Optimization of Lipase Production from Local Bacteria Isolate with Palm Oil Inducer H )

Received: 28th June 2020 Revised: 26th November 2020 Accepted: 11th February 2021 Online: 15th March 2021 A lipase is a group of hydrolase enzymes with multi-functional abilities such as catalyzing hydrolysis, alcoholysis, esterification, interesterification, and synthesis of organic compounds. The purpose of this study was to obtain the optimum conditions for lipase production and to study the effect of adding a palm oil inducer on the production of lipase from local bacterial isolates. The method used in this study included the rejuvenation of local bacterial isolate cultures, optimization of production, which included pH, palm oil inducer, and incubation time. The lipase activity test was carried out by using the acid-base titration method with the phenolphthalein indicator. The results showed that the optimal conditions for lipase production occurred at pH 5 with an activity value of 7.5 U/mL and an incubation time of 18 hours with an activity value of 13.83 U/mL. The addition of a 10% (v/v) palm oil inducer increased the value of lipase activity to 8.75 U/mL.


Introduction
Lipase is a group of hydrolase enzymes with the IUPAC name triacylglycerol ester hydrolases (E.C. 3.1 . 1.3 ), which function to hydrolyze triglycerides to become fatty acids and glycerol. Other reactions that lipase can catalyze interesterification, and synthesis in organic reactions [1]. Lipase also plays a role in separating fatty acids and dissolving the oil in industrial equipment to mix with water [2]. Due to the extensive function of lipase catalysis, lipase is widely marketed and used in various fields of industry and biotechnology [ 3 , 4 ].
Lipase can be isolated and purified from fungi, yeast, bacteria as well as plants and animals. However, lipase derived from microbes is most widely used in the industrial world because it is more economical, stable, easy to cultivate, and generally resistant to heat, although the bacteria that produce it are not resistant to heat [ 4 ]. Lipase produced by microbes can come from different environments, including industrial waste disposal environments, factories processing vegetable oil, oil-contaminated soil, oil derived from seeds, food spoilage processes, coal, and compost piles [ 5 ].
Initial studies conducted on eight isolates, obtained from the mesophilic phase of the domestic waste composting process with a composting temperature of 37°C , found that there were isolates with lipase activity, which was LKMA 3 isolates. However, this isolate still had a relatively low lipase activity of 0.1666 U / mL [6]. To obtain lipase with relatively better activity, optimum growth media is needed to develop lipase-producing microbes. The influencing factors include the media ' s nitrogen content and the physicochemical factors such as pH, temperature, and incubation time [2]. Lipase production will also increase if a suitable inducer is present in the medium. An inducer is a substance added to the bacterial growth medium, which aims to stimulate bacteria ' s growth in producing lipase. Without an inducer, lipase is still produced but in small amounts [ 5 ]. Therefore, to get a relatively better value of enzyme activity, it is necessary to optimize the conditions, including pH, inducer addition, and incubation time.
alcoholysis esterification are In this study, the optimization of lipase production from local bacterial isolates -LKMA 3 , was obtained from the mesophilic phase of the composting process of domestic waste. The optimization phases carried out include pH optimization, incubation time, and the addition of palm oil inducers. The measurement of lipase activity resulting from each optimization stage was determined by the titration method [ 7 ].

Methodology
This research is an experimental study conducted to obtain the optimum conditions for lipase production and study the effect of adding palm oil inducers in lipase production from local bacterial isolates.
The materials used in this study were local isolate lipolytic bacteria from the domestic waste composting process with the LKMA 3 code, nutrient agar (NA) and Nutrient Broth (NB) for bacterial growth media, distilled water, olive oil (Herborist brand), Tween 80, phenolphthalein indicator, palm oil as the inducer, NaOH, 70 % alcohol, phosphate buffer, Na2C 03 , CuS 04 , acetone, and ethanol.

pH optimization
The pH optimization was carried out by growing LKMA 3 isolates on NB media using 0.1 M phosphate buffer with a pH range of 4 -6. Isolates were grown for 24 hours in an incubator shaker at a speed of 150 rpm at 37°C , then centrifuged. The supernatant obtained was determined by its lipase activity by the titration method

. Lipase activity test
The lipase activity test was carried out using the acid-base titration method by mixing 2 mL of olive oil, 1 mL of 0.1M phosphate buffer, and 1 mL of an enzyme, then incubated at 37°C for 30 minutes. The enzymesubstrate was inactivated with 1 mL of a mixture of acetone: ethanol (1: 1), then two drops of 1% phenolphthalein indicator were added, then titrated with 0.1 M NaOH. Titration was stopped when the solution changes color to pink [6]. Activity measurements were performed in duplicate for each sample. Determination of the blank was carried out with the same solution composition. After the enzyme was added, 1 mL of acetone: ethanol mixture (1: 1) was immediately added to inactivate the enzyme and then incubate at 37°C for 30 minutes. The same procedure performed titration as for sample analysis [ 4 ].

. Results and Discussion
The local isolate LKMA 3 bacteria were isolated from the mesophilic phase in the composting process of domestic waste at 37°C [6]. The results of the morphological test showed that the isolates obtained were gram-negative, bacilli-shaped, and motile.
Preliminary test results on the crude extract of the enzyme product before optimization showed a relatively low lipase activity of 0.166 U / mL. One way to increase the number of lipase products produced by bacteria is to add an inducer. The study results reported that the addition of palm cooking oil as an inducer was proven to increase the production of lipase from Aspergillus niger [ 7 ].
In this study, the optimization process of lipase production was carried out through variations in pH, incubation time, and the addition of an inducer to the LKMA 3 bacterial growth medium. The pH variation is carried out in the pH range 4 -6. Determination of the optimum pH for optimization of lipase production is presented in Figure 1.

. Optimization of additional inducers
The addition of an inducer was carried out at optimum pH conditions. Each bacterial growth medium was added with palm oil inducer with a concentration of 6%, 8%, 10%, 12%, 14 %, 16%, 18%, 20% (v / v). LKMA 3 isolates were grown in NB media that had been given an inducer and incubated in the same way as in Section 2.2.

Optimization of incubation time
LKMA 3 isolate bacteria were grown on NB media with optimum pH and inducer concentration.
Optimization of growth time was carried out for 48 hours in an incubator shaker at 37°C at 150 rpm. Each 6-hour interval sampling was carried out to determine the OD (Optical Density) value at a wavelength of 600 nm. Lipase activity testing was carried out by the titration method [ 4 ] Figure 1 shows that the optimum pH of LKMA 3 isolate bacteria originating from the mesophilic composting phase in producing lipase is at pH 5 with a lipase activity value of 7.50 U / mL. Meanwhile, at pH 4 and 6, the lipase activity values were 3.33 U / mL and 5.00 U / mL. An increase in bacterial lipase activity originating from the mesophilic environment of oil-contaminated industrial areas has also been reported. There was an increase in lipase activity from 7.5 U / mL to 8 U / mL at pH 6 after optimization [8].
In the optimization stage of lipase production, palm oil inducer was also added with a concentration range of 6%, 8%, 10%, 12%, 14 %, 16%, 18%, and 20% (v / v). Each inducer was added to the production medium at pH 5 and incubated for 24 hours at 37°C at 150 rpm. The lipase activity results by the acid-base titration method at various inducer concentrations are presented in Figure 2. palm oil inducer Figure 3 shows that the addition of a 10% palm oil inducer significantly increased local isolate bacteria LKMA 3 ( 24.67 %). The optimum condition occurs at the 18th hour (OD value = 2.6550 ), which is an exponential phase of bacterial growth of LKMA 3 isolates with the addition of an inducer. The bacterial growth of LKMA 3 isolates without palm oil inducers experienced slower growth, as indicated by the lower OD value of the exponential phase (OD = 0.6549 ). In the exponential phase, bacterial growth is very fast, so that the production of enzymes, including lipase, is very much produced by bacteria [6]. Enzymes function in metabolic processes that support cell growth and have been produced from the beginning of bacterial growth, which increases in the exponential phase and then decreases according to the cell growth phase [  The incubation time ' s optimization phase aims to obtain the optimum time required by the local isolate bacteria LKMA 3 to produce the maximum amount of lipase. In this study, the incubation time range started from the oth hour to the 48 th hour. During the incubation time, every 6 hours, optical density (OD) measurements were carried out at a wavelength of 600 nm, and determination of lipase activity by the acid-base titration method.
Optical density is one way to determine the number of bacterial cells by using the spectrophotometric method by measuring the OD value. The OD value is a measure of turbidity, which indicates the number / density of bacterial cells in the growth medium.
Measurement of OD values at certain times during the incubation period aims to determine the profile of bacterial growth [ 9 ]. The growth profile of local bacterial isolates-LKMA 3 in the conditions without and with the addition of palm oil inducer during the growth period is presented in Figure 3 .   Figure 4 shows that the optimum incubation time for bacteria to produce lipase was obtained at the 18th hour. The production of lipase by local isolate bacteria LKMA 3 at optimum conditions at 18 hours with 10% palm oil inducer was much more significant than without inducer. At 18 hours, the lipase activity with an inducer ' s addition was 13.83 U / mL, an increase of about six times greater than that without an inducer, which was 2.17 U / mL. This study indicates that the local isolate LKMA 3 bacteria can use palm oil as an inducer in increasing lipase production. In general, compounds that can be used as inducers contain long-chain fatty acids such as oleic, palmitic, stearate [10]. Palm oil contains high levels of oleic and palmitic acids to be used as an inducer to produce lipase. The addition of palm oil inducers can increase the growth and number of cells which causes the production of lipase to increase.