Potential Yeast from Indonesian Wild Forest Honey Showing Ability to Produce Lipase for Lipid Transesterification

Microbiol. Biotechnol. Lett. (2019), 47(4), 555–564 http://dx.doi.org/10.4014/mbl.1907.07008 pISSN 1598-642X eISSN 2234-7305 Microbiology and Biotechnology Letters Potential Yeast from Indonesian Wild Forest Honey Showing Ability to Produce Lipase for Lipid Transesterification Prayolga Toban Palilu, Rina Sri Kasiamdari, and Miftahul Ilmi* Faculty of Biology, Universitas Gadjah Mada, Jl. Teknika Selatan, Sekip Utara, Yogyakarta 55281, Indonesia Received: July 18, 2019 / Revised: September 13, 2019 / Accepted: September 16, 2019 Biodiesel is produced through the transesterification process in the presence of alcohol and a catalyst that catalyzes the conversion of triglycerides to esters and glycerol compounds. A more optimal product conversion can be achieved using enzymes, such as lipase. Lipase is reported to be produced in osmophilic yeasts due to the low water content in their natural habitats. Wild forest honey is one of the osmophilic natural habitats in Indonesia. However, lipase-producing yeast has not been reported in the Indonesian honey. In this study, we screened the lipase-producing yeasts isolated from wild forest honey collected from Central Sulawesi. The production profile and activity of lipase were determined at different pH values and temperatures. One promising yeast was isolated from the honey, which was identified as Zygosaccharomyces mellis SG 1.2 based on ITS sequence. The maximum lipase production (24.56 ± 1.30 U/mg biomass) was achieved by culturing the strain in a medium containing 2% olive oil as a carbon source at pH 7 and 30℃ for 40 h. The optimum pH and temperature for lipase activity were 6 and 55℃, respectively. The enzyme main- tained 80% of its activity upon incubation at 25℃ for 4 h. However, the enzyme activity decreased by more than 50% upon incubation at 35 and 40℃ for 2 h. This is the first study to report the lipase producing capa- bility of Z. mellis. Further studies are needed to optimize the enzyme production. Keywords: Osmophilic yeast, production profile, screening, Sulawesi, Zygosaccharomyces mellis Introduction catalyst in biodiesel production as well as modifications of fats by transesterification reactions [3]. Lipase (triacylglycerol acyl hydrolases, E.C 3.1.1.3) Biodiesel can be produced by esterification and esteri- catalyses the hydrolysis of triglycerides in a medium fication to produce esters. Esterification is catalysed with high water availability. However, under low water using acids, whereas transesterification catalysed by conditions, the enzyme tends to catalyse the esterifica- acids, bases, or enzymes [4]. Transesterification is a tion reaction. reaction of a triglyceride with alcohols in the presence of The ability of lipase to carry out various types of reac- a catalyst to produce esters with glycerol as the by-prod- tions makes it widely useful in various industrial appli- uct. Bases are the most used catalyst because it is cation such as additives in detergents, the elaboration of cheaper than the other catalyst and gives high reaction dietetic foods for use in the food industry and synthesis rates [5]. However, base catalysts are known to lead to chemical processes [1, 2]. Lipase also can be used as a saponification of FFAs, generating emulsions which are difficult to break and lead to serious issues in down- *Corresponding author stream processing [4]. Enzymes can also be used to con- Tel: +62 81 392709667 E-mail: [email protected] vert plant oils into biodiesel and give a higher product © 2019, The Korean Society for Microbiology and Biotechnology purity, require less energy input and enable the use of a December 2019 | Vol. 47 | No. 4 556 Palilu et al. wider range of feedstocks [3, 4]. glucose placed in 250 ml of Erlenmeyer flasks. The Lipases occur widely in microbes [6, 7] and considered medium was incubated at room temperature and as the easiest handle than the other producers. Microbes 200 rpm shaking for 3 days. The cell density was tested from the osmophilic environment, such as honey, poten- with a spectrophotometer with a wavelength of 600 nm. tially to produce lipase with high transesterification Cultures with the absorbance of 0.8 were inoculated on reaction rate considering the low water availability in liquid production media. habitat [8]. Most of the lipase research focuses on the The composition of the production medium to grow production of extracellular lipases through a wide vari- yeast and produce lipase was modified from a previous ety of microorganisms [9, 10]. study [15]. The medium containing 3% (w/v) peptone, Indonesia is one of the world’s wild forest honey pro- 0.05% (w/v) MgSO4, 0.05% (w/v) KCl, 0.2% (w/v) ducers due to its high diversity of native flowers and K2HPO4, 1% (v/v) olive oil, and 1% (w/v) glucose was bees [11]. The honey is a potential habitat for various placed in 250 ml Erlenmeyer flasks. The flasks were microorganisms, especially lipase-producing yeasts. The inoculated with 10% (v/v) seed culture and incubated at previous study was able to isolate 26 yeast isolates from room temperature with 100 rpm shaking. Culture har- wild forest honey of Central Sulawesi [12]. However, the vested every 12 h for 3 days to determine the best time ability of isolated yeasts to produce lipase is unknown. production of each isolate. All experiments were done in In this study, we screened lipase producing ability triplicate. form the forest honey yeasts using qualitative and quantitative approaches. We also determined the enzyme Lipase production and activity measurement production profiles of the potential isolate. Further, The yeast biomass was harvested from 10 ml medium activities of the produced lipase on different pH and tem- by centrifugation (4000 rpm for 20 min) and dried in an peratures were measured. oven at 60℃ for 5 days. The weight of the biomass was measured in milligrams. The content of extracellular Materials and Methods lipase was estimated according to the lipase activity in the supernatant. Microorganism and culture condition Lipase activity of the supernatant was determined by Twenty-six yeast isolates from wild forest honey of incubating 100 µl filtrate with 1 ml isooctane containing Central Sulawesi were provided by Laboratory of Micro- 0.25 M oleic acid and 0.25 M ethanol for 20 min at 30℃. biology, University of Gadjah Mada (UGM), Yogyakarta. The amount of oleic acid at 0 min and after 20 min was Pure isolates were kept in potato dextrose agar medium determined using the cupric-acetate pyridine colorimet- (PDA). ric assay [16]. One unit of activity (U) is defined as the amount of oleic acid (µmol/ml) converted to product per Screening of lipase production min. Yeast lipase productivity (U/mgbiomass) is the ratio Primary screening. The screening was done using phe- of lipase activity (U/ml) and cell biomass (mg/ml). nol red with 0.04% olive oil as carbon source and 0.1% Tween 80 as an inducer. Yeast that grows and forms the Molecular identification yellow zone around the colonies is considered as lipolytic Molecular identification was carried out on 1 isolate [13]. Lipolytic index (IL) is expressed as a comparison with the highest productivity. DNA isolation was carried between the diameter of the clear zone formed with the out with the Zymo Research Kit, USA. The qualitative diameter of the colony [14]. DNA testing was carried out by electrophoresis and quantitative DNA testing with nanodrop. The quantita- Secondary screening. The screening was carried out tive test of DNA is the measurement of DNA concentra- by growing isolates with IL > 2 in the seed culture first tion with the sample concentration needed is 10−250 to induce lipase production. Seed culture media also (ng/µl). known as YPMG media contain 3% (w/v) yeast extract, DNA was amplified by PCR using ITS 1F 3'-TCC 5% (w/v) peptone, 3% (w/v) malt extract and 10% (w/v) GTA GGT GAA CCT G-5 GCG and ITS 4R 3'-TCC http://dx.doi.org/10.4014/mbl.1907.07008 Lipolytic Yeast from Indonesian Honey 557 TCC TAT TAT GC-5 TCC GC-5’ as primers. The tem- overnight. The precipitate was obtained by centrifuga- perature program on PCR process are pre-denaturation tion (14,000 g, 20 min at 4℃). The precipitates were 95℃ for 3 min, denaturation 95℃ for 30 s, annealing resuspended in a minimal amount of distilled H2O and 63.5℃ for 1 min, extension 72℃ for 1 min 40 s, and final dialyzed against distilled water using a successive large extension at 72℃ for 1 min 40 s. volume of H2O. The total protein after dialysis was PCR products in the form of amplicons are sequenced determined using Bradford method [17]. to find out the number and sequence. The amplicon was sent to PT. Genetika Science, Jakarta. The sequences Enzyme activity in different pH and temperatures. The obtained are edited with GeneStudio TM Version 2.2.0.0. effect of pH was determined by measuring lipase activity The analysis of gene sequence alignment obtained was at different pH ranging from 4 to 10. The pH of the reac- done by the Basic Local Alignment Search Tool (BLAST) tion mixture was set using different buffers (citrate program on the NCBI gene bank to determine the per- buffer for pH 3−5, phosphate buffer for pH 5−7 and centage of similar base pairs with references found in borate buffer for pH 7−9). The effect of temperature on GenBank. The similarity value calculated by Phydit lipase activity was determined by measuring lipase 3.1. activity at different temperatures ranging from 25 to 45℃. Lipase activity was determined using previously Profiles of enzyme production described methods. Lipase specific activity (U/mg) is the The profile of enzyme production was determined from ratio of lipase activity (U/ml) and total protein in enzyme 1 isolate with the highest productivity grown in seed cul- solution (mg/ml).

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