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Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci. (Jan–Mar 2018) 88(1):373–381 https://doi.org/10.1007/s40011-016-0763-6

RESEARCH ARTICLE

Purification and Characterization of Extracellular Lipase from Serratia marcescens VITSD2 V. Mohanasrinivasan1 • C. Subathra Devi1 • D. Jayasmita1 E. Selvarajan1 • S. Jemimah Naine1

•

Received: 28 September 2015 / Revised: 10 June 2016 / Accepted: 12 July 2016 / Published online: 22 July 2016 Ó The National Academy of Sciences, India 2016

Abstract Lipases represent a group of extracellular enzymes and hold a prominent place among the enzymes. In the present research work Serratia marcescens isolated from soil was selected for lipase production. Effects of various parameters such as substrate, pH, temperature, incubation time and inoculum size were analysed for the enhanced lipase production. The optimum substrate was found to be olive oil with the concentration of 0.7 mL in 100 mL of production medium. Serratia marcescens produced maximum lipase after 48 h of incubation at 30 °C with the optimum pH as 7.0. The inoculum concentration of 4 % was more favourable for the production of lipase. The extracted lipase was purified by two steps: ultra-filtration and Carboxymethyl(CM)-Cellulose chromatography. The specific activity of the purified enzyme was determined to be 65.0 U/mg with 2.55 purification fold and yield of 67.5 %. Polyacrylamide gel electrophoresis (SDSPAGE) analysis revealed the presence of a single band with a molecular weight of around 35 kDa. Based on Fourier transformation-infrared spectroscopy analysis the functional groups of lipase were determined and high performance liquid chromatography analysis with the retention time of 1.995 min reveals the presence of lipase in the purified extract. Keywords Serratia marcescens Lipase Purification Optimization

& C. Subathra Devi [email protected] 1

School of Biosciences and Technology, VIT University, Vellore, Tamil Nadu, India

Introduction The advent of enzymology represents an important breakthrough in the biotechnology industry, with the worldwide usage of enzymes being nearly U.S. $ 1.5 billion in 2000 [1]. The major share of the industrial enzyme market is occupied by hydrolytic enzymes, such as lipases, proteases, amylases, amidases, and esterases. In recent times, lipases (triacylglycerolsacylhydrolase, E.C.3.1.1.3) have emerged as key enzymes in swiftly growing biotechnology, owing to their multifaceted properties [2–4]. Lipases are produced from microbes and specifically bacterial lipases play a vital role in commercial ventures [5]. Microbial enzymes are often more stable and their production is more convenient and safer. Serratia sp. has been studied for its ability to produce lipase [6]. Serratia sp. is motile, gram negative, rod shaped facultative anaerobe; it grows in temperature ranging from 5 to 40 °C. The ability to form prodigiosin is one of its unique characteristics [7–9]. This bacterium grows fast and produces a high cell yield in a simple medium due to its very high sugar-assimilating activity. Current applications of lipases are the use of the enzyme as supplements benefit in digestion of fats, conversion of fats to energy, cleaning the clogged veins and arteries and treatment the pancreatic insufficiency, cystic fibrosis, spastic colon, Crohn’s disease and celiac disease [10]. The exponential increase in the application of lipases in various fields in the past few years demands both qualitative and quantitative improvement. The quantitative enhancement requires strain improvement and medium optimization for mass production [11]. Since lipases have the remarkable ability to carry out a wide variety of chemo selective transformations, they are the tools of choice for organic chemists. Due to huge importance of

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lipase in organo-chemical reactions and its enormous application in several industries, the current study was undertaken to produce, optimize and purify lipase, which is produced by Serratia marcescens VITSD2 on relatively low cost medium.

Material and Methods Chemicals All the chemicals and media used in this study were from HiMedia chemicals, Mumbai, India. Maintenance of Serratia sp. Throughout the study Serratia marcescens VITSD2 (Accession No.-KC961637) was used. The culture of Serratia marcescensVITSD2 was maintained at 4 °C and sub cultured every 2 weeks. Nutrient agar medium was prepared and the pH of the medium was adjusted to 7.0 [12]. Screening for Lipolytic Activity Serratia marcescens VITSD2 was streaked on Tributyrin agar (Peptic digest of animal tissue 5 g, Yeast extract 3 g, Agar 15 g for 1000 mL, Final pH 7.5 ± 0.2). The plates were incubated at 25 °C for 3–4 days. After incubation the plates were observed for the zone of clearance around the colonies [6, 8]. Preparation of Seed Inoculum A loop full of Serratia marcescens VITSD2 culture was taken from nutrient agar plate having 0.5 % Peptone, 0.3 % beef extract/yeast extract, 1.5 % agar, 0.5 % NaCl and 100 mL distilled water. Its pH was adjusted to neutral (7.4) at 25 °C. Further, it was aseptically transferred to 50 mL of TGE broth (Enzymatic digest of casein 1 g, beef extract 0.6 g, dextrose 0.2 g). The seeded broth was incubated at 30 °C in a shaking incubator at 150 rpm up for 24 h. This inoculum was used to prepare glycerol stocks as well as to inoculate the production medium in the experiment performed. Assay of Lipase Lipase activity was determined by the quantitative titration method [13] with three different substrates (olive oil, tributyrin, Tween80). One unit of lipase activity is defined as the amount of enzyme required to liberate l lmol of fatty acid per mL per minute under the specified conditions (such as reaction time, temperature etc.)

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Optimization of Process Parameters for Lipase Production Various process parameters influencing lipase production was optimized. The effect of pH (6, 6.5, 7, 7.5 and 8), incubation temperature (20, 25, 30, 35 and 40 °C), inoculum size (1, 2, 3, 4, 5 %), substrates (Tween80, Tributyrin oil and olive oil) and substrate concentration on lipase enzyme synthesis were analysed. The enzyme extracted from the broth was checked for activity. Production of Lipase Production of lipase was carried out in optimized medium. One L Rhan broth medium (Olive oil 0.7 %, peptone 5 g, yeast extract 5 g, glucose 5 g, NaCl 0.25 g and MgSO47H2O 0.5 g) was prepared in a 2 L Erlenmeyer’s flask and the pH was adjusted to 7. After proper mixing of the media with the substrate, it was inoculated with 4 % overnight seed culture of Serratia marcescens VITSD2. The medium was incubated at 30 °C for 48 h on rotatory shaker. After 48 h of incubation period, the medium was centrifuged at 8000 rpm for 15 min at 4 °C. The lipase activity and protein concentration were determined according to Fox and Stepaniak [14]. Purification Ultra Filtration Purification of lipase was carried out using ultra-filtration and ion-exchange chromatography. The extract was subjected to ultra-filtration with a 10 kDa cut off membrane and assayed for protein content and lipase activity. The retentate was used as a sample for ion exchange chromatography. CM-Cellulose Chromatography The ultra-filtered sample was first applied to a column (20 9 2.5 cm) of CM-cellulose (Sigma Chemical Co.) which was pre-equilibrated with 10 mM sodium phosphate buffer at pH 7.5. The lipase was allowed to bind to the gel for 2 h at 4 °C and eluted with a linear gradient of TritonX100 (0–1 %; 200 mL). The flow rate was 60 mL/h, and the fractions of 5 mL were collected. After the column chromatography, the fraction containing lipase was analysed on polyacrylamide gel electrophoresis (Native-PAGE) [15]. Determination of Lipase Activity The activity of the lipase was determined using the following formula [16].

Purification and Characterization of Extracellular Lipase from Serratia marcescens VITSD2

Lipase activity ¼

Vol: of NaOH consumed ðmLÞ Molarity of NaOH Vol: of lipase ðmL) Reaction time ðmin)

One unit of lipase activity was defined as the amount of enzyme that liberated 1 lmol fatty acid min-1 at 30 °C and pH 7 under the assay conditions. Measurement of Protein Concentration The total soluble protein estimation was carried out by the modified Folin–Lowry method with bovine serum albumin (BSA) as a standard [17]. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS PAGE) The partially purified enzyme was subjected to sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS PAGE) with lower separating gel (pH 8.8), upper stacking gel (pH 6.8) and 12 % acrylamide concentration. A broad range protein molecular weight marker was used to compare the protein bands with the standards, for the confirmation of the enzyme. High Performance Liquid Chromatography The purified lipase was analysed for homogeneity by HPLC (Waters) under the following conditions: column B on a pack (preparative scale; 7.8 9 300 mm); flow rate 1 mL/min; initial solution (sol A): 0.1 % TFA in water, elution: linear gradient with solution B (60 % acetonitrile in solution. A); total volume: 50 mL. Protein was detected at 280 nm.

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Fourier Transformation Infrared Spectroscopy The FT-IR (Perkin-Elmer) spectrum of the lipase was obtained at a resolution of 4 cm-1. The sample was incorporated into KBr (spectroscopic grade) and pressed into a 2 mm pellet. IR spectra was recorded in the transmittance mode from 4000 to 400 cm-1 [18, 20].

Results and Discussion Lipolytic Activity of Serratia marcescens VITSD2 The lipolytic activity of Serratia marcescens VITSD2 was observed on Tributyrin agar medium. A clear zone was observed around the horizontal streaking of the organism. The zone of hydrolysis was measured, to determine the lipase activity. The clear zone produced by Serratia marcescens VITSD2 was observed after 24 h and the maximum zone of hydrolysis was observed after 72 h. After 48 h of incubation the zone of hydrolysis was found to be 1 cm. After 72 h, the zone was found to increase from 1 cm to 4 cm (Fig. 1a, b). Effect of pH on Lipase Activity Lipase activity has been shown to be markedly dependent on pH in different species of microorganisms. The microorganism was grown in 50 mL of production medium with a pH range of 6.0–8.0 at 30 °C under shaking conditions for 48 h. Maximum lipase activity was observed at pH 7.0 and found to be 12 U/mL respectively. From pH 6 to 7, an increase in enzyme activity as well as specific enzyme activity was observed. But, with further increase in pH there was a decrease in both enzyme activity and

Fig. 1 a Lipolytic activity at 48 h, b lipolytic activity at 72 h

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Fig. 2 Effect of a pH, b temperature, c incubation period, d inoculum %, e substrates on lipase activity

specific enzyme activity as shown in Fig. 2a. Recently researchers [19] produced lipase with B. licheniformis MTCC-10498, which was grown in 50 mL of production

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medium with a pH range of 7.0–10.0 at 30 °C under shaking condition for 36 h. Maximum lipase production was observed at pH 7.5 (*0.4 U/mL).


Effect of Temperature on Lipase Activity The effect of different temperatures on lipase activity at various time intervals is presented in the Fig. 2b. After 48 h of incubation at 30 °C, under shaking conditions the maximum lipase activity was observed as 8.4 U/mL. The lowest activity was observed at 20 °C. As the temperature was increased above 30 °C there was a decrease in the enzyme activity. The B. licheniformis MTCC-10498 in the MB broth showed maximum activity at 55 °C, reaching the highest enzyme activity of 0.327 ± 0.05 U/mL after 48 h post inoculation. A lower or higher cultivation temperature caused a decline in lipase production. The lipase activity was found to be lower than the other parameters tested, which suggests the lower affinity of the enzyme for its substrate. Lipase activity was observed at broad range of temperature. Out of these, a maximum lipase enzyme activity was observed at 36 °C by lipase producing bacteria from oil contaminated soils [20].

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Olive oil showed the highest enzyme activity of 21 U/mL. Minimum lipase activity was observed with Tween 80 as shown in Fig. 2e. In Pseudomonas sp, Tembhurkar et al. [21] found mustard oil as a better stimulant for lipase production than other substrates. The lipase yield in medium supplemented with mustard oil was found to be 0.276 lM min-1 mg-1. Effect of Amount of Substrate as Olive Oil The lipase activity has been shown to be highly dependent on the amount of substrate added in the production medium. The maximum lipase activity of 14.2 U/mL was observed with 0.7 mL of olive oil in the Rhan broth production medium. Negligible lipase activity was observed in the absence of olive oil in the production medium. With further increase in the amount of substrate the activity decreased as shown in Fig. 2d. Production of Lipase by Serratia marcescens VITSD2

Effect of Incubation Period on Lipase Activity When, 24 h seed culture of Serratia marcescens VITSD2 was inoculated in the Rhan broth production medium and harvested at 24 h of interval, maximum enzyme activity was observed at 48 h (16.2 U/mL). At 72, 96 and 120 h there was a decrease in activity of enzyme. The lowest enzyme activity was observed at 120 h (11 U/mL) (Fig. 2c). A similar result was obtained [21] where the maximum lipase activity was observed at 72 h of incubation by Pseudomonas sp. Gerritse et al. [22] reported maximum lipase activity at 96 h in Pseudomonas alcaligenes. Effect of Inoculum Size on the Lipase Activity When a 4 % (v/v) inoculum of 24 h seed culture was employed in the production broth, at 30 °C, maximum enzyme activity was observed (15.9 U/mL). The lowest enzyme activity was observed with 5 % inoculum (15.3 U/ mL) (Fig. 2d). Sharma et al. [19] observed maximum enzyme activity (0.481 ± 0.05 U/mL) with 5 % (v/v) inoculum of B. licheniformis. Increase in inoculum concentration (above 5 %) showed slight decrease in lipase activity. Effect of Different Types of Substrates on Lipase Activity In the present study, Tween80, Olive oil and Tributyrin oil were used as substrate and among these three substrates,

The specific enzyme activity of the crude enzyme was found to be 23.4 U/mg whereas in the ultra filtered sample it was determined as 25.4 and 65.0 U/mg for purified sample. The purified enzyme showed the yield of 67.5.2 % with the purification fold of 2.5. Similarly previous reports on lipase activity by B. licheniformis MTCC-10498 in optimized conditions [19] have been shown as 2.0 U/mL and specific activity as 1.01 U/ mg. Purification The extracellular lipase from Serratia marcescens VITSD2 was purified using a two-step procedure: Ultra filtration and CM-Cellulose chromatography. The specific activity of the ultra-filtered enzyme was observed to be 25.42 U/mg with 1.08 purification fold and yield of 77.92 %. The highest specific activity was eluted in fractions 5 with 65.0 U/mg in CM-Cellulose chromatography with 2.55 purification fold. The yield was found to be almost 68 % (Table 1) High levels of lipase production were reported from various microbes in the presence of olive oil as carbon source in the culture medium [23, 24]. The presence of carbon in the cultivation medium could have depressed the production of lipase as compared to olive oil carbon supplementation to the basal production medium which inhibits lipase production, perhaps by catabolic repression. This has been shown for other lipase-producing organisms for which a high glucose concentration reduces the lipase production [25, 26].

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Table 1 Purification of lipase Purification step

Enzyme activity (U/mL)

Protein concentration (mg/mL)

Specific activity (U/mg)

Purification fold

% yield

Crude

77

3.28

23.47

1

100

Ultra filtered

60

2.36

25.42

1.08

77.92

CM-cellulose

52

0.8

65.0

2.55

67.5

(6–205 kDa). It was indicated that the molecular weight of Serratia marcescens VITSD2 lipase was 35 kDa (Fig. 3). High Performance Liquid Chromatography

Fig. 3 SDS-PAGE (Lane 1—Purified fraction 5, Lane 2—purified fraction 4, Lane 3—standard lipase, Lane 4—Marker)

SDS-PAGE SDS PAGE revealed the presence of a single band in Lane 1 with a molecular weight of around 35 kDa by comparing with standard lipase with known molecular weight

Fig. 4 HPLC Chromatogram of standard lipase

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The purified fractions showing highest specific activities were characterized by HPLC. The retention time of the purified fractions were compared with the standard lipase (Hi Media). The retention time of the standard lipase was found to be 2.267 min (Fig. 4). The retention time of the purified lipase was found to be 1.995 min for 5th fraction and 1.977 for 4th fraction respectively (Fig. 5). In case of ultra-filtered lipase the retention time was found to be 1.95 min. After comparing with the retention time of standard, the presence of lipase was confirmed in the purified fractions. As the standard is not seen in Serratia lipase, so there was a slight difference in the retention time. The interference of other peaks in the chromatogram of CM-Cellulose purified fractions can be avoided by further purification. Lipase from rice bran was characterized with HPLC and its retention time was found to be between 7 and 8 min [27]. Lipase, produced by Bacillus pumilus was analysed using HPLC, and its retention time was found to be 5.57 min [28].


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Fig. 5 HPLC Chromatogram of purified lipase

100

1633.71

80

1085.92

1402.25

%T

989.48

Fig. 6 FT-IR spectroscopy of purified lipase

3242.34

60

3415.93

40

4000 4

3000

Fourier Transformation-Infra Red Spectroscopy (FT-IR) FT-IR spectroscopy of the standard lipase (Hi Media) was compared with the crude, ultra-filtered and CM-Cellulose purified lipase. The characteristic signal around 3600–3100 cm-1 identifies the existence of–OH functional group, and the stretch of 1690–1630 cm-1 corresponds to C=O functional group [29]. The peak around 1400–1200 cm-1 relates to the N–H bending. The absorption band around 1200–1000 cm-1 corresponds to C–O stretch [30]. The presence of peak between 1000 and 650 cm-1 corresponds to = C–H bends. All these peaks are present in the standard lipase as well as CM-Cellulose purified lipase,

2000

1500

1000

500 1/cm

which confirm the presence of lipase in the purified fraction (Fig. 6). In case of ultra-filtered and crude lipase the IR spectra reveal additional peaks along with the peaks present in the standard lipase (Fig. 7). The presence of additional peaks implies the interference due to other functional groups. Future Perspectives In view of the immense potential, the ability of lipase enzymes from natural resources is increasingly renowned. A large number of novel classes of lipase enzymes from bacteria particularly from Serratia have evolved with the greatest diversity. Efforts should be directed towards exploring the enzyme with high throughput screening.

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1643.35

1402.25

%T 75

989.48

90

1087.85

Fig. 7 FT-IR spectroscopy of standard lipase

45

3531.66 3450.65

60

4000 SL-

3000

Conclusion Recently, lipases have been receiving attention because of their possible applications in biodegradation with various applications in the environmental, agricultural, food technology and cosmetics. Although lipases have been isolated and characterized from a wide variety of sources, it is still important to screen new sources for production of lipases with more economical values and enhanced properties to expand their usefulness. Pilot scale production and kinetics of lipase is yet to be adopted for maximum production. Acknowledgments Authors are thankful to VIT University for the constant encouragement, help and support for extending necessary facilities. Compliance with Ethical Standards Conflict of Interest Authors have no conflict of interest.

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