response surface curve (Figure 1B) showed the optimal predicted lipase activity at (x= 1.87 and y= 7.71). The production of lipase using the optimum conditions obtained from OFAT followed by FCCCD increased by 4.23-folds and 1.67-folds compared to the production obtained by PB (94.13 U/gds) and OFAT (250 U/gds), respectively. Moreover, the fermentation time decreased to 72 hrs instead of 144 hrs in OFAT experiments. Table 1 Experimental design using FCCCD of two parameters with actual and coded value, centre points, actual and predicted lipase activity B: Inoculum concentration (% v/w) 7.00 (0)

Actual

Predicted

1

A: Yeast extract concentration (% w/v) 0.6 (-1)

256.33

267.2915

4.100

2

0.6 (-1)

10.0 (0)

286.77

279.0259

-2.775

3

1.5 (0)

7.00 (0)

394.26

394.2521

-0.002

4

0.6 (-1)

4.00 (-1)

241.00

237.7826

-1.353

5

2.4 (+1)

10.0 (+1)

372.03

369.4126

-0.785

Run order

Lipase Activity U/g dry PKC Error%

6

1.5 (0)

7.00 (0)

400.22

394.2521

-1.514

7

2.4 (+1)

4.00 (-1)

375.00

376.9093

0.507

8

1.5 (0)

4.00 (-1)

375.62

376.9282

0.347

9

1.5 (0)

7.00 (0)

402.33

394.2521

-2.489

10

1.5 (0)

7.00 (0)

393.00

394.2521

0.3176

11

2.4 (+1)

7.00 (0)

381.34

382.0482

0.1854

12

1.5 (0)

7.00 (0)

393.12

394.2521

0.2872

13

1.5 (0)

10.0 (+1)

383.44

393.8015

2.6311

(A)

(B)

Figure 1: (A) Contour plot of the interaction between yeast extract concentration % (w/w) and inoculum concentration % (v/w). (B) 3D response surface curve of the interaction effects of yeast extract and inoculum concentrations on lipase activity by C. cylindracea.

Characterization of the PKC- lipase Optimal pH and temperature for PKC-lipase activity were found at pH 8.0 and 37°C. As for stability, the enzyme was stable at pH 7.0-8.0 after 24 hrs. 60.0% of the activity remained after 24 hrs at pH 9.0. At lower pH, the activity of PKC- lipase dropped to 50.0% or less. Nevertheless, at pH 5.0- 6.0, more than 50% of the activity remained after 12 hrs. At different temperatures, PKC-lipase exhibited different stability. It retained its full activity at 25 - 45°C after 90 minutes of the incubation. At 50°C, 73% of the enzyme activity was retained while 68% of the activity remained after 90 min of incubation. The PKC-lipase obtained in this study was stable at 45°C for 90 min with full activity, 50°C and 55°C for 60 min with 95% and 83% of its initial activity, respectively. The enzyme was also stored for 3 months with no lost of its activity at -20°C. After stored for 7 days at room temperature, around 60% of the activity remained where 92.6% remained when stored at 4.0°C.

The results obtained showed that the enzyme retained 94.25% and 93.43% of its initial activity in 25% methanol and ethanol, respectively. Increasing the concentration inhibited the activity. 55.4% of the activity remained after one hour in 50% of methanol. 2-propanol and acetone was found to have more inhibitory effect where 85.6% and 82% of the activity was retained after the incubation in 25% of those solutions. At higher concentrations, all water-miscible solvents showed inhibitory effect, where less than 10% of the lipase activity was detected. Meanwhile at low concentrations of water immiscible solvents (n-hexane and toluene), the enzyme retained 52% and 48.18% of its activity. Unlike other solvents, higher concentrations of n-hexane and toluene did not completely inhibit the activity where more than 30% of the activity remained after 1hr. Lipases can work in both aqueous solutions as well as in nearly anhydrous solvents [15] The stability of lipases in organic solvents has its benefits particularly in the reversible reactions which involve synthesis such as esterification [18] and transesterification [19]. PKC-lipase lipase showed changes in its activity when it was incubated with several metal ions. Fe3+ was the only metal among the group that inhibited the activity where 17.0% of the activity remained after 1hr. Calcium and magnesium ions stimulated the enzyme the most while cobalt showed no considerable effect. On the other hand, 10 mM EDTA enhanced the activity by 15%, where 74% of the activity retained in a solution contained 1.0% (v/v) of Triton X-100. SDS and Tween-80 almost inhibited the activity. Ca2+, Mg2+and other metal ions enhance the activity because of their contribution as cofactors as well as their role in stabilization of the tertiary structure [20].

Enzymatic hydrolysis of lipids using the PKC- lipase Oleic acid is the major constituent (45- 80%) of the oils that have been chosen in this study (palm, olive and canola oil). Hence, the degree of hydrolysis has been expressed as percentage of oleic acid as suggested by Serri et al [16]. As shown in Figure 2, the results proved that the PKC-lipase has the ability to successfully hydrolyze the triglycerides into FFA and glycerol of different oils with different degrees. The highest percentage of hydrolysis (50.48%) after 24 hrs was observed in the presence of canola oil, while the lowest was with palm oil (40.11%). On the other hand, hydrolysis of olive oil was not to be neglected (45.98%). PKC- lipase was not purified in at this stage and so might contain other impurities. Considering that, the results from hydrolysis were quite reasonable.

Figure 2: Degree of hydrolysis using PKC- lipase, expressed as % oleic acid in various lipids.

Conclusion By optimizing the process conditions, the maximum lipase production (400±2 U/gds) was achieved within 72 hrs. The production was 4.23-fold higher compared to PB design (94.13 U/gds). This study developed a combination of media components and process conditions for lipase production using SSB in order to reduce the production cost. The process considered to be cost effective compared to submerged fermentation. Furthermore, the data obtained from characterization gave an insight in industrial applications of lipases in detergents, food and biodiesel production. References 1.

2.

3.

4.

Pandey, A., Soccol, C.R., Rodriguez-Leon, J.A., Singh-Nee Nigam, P. (2001). Solid State Fermentation in Biotechnology: Fundamentals and Applications, Asiatech Publishers Inc, New Delhi. Wu, Y.T., Mohammad, A.W., Jahim, J.M., Anuar, N. (2009). A holistic approach to managing palm oil mill effluent (POME): Biotechnological advances in the sustainable reuse of POME, Biotechnol. Adv. (27) pp. 40–52. Imandi, S.B., Karanam, S.K., Garapati, H. (2010) Optimization of media constituents for the production of lipase in solid state fermentation by Yarrowia lipolytica from palm kernel cake (Elaeis guineensis), Adv. Biosci. Biotechnol. (1), pp. 115-121. Economics & Industry Development Division, Malaysian Palm Oil Board (MPOB), Statistics, http://bepi.mpob.gov.my/index.php/statistics/production/71-production-2012/520-production-ofpalm-kernel-cake-2012.html [Accessed 8 June, 2013].

5.

6. 7.

8. 9.

10.

11.

12.

13.

14. 15.

Anusha, N.C., Umikalsom, M.S., Ling, T.C., Ariff, A.B. (2012) Relationship between fungal growth morphologies and ability to secrete lipase in solid state fermentation, Asian. J. Biotechnol. (4), pp. 15-29. Hishamuddin, M.A. (2002) Malaysian palm kernel cake as animal feed. Malaysian Palm Oil Board, Palm Oil Developments (34), pp. 4-6. Rashid, S.A., Darah, I., Omar, I.C. (2011) Utilization of palm kernel cake for the production of mannanase by an indigenous filamentous fungus, Aspergillus Niger USM F4 under solid substrate fermentation, Internet J. Microbiol. (9), pp. 1-9. Shukla, P., Gupta,K. (2007) Ecological screening for lipolytic molds and process optimization for lipase production from Rhizopus oryzae KG-5, J. Appl. Sci. Environ. Sanit. (2), pp. 35–42. Yang, X.X., Lin, X.Z., Bian, J., Sun, X.O., Huang, X.H. (2004) Screening and phylogenetic analysis of Antarctic bacteria producing low-temperature lipase, Acta Oceanologica Sinica. (23), pp. 717–723. Rekha, K.S.M Lakshmi, C.M.V., Sri Devi, V. Kumar, S.M. (2012) Production and optimization of lipase from Candida rugosa using ground nut oil cake under solid state fermentation, Int. J. Eng. Res. Appl. (1), pp. 571- 577. Rigo, E., Polloni, A.E., Remonatto, D., Arbter, F., Menoncin, S., Oliveira, J.V., Oli-veira, D., de Treichel, H., Kalil, S.J., Ninow, J.L., Luccio, M.D. (2010) Esterification activity of novel fungal and yeast lipases, Appl. Biochem. Biotech. (162), pp. 1881-1888. Castilho, L.R., Polato, C.M., Baruque, E.A., Sant, A., Freire, D.M. (2000) Economic analysis of lipase production by Penicillium restrictum in solid-state and submerged fermentations, Biochem. Eng. J. (4), pp. 239–247. Salihu, A., Alam, Z. M., Abdulkarim, M. I., Salleh, H. M. (2011) Optimization of lipase production by Candida cylindracea in palm oil mill effluent based medium using statistical experimental design. Journal of Molecular Catalysis B: Enzymatic (69), pp. 66-73. Gopinath, S. C., Anbu, P., Hilda, A. (2005). Extracellular enzymatic activity profile in fungi isolated from oil- rich environments. MycoScience (46), pp. 119-126. Salihu, A., Alam, Z. M., Abdulkarim, M. I., Salleh, H. M. (2012). Characterization of Candida cylindracea lipase produced from Palm oil mill effluent based medium. International Journal of Chemical and Biochemical Sciences (2), pp. 24-31.

16. Serri, N.A., Kamarudin, A.H., Abdul Rahaman, S.N. (2008) Preliminary studies for production of fatty acids from hydrolysis of cooking palm oil using C. Rugosa lipase. J Phys Sci. 19 (1), pp. 79–88.

17. Qian, M.C., Oscar, A.P. (2010) Fat Characterization, Nielsen SS, editor. Food Analysis Laboratory Manual, New York: Springer, pp.108-109. 18. Marlot, C., Langrand G, Triantaphylides C, Baratti J. (1985) Ester synthesis in organic solvent catalyzed by lipases immobilized on hydrophilic supports. Biotechnol. Lett. (7), pp. 642-647. 19. Macrae A.R. (1985) Biocatalysts in Organic Synthesis. In: Tramper J, Van der Plaas HC, Linko P (editors). Studies in Organic Chemistry, Amsterdam, UK: Elsevier; p. 195-208. 20. Lima, V.M.G., Krieger, N., Mitchell, D.A., Fontana, L.D. (2004) Activity and stability of crude lipase from Penicillium aurantiogriseum in aqueous media and organic solvents. Biochem Eng J (18), pp. 65–71.