4.MI-Trismillah Available online at http://jurnal.permi.or.id/index.php/mionline DOI: 10.5454/mi.10.4.4ISSN 1978-3477, eISSN 2087-8575 Vol 10, No 4, December 2016, p 139-148 *Corresponding author; Phone:+62-8161435792, Fax: - 7566922, Email: trismilah@bppt.go.id +62 Lipases are triacylglycerol acyl-hydrolases (EC 3.1.1.3) that catalyze the hydrolysis of triacylglycerol to glycerol and fatty acids (Verma et al. 2012). The enzymes are capable of hydrolyzing the ester bonds of water-insoluble substrates at the interface between the substrate and water. It is well known that the reaction is reversible and the enzymes catalyze both ester synthesis and transesterification. Since lipases catalyze a number of different reactions, they have been widely used in such industries as food, chemical, and pharmaceutical, detergent industries as well as in leather processing industry, and also for producing better quality products as part of an eco-friendly process (Selvamohan et al. 2012; Hanifi et al. 2013; Francoiset al. 2014). Lipases are ubiquitous enzymes Lipase is a lipids hydrolyze enzyme which are widely used in various industries such as chemical, pharmaceutical, food industries, and detergents. Bacillus licheniformis F11.4 is one of the bacteria with potential source of lipase. This study aimed to obtain optimum production of lipase from B. licheniformis F11.4 by optimizing the composition of media and pH values w​​ ith fish flour as a replacement for peptone and yeast extract based medium. Selection of the significant factors used a 2-level factorial design. The upper limit and lower limit of the selected factors was optimized using Central Composite Design (CCD) and the data analysis was performed using the Response Surface Methodology (RSM). Fermentation was carried out in erlenmeyer at initial pH 8 and a temperature of 37 °C, using a shaker incubator at 150 rpm. A fermentation system for lipases production is considered optimal when its desirability value closes to 1. By using numerical optimization, an optimal medium could be obtained, i.e. consisting of olive oil (OO): crude palm oil (CPO) 0.14 % (w/v) and fish flour 2% -1 (w/v), at pH 8 and 150 rpm, which produced lipase with enzyme activity of 1.563 U mL and protein level of 0.08 -1 mg mL . Furthermore, the results are verified in the Erlenmeyer, working volume of 50 mL, pH = 8, T = 37 °C, -1 agitation 150 rpm, t=18 hours, the activity of lipase and protein levels are 1.568 ± 0.014 U mL and 0.072 ± 0.006 -1 -1 mg mL respectively.The results showed that the optimum condition lipase activity was 1.568 U mL so that the the activity of only 75% compared to that before optimization. Key words: Bacillus licheniformis F11.4, Central Composite Design, enzyme activity, protein content, Response Surface Methodology Lipase merupakan enzim penghidrolisis lipida yang banyak digunakan di berbagai industri seperti kimia, farmasi, makanan, maupun aditif detergen. Bacillus licheniformis F11.4 adalah salah satu bakteri yang potensial sebagai sumber lipase. Penelitian ini bertujuan untuk mendapatkan produksi lipase yang optimal dari B. licheniformis F11.4 melalui optimasi komposisi media dan nilai pHdengan tepung ikan sebagai pengganti pepton dan yeast ekstrak dalam medium. Pemilihan faktor-faktor yang berpengaruh secara signifikan menggunakan desain faktorial 2 level. Batas atas dan batas bawah faktor-faktor yang terpilih selanjutnya dioptimasi menggunakan Central Composite Design (CCD) dan analisis data dilakukan dengan menggunakan Respone Surface Methodology (RSM). Fermentasi dilakukan di dalam erlenmeyer pH awal 8 dan suhu 37 °C, menggunakan shaker inkubator 150 rpm. Sistem fermentasi untuk produksi lipase dikatakan optimal jika nilai desirability mendekati 1. Dengan optimasi numerik nilai desirability mendekati satu, komposisi media fermentasi terbaik adalah minyak olive (OO) : minyak sawit mentah (CPO) = 0,14 % (b/v); tepung ikan = 2% (b/v) dan nilai pH = 8, agitasi 150 rpm, aktivitas enzyme dan kadar protein yang akan diperoleh masing- -1 -1 masing adalah 1,563 U mL , dan 0,08 mg mL . Selanjutnya hasil tersebut diverifikasi di dalam Erlenmeyer, volume kerja 50 mL, pH = 8, T = 37 °C, agitasi 150 rpm, t = 18 jam, memberikan nilai aktivitas lipase dan kadar -1 -1 protein masing-masing adalah 1,568 ± 0,014 U mL dan 0,072 ± 0,006 mg mL . Hasil penelitian menunjukkan -1 bahwa pada kondisi optimum aktivitas lipase adalah 1,568 U mL sehingga aktivitas hanya 75% dibandingkan dengan kondisi sebelum optimasi. Kata kunci: aktivitas enzim, Bacillus licheniformis F11.4., Central Composite Design, aktivitas enzim, kadar protein, Response Surface Methodology Optimization of Lipases Production byBacillus licheniformis F11.4 using Response Surface Methodology 1 2 1 TRISMILAH *, PRIH SARNIANTO , AND EDI WAHJONO 1 Center for Bioindustrial Technology, Agency for the Assessment and Application of Technology (BPPT), LAPTIAB, Bld.610-612, PUSPIPTEK, South Tangerang , 15314, Indonesia; 2 Faculty of Pharmacy, Universitas Pancasila, Srengseng Sawah, Jagakarsa, Jakarta 12640, Indonesia synthesized by all biological system, including animals, plants, and microorganisms (bacteria, fungi, yeast, and actinomycetes). Microbial lipases have gained special attention due to their ability towards both extreme temperature and pH, organic solvents as well as chemo-, region-, and enantio-selectivity (Devi et al. 2012; Verma et al. 2012; Kumar et al. 2007). Among lipases producing bacteria, Pseudomonas aeruginosa, Enterobacter aerogenes and some species of Bacillus such as B. subtilis, B. pumilis, B. licheniformis, B. thermoleovorans, B. stearothermophilus, and B. sphaericus produce lipases suitable for biotechnologi- cal applications. Widely known as a multipurpose organism, B. licheniformis has gained popularity along with B. subtilis. B. licheniformis, which is commonly found in soil and on the feathers of ground dwelling birds (Maria et al. 2013), produces extracellular lipases. Lipases production by B. licheniformisis affected by many physicochemical (particularly pH and temperatu- re) and nutritional factors (the nature and availability of carbon and nitrogen substrates as well as lipids). In bacterial fermentation, peptone is usually used as the main source of nitrogen with yeast extract as the source of growth factors, including vitamins.To increase the production of lipases, olive oil is often used both as inducer and carbon substrate (Niyonzima et al. 2013). However, peptone, yeast extract, and olive oil are quite expensive and not very easy to obtain. In the present study, peptone and yeast extract were entirely substituted with fish flour as a source of nitrogen, then olive oil and crude palm oil (CPO) can be used as a carbon source or inducer in media production lipase (Limpon et al, 2012). To determine the optimal substrate composition as well as fermentation condition (pH, temperature), Response Surface Methodology (RSM) was used along with Central Composite Design (CCD) technique. RSM is the most popular optimization method used in recent years. There are so many works based on the application of RSM in chemical and biochemical process (Deniz et al. 2007). RSM experimental design is an efficient approach to determine which explanatory variables (e.g. medium componentand/or fermentation condition) have impacts to the response variables of interests (e.g. growth rate, protein level or productivity) (Box et al. 2005), and there are several reports on application of RSM for the production of primary and secondary metabolites through microbial fermentation (Devi et al. 2012; Dash et al. 2002). CCD technique contains an imbedded factorial or fractional factorial design with “center points” that is 140 TRISMILAH ET AL. Microbiol Indones augmented with a group of “star points”, allowing estimation of curvature (Sematech 2010). This study aimed to obtain optimum production of lipase from B. licheniformis F11.4 through optimization of media composition and pH values ​​with fish flour as a replacement for peptone and yeast extract based on Sharma media that has been modified (Sharma et al. 2012). MATERIALS AND METHODS Materials. B. licheniformis F11.4 is a collection of the Laboratory of Bioindustrial Technology- LAPTIAB, BPPT, Serpong. Fish flour from CV Pratama Hikmah Karya, Cimahi, Bandung, Indonesia; olive oil (OO) a trademark of Le Riche; crude palm oil (CPO) a product of PT Sinar Mas, Indonesia; tryptone, agar, peptone, yeast extract, NaCl from HiMedia Laboratories Pvt. Ltd., Mumbai, India. Other ingredients and equipment are commonly used for bioprocess research. Regeneration of the Microorganisms. A colony of B. licheniformis F11.4 was transferred from the stock culture onto Luria Bertani Agar and incubated at 37 °C or 50 °C, pH 7, for 16 h. Propagation of the Microorganisms. One colony of regenerated B. licheniformis F11.4 was innoculated into Luria Bertani (LB) liquid media and incubated at 37 °C in a shaking incubator (Kuhner) with constant agitation at 150 rpm, for 8-16 h, up to optical density (OD) of 0.6-0.8. The suspension of bacterial cells were then used as bacterial starter in the production of lipases. The LB liquid media were prepared by dissolving into RO water adjusting its pH to 7 by adding HCl 1M or NaOH 1M, and sterilizing the mixture in autoclave for 20 min at 121 °C. Production of Lipase. The bacterial starters (100 mL) were innoculated into liquid medium for lipase production (900 mL), incubated at 37 °C with initial pH of 8 and agitation at 150 rpm, for 18 h. The number of 7 -1 bacteria were minimum 10 cells mL , determined prior to the incubation (hour zero). The fermentation medium used was described by Sharma with some modification (Sharma et al. 2012) consisted of 3% pepton ,1% yeast extract, 0.3% NaNO ,0.01% KH PO , 3 2 4 1% olive oil (OO), and 1% CPO. In the modification, peptone and yeast extract were substituted with fish flour of equal protein content. The protein content was determined by using modified Lowry’s method. In the present study, two kind of fish flours, one made of bony fish (P) and the other of more-fleshy fish (N), were compared. Regarding the inducer, the proportion of OO and CPO was modified to 0.25% (w/v) and 0.75% (w/v), respectively, in accordance with the results of the preliminary study on OO and CPO requirementf o r lipase production by B. licheniformis F11.4. Harvesting of Lipase: The enzyme harvesting were carried out by separating the supernatant from biomass through centrifugation at 10 000 rpm for 15 min at 4 °C. The supernatant, termed as crude (extracellular) enzyme fraction, was assayed for its lipase activity. Assay for Lipase Activity. The crude enzyme’s lipase activity was determined using spectrophotome- tric method. As the substrate, 3 mg of paranitrophenyl- palmitate (p-NPP) was dissolved in 1 mL isopropanol and mixed with 10 mg of arabic gum and 40 mg of triton X-100 in 9 mL of 0.05M Tris-HCl buffer solution of pH 8. Into 0.9 mL of substrate, 0.1 mL crude enzyme was added and then incubated at 37 °C in a thermoshaker with constant agitation at 300 rpm, for 30 min. The absorbance of the incubated mixtures was measured at λ410 nm against blank. Lipase activity was calculated using the following formula (Silva et al. 2005): Where C = concentration of p-NPP (mg mL-1) , t = time (minute), V = Volume of crude enzyme (m), and molecular mass of p-NPP (MR p-NPP) = 377.52. Protein content of the crude enzyme was analyzed by spectrophotometric method using Bradford reagent (Bradford 1976). Optimization Using RSM. In the present study, optimization using RSM was performed in two stages. In the first stage, two-level factorial design, the proportion of OO and CPO (0.1-0.5% w/v) as inducers, fish flour (1.8-5.4%) as the main source of nitrogen, and pH (8-10) were treated as independent variables with lipase activity as the response variable. The fermenting media also contained 3% NaNO and 0.1% 3 KH PO ; and B. licheniformis F11.4. innoculated were 2 4 incubated at 37 °C with constant agitation at 150 rpm. The data were analyzed with Design Expert (version 7.1.5, Stat-Ease Inc., USA) software. Media composition that gave the highest lipase activities were used to determine the upper limit and lower limit of independent variables for the second stage optimization experiment that used central composite design (CCD). 3 In the second stage, factorial design 2 which was expanded with 6 starting points and 6 center points were used, allowing 20 combinations of OO, CPO, and fish flour. For each combination, fermentation was carried out in a 125 mL erlenmeyer flask using 25 mL fermenting media of pH 8, containing 3% NaNO and3 0.1% KH PO , at 37 °C and 150 rpm for 18 h. 2 4 Numerical optimization of the results was performed using the Design Expert software.The ideal desirability value in numerical optimization is 1.0. The results of the numerical optimization that close to the ideal desirability value are verified in the laboratory with a minimum of 6 replicates. RESULTS Optimization Using Response Surface Metho- dology. In the first stage, optimization of lipase production was conducted by using a two-level factorial design experiment toward the three affecting factors, i.e. OO:CPO was coded A, fish flour was coded B, and pH was coded C, with lipase activity as the variable response. The Design Expert software was used to generate the experimental designs, of which produced 12 experimental design models (Table 1). The statistical significance of the models in lipase activities of crude enzymes produced were evaluated using analysis of variance (ANOVA) and the results are presented in Table 2. From the first stage of research the two-level factorial, used to specify an upper limit and a lower limit on the central composite design (CCD) as shown in Table 3. Based on the data in Table 3 of research second stage using composite design center (CCD) with 3 factors A, B, and C and two response variables are the activity and protein levels and resulting 20 combination of treatments as shown in Table 4. Further data enzyme activity and protein levels of 20 combinations of research results incorporated into the program Design Expert 7.1.5. and the results of the analysis can be seen in Fig 1 and Fig 2. The results of ANOVA for lipase activity response can be seen in Table 5, and the interaction between the media composition and pH value on the response of lipase activity can be seen in Fig 3a, Fig 3b, and Fig 3c. The test results of analysis of variance (ANOVA) response protein levels can be seen in Table 6, while the interaction between the media composition and pH value on the response of lipase activity can be seen in Fig 4a, Fig 4b, and Fig 4c. The results of numerical calculation models media optimization and the pH value based on the central Volume 10, 2016 Microbiol Indones 141 -1 Lipase activity (U mL ) = C × 1000 MRpNPP × t × v Microbiol Indones142 TRISMILAH ET AL. model is larger than F table (28.96> 4.87) and p-value = 0.0094 <α = 0.05 shows a model of a significant or unacceptable to see the estimated effect of each variable and interaction factor with the response. The 2 coefficient of determination (R ) 0.9854 shows a high correlation between the influence of factors on the response variable. Point arc curve (curvature) also showed a significant result so that the model can be used to define the design of the central composite design of experiments process (Central Composite Design/CCD) to determine the optimal media composition in the production of lipase B. licheniformis F11.4. Of the two- level factorial experiments obtained the highest lipase -1 activity of 1.198 U mL in the combined treatment of 0.1% OO:CPO; 1.8% fish flour and 8 pH value is then used to determine an upper limit and a lower limit on CCD (Table 3). From the central composite design (CCD) by Design Expert 7.1.5 program provides 20 combined treatment (Table 4). Results of analysis of variance Design Expert 7.1.5 in Table 5 and Table 6 is the response to enzyme activity and protein levels indicate that a significant model analysis with P values ​​<5%, which means that the model can be used for process composite design (CCD) and Response Surface Method (RSM) using Design Expert 7.1.5 program delivering provide desirability 0.764 with composition 0.1% (w/v) OO:CPO; v2.04% (w/v) fish flour and pH 8. Desirability value approaching 1 (one) obtained by trial and error using Design Expert 7.1.5 program. From the trial and error were obtained twelve combination with desirability value = 0.921. DISCUSSION Factors that influence the production of lipase B. licheniformis F11.4. were determined by two level factorial experimental design. Response lipase B. licheniformis F11.4. was observed on hour to 8, 14, and 18. The result of the interaction between the concentration OO:CPO, the concentration of fish flour and pH values ​​were analyzed using Analysis of Variance (ANOVA), selected to give the highest activity on the observation hours to 18. The analysis results in Table 2 show that the model of interaction between the concentration OO:CPO, the concentration of fish flour and the pH value to yield significant results. From the analysis of variance, the value of F of the Table 1. Generated experimental design models for lipase production by B. licheniformis F11.4 Run OO:CPO [A] Fish flour [B] pH [C] -1 Activity, U mL 1 0.50 5.40 10.00 0.969 2 0.50 5.40 8.00 0.511 3 0.10 1.80 8.00 1.198 4 0.30 3.60 9.00 0.695 5 0.50 1.80 10.00 0.463 6 0.50 1.80 8.00 1.150 7 0.30 3.60 9.00 0.691 8 0.10 1.80 10.00 0.618 9 0 .30 3.60 9.00 0.751 10 0.10 5.40 8.00 0.875 11 0.10 5.40 10.00 0.933 12 0.30 3.60 9.00 0.801 Factors affecting lipase production by B. licheniformis F11.4: A = ratio of olive oil and CPO = OO/CPO, upper limit 0.5% (w/v), lower limit 0.1% (w/v) B = fish flour P, upper limit 5.4 % (w/v), lower limit 1.8% (w/v) C = pH, upper limit 10, lower limit 8 Volume 10, 2016 Microbiol Indones 143 Second equation (quadratic) was obtained from Design Expert 7.1.5 (Equation 2), which explains the data response on protein content. The accuracy of the model can also be seen from a comparison of the actual value of research with model predictions. Prediction model (Predicted) expressed as a straight line and the actual results of the study (Actual) expressed as a box. From Fig 1 and Fig 2 can be seen that the actual value (of the box) scattered approach the predicted values (​​ straight line), it indicates the optimization of lipase production. Addition of test results Lack of Fit to the model can be seen that there are no inaccuracies models, it can be proved of value for Lack of Fit obtained P value 0.052 (Not significant) for the response to the activity and the P value 0.2136 (Not significant) for response to the protein content, meaning that the regression model is accepted. Second equation (quadratic) was obtained from Design Expert 7.1.5 (Equation 1), which explains the data response on enzyme activity. Table 2. ANOVA of the factors affecting lipase production by B. licheniformis F11.4 Table 3 The upper limit and lower limit factors for central composite design (CCD) Factor Name Low Actual High Actual A OO:CPO (% w/v) 0.05 0.18 B Fish flour (% w/v) 0.8 3.1 C pH 7 9 Y = + 2.197 - 8.314 A + 0.209 B - 0.411 C + 3.512 AB + 0.827 AC - 0.032 BC + 5.702 A2 + 8.122 B2 – 0.021 C2.................................................... (Eq.2) Y = 20.024 + 5.773A + 0.338 B +5.219 C + 0.148 AB + 0.189 AC + 0.075 BC – 25.056 A2 – 0.255 B2 – 0.337 C2................................................... (Eq.1) – Microbiol Indones144 TRISMILAH ET AL. -1 Table 4 The data activity (U mL ) and protein content (mg ) of were combined treatments of OO:CPO, Fish flour and the pH value, the result of the central composite design (CCD) by the program Design Expert 7.1.5. -1 mL Fig 1 Distribution of actual and predicted values in the lipase production activity respon. Volume 10, 2016 Microbiol Indones 145 saddle (saddle point) so that the optimum point of the curve a little bit difficult to determine. The calculation numerical models of media optimization and the pH value is based on the CCD and the analysis of RSM provides desirability value 0.764. Trial and error using Design Expert 7.1.5 program there are twelve combinations that provide value desirability 0.921. (w/v) (w/v) f -1 mL -1 mL (w/v) (w/v) f Validation study was conducted in 125 mL Erlenmeyer, initial pH 8 fermentation time 18 h with the addition 0.3% NaNO and 0.1% KH PO with six 3 2 4 replications, providing value lipase activity 1.568 ± -1 0.014 U mL and 0.072 ± lipase protein content of -1 0.006 mg mL or the value of a specific activity is -1 21.210 U mg . From India reported by Sharma et al. 2012, produce lipase from B. licheniformis MTCC- 10498, inducers tween 80 0.5% w/v, pH 7.5, a temperature of 55 °C, 150 rpm, fermentation time of 72 -1 h the enzyme activity of 2.1 U mL . From India was also reported by Devi et al. 2012, that for the As for the lower limit and upper limit, 0.01- 0,14 % OO:CPO; 0.8 to 2 % ish flour and pH 7.8 to8; value will be the activity of 1.563 U and the protein content of 0.08 mg with media compositions 0.14% OO:CPO; 2 % ish flour and pH 8. deviation is low both in response to the activity as well as on the response of the protein content is evidenced by the standard deviation of ANOVA each is 0:21 and 0:06. A low standard deviation shows that the model has good accuracy or the model correspond (fit model). According Gaspersz (1995) the model is said to be proper if the assumption residual meet the assumption of normal distribution. If the residual plot spread randomly around zero and likely to approach a straight line (linear line) so as to be normally distributed. ANOVA test results in Table 5 obtained determina- 2 tion coefficient (R ) 0.8824 for the response to enzyme activity and in Table 6 for a response to the protein 2 content obtained determination coefficient (R ) 0.7312. It shows that 88% correlation activity response variable and 73.12% variable response to the production of lipase protein levels are influenced by independent variables (OO:CPO; fish flour: pH value). The optimum area of independent variables (factors) that produces the maximum response can be seen from the three- dimensional contour plot curves in Fig 3a that the interactions between fish flour, the pH of the enzyme activity showed that the parabolic curve is obtained. In Fig 3b; 3c and Fig 4a; 4b; 4c curve obtained shaped Fig 2 Distribution of actual and predicted values in the lipase production protein content respon. Microbiol Indones146 TRISMILAH ET AL. -1 specific enzyme activity of 32.2 U mg . The results of the optimization study B. licheniformis F11.4 lipase production gives the -1 enzyme activity 1568 ± 0014 U mL , lipase protein -1 content of 0.072 ± 0.006 mg mL and a specific enzyme -1 activity of 21.77 U mg . The optimum medium composition of fishflour 2% (w/v), olive oil 0.07% (w/v), CPO 0.5% (w/v), NaNO 0.3% (w/v), and 3 KH PO 0.1% (w/v) in the working volume of 50 mL 2 4 production of lipase B. subtilis with inducers tween 80 and using the method RSM. Media optimization -1 results with yeast extract 9.3636 g mL , CaCl 0.8986 g -1 mL and incubation periods 1.813 d, gives the activity -1 -1 enzyme 16.627 U min mL . Anahita et al (2011) from Malaysia also reported that the research of production the lipase Acinobacter sp. in submerged fermentations using RSM at the optimum condition at the fermentation time 24 h, T = 29 °C, pH = 6, the value of a Table 5 ANOVA lipase activity response of experimental design CCD Fig 3 Interaction OO: CPO, fishflour, pH on lipase activity response analysis results Surface Method (RSM) using the program Design Expert 7.1.5 Volume 10, 2016 Microbiol Indones 147 assistance during the completion of this study and PTB-BPP Technology, which has provided funding and laboratory facilities for this study. REFERENCES Anahita K, Afshin E, Boon KB, and Oi ML.2011. Production of a solvent, detergent, and thermotolerant lipase by a newly isolated Acintobacter sp. in erlenmeyer with the operating conditions of pH 8, temperature 37 °C, agitation 150 rpm and fermentation time of 18 h. The increase in the activity of only 75% -1 compared to before the optimization ie 1,176 U mL . 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