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Tween 80 influences the production of intracellular lipase by Schizochytrium S31 in a stirred tank reactor
Accepted Manuscript Title: Tween 80 influences the production of intracellular lipase by Schizochytrium S31 in a stirred tank reactor Author: Avinesh R. Byreddy Nalam Madhusudhana Rao Colin J. Barrow Munish Puri PII: DOI: Reference:
S1359-5113(16)30992-8 http://dx.doi.org/doi:10.1016/j.procbio.2016.11.026 PRBI 10871
To appear in:
Process Biochemistry
Received date: Revised date: Accepted date:
26-2-2016 17-11-2016 30-11-2016
Please cite this article as: Byreddy Avinesh R, Rao Nalam Madhusudhana, Barrow Colin J, Puri Munish.Tween 80 influences the production of intracellular lipase by Schizochytrium S31 in a stirred tank reactor.Process Biochemistry http://dx.doi.org/10.1016/j.procbio.2016.11.026 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Manuscript for Process Biochemistry
Tween 80 influences the production of intracellular lipase by Schizochytrium S31 in a stirred tank reactor
Avinesh R. Byreddya, Nalam Madhusudhana Raob, Colin J. Barrowa,*, Munish Puria,* a
Centre for Chemistry and Biotechnology, Deakin University, Waurn Ponds, Geelong,
Australia. 3217 b
Centre for Cellular and Molecular Biology, Council of Scientific and Industrial Research,
Uppal road, Hyderabad, Telangana 500007, India
*Corresponding authors Dr Munish Puri CCB, Deakin University, Victoria 3217, Australia. E-mail addresses: [email protected] (M. Puri).
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Graphical abstract
Lipid pH
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Research Highlights
Tween 80 influenced lipase productivity in Schizochytrium S31
Sonication was optimised for the extraction of intracellular lipase from Schizochytrium S31
Fatty acid chain length selectivity of crude lipase was evaluated using p-NP esters
The extracted lipase showed preferential hydrolysis of DHA over EPA
Abstract Marine microorganisms are a potential source of enzymes with structural stability, high activity at low temperature and unique substrate selectivity. Thraustochytrids are marine heterotrophic microbes, well known for the production of omega-3 fatty acids. In this study the effect of Tween 80 as a carbon source was investigated with regard to biomass, lipase and lipid productivity in Schizochytrium sp. S31. Tween 80 (1%) and 120 h of incubation was the optimum period for biomass, lipid and lipase productivity in a stirred tank reactor. The yields obtained were 0.9 g L-1 of biomass, 300 mg g-1 of lipid and 39 U/g of lipase activity. Sonication was optimised in terms of time and acoustic power to maximise the yield of extracted lipase. The extracted lipase from Schizochytrium S31 was observed to hydrolyse long chain polyunsaturated fatty acids DHA and EPA.
Key words: lipase, DHA, Omega-3 fatty acids, marine, bioactives
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1. Introduction Lipases (triacylglycerol lipases, EC 3.1.1.3) belongs to the hydrolase family of enzymes that catalyse the hydrolysis of triglycerides into free fatty acids and glycerol. However, under certain conditions, they are also able to catalyse synthetic reactions [1]. In addition, lipases have some important functional properties such as substrate selectivity, regio-selectivity and high enantio-selectivity. These properties make them potential catalyst in numerous industrial processes, such as food, detergent, chemical, pharmaceutical industries and lipid modification [2, 3]. Lipases are ubiquitous enzymes widely distributed in plants, animals and microorganisms. Among them, microbial lipases have displayed a broad range of substrate specificities. This property might have evolved through the access of microbial lipases to different carbon sources [4]. There is a growing demand for the novel lipases from a microbial sources, because of their ease in production and access to genetic manipulations. Also because they work under mild reaction conditions without requirement of any co-factors [5]. The health benefits of omega-3 fatty acids have been evaluated by a number of clinical studies [6]. There is strong evidence for the efficacy of omega-3 fatty acids in the prevention of sudden death from cardiovascular disease and rheumatologic conditions [7]. The anti-cancer activity of omega-3 fatty acids, particularly against colorectal cancer, has recently been shown [8]. Infant brain and eye development is influenced by DHA, which has led to the addition of DHA to the infant formulae. Learning capacity of school going children has been shown to increase by taking DHA enriched food supplements [9]. Oily marine fish are the primary source for the industrial production of omega-3 fatty acids. These fish derived omega-3 fatty acids from their diet, primarily from phytoplankton. Researchers have implemented different methods for concentrating omega-3 fatty acids [10]. A promising green approach for concentration of omega-3 fatty acids is the use of lipases. Generally the
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enzymatic methods are faster than other methods and can be carried out under milder conditions then methods involving chromatographic separation, molecular distillation or urea complexation [11]. Commercially available lipases have been exploited for omega-3 fatty acid concentration [12-14]. However, selectivity of fatty acid hydrolysis is poor and no lipases were detected which selectively hydrolyse DHA and EPA, a property that would be useful in selective concentration of these fatty acids from fish or microbial oils. Thraustochytrids are marine heterotrophic microbes, well known for the production of omega-3 fatty acids, carotenoids and enzymes [15]. Thraustochytrids produce lipases and because of their high productivity of omega-3 fatty acids, particularly DHA, they are potential sources of omega-3 selective lipases, although none have yet been characterised [16, 17]. In this work, we optimised culture conditions to enhance lipase production from Schizochytrium sp. S31 in a bioreactor and optimised the extraction of this lipase using sonication. Using a recently developed p-nitrophenyl esters (p-NP) assay we evaluated the substrate selectivity of lipase extracted from Schizochytrium S31. This is an initial attempt to understand the shift in lipid change during lipase production and optimization of lipase extraction from Schizochytrium sp. 2. Materials and methods 2.1. Microorganism and maintenance of culture Schizochytrium S31 (ATCC 20888) was procured from the American type culture collection (ATCC). The stock culture was maintained on GYP medium consisting of glucose 5, yeast extract 2, mycological peptone 2, agar 10 g L-1 and artificial seawater 50% at 25 °C and sub-cultured after 15 days. All medium components and chemicals including p-NP were obtained from Sigma, Australia. The p-NP fatty acid esters of linoleic acid (C18:2), αlinolenic acid (C18:3), eicosapentaenoic acid (C20:5) and docosahexaenoic acid (C22:6)
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were synthesised based on a recently published protocol. The p-NP-EPA and p-NP-DHA yields were 90% and remained stable for 6 months [18].
2.2. Schizochytrium S31 growth and lipase production Schizochytrium S31 was grown in a medium containing peptone 0.5, yeast extract 0.5 g L-1 and Tween 80 (1% w/v; as sole carbon source). 50 ml of medium in a 250 ml Erlenmeyer flask was inoculated with a loop full of culture from the plates. This culture flask was incubated for 48 h at 20 oC and shaken at 150 rpm. A 48 h old 5% seed culture (v/v) was used to inoculate sterilised production medium (100 ml in 500 ml Erlenmeyer flask) containing peptone 0.5, yeast extract 0.5 g L-1 and Tween 80 for the production of lipase. The flasks were incubated in a rotary shaker for up to 9 days. Samples were withdrawn aseptically at regular time intervals and then dried and weighted and lipase activity determined. For dry weight determination, the entire content of the liquid culture was transferred to a pre-weighed centrifuge tube and harvested by centrifugation at 3500 rpm for 10 min and the supernatant was discarded. Harvested cells were washed thoroughly with phosphate buffer (pH 7.0) and then freeze-dried for 24 h. The biomass weight was determined gravimetrically. Tween 80 concentrations were estimated based on a colorimetric method [19]. A standard curve was prepared by varying the Tween 80 concentration and the unknown sample concentration was determined using the standard curve. Temperature (15 to 30 oC) and initial pH (5-8) of the medium were optimised to maximise lipase activity. All experiments were carried out in triplicates. Fermentation of thraustochytrids was carried out in a 2 L bioreactor (New Brunswick, USA) with a working volume of 1.4 L at 25 oC with aeration of 0.5 vvm and agitation of 150 rpm. Varying concentration of Tween 80 (0.5 to 1.5%, v/v) was used to optimise biomass, lipase and lipid production in the bioreactor. Samples were withdrawn at regular time 6
intervals and analysed for residual substrate. Changes in pH were also monitored during the course of fermentation.
2.3. Estimation of Schizochytrium S31 growth The growth of the organism was estimated by taking 1-ml aliquot periodically and measuring the optical density (OD) at 600 nm in a UV-visible spectrophotometer (Shimadzu1601, Japan) with medium as a blank. The OD value was converted into cell mass concentration (mg L−1) using a standard curve. 2.4. Optimization of cell disruption through sonication Cell disruption of Schizochytrium S31 biomass was performed as previously described [20]. Thraustochytrid cell biomass (50 mg) was suspended in 3 mL of solvent in a 15-mL centrifuge tube. Sample was sonicated at 20 kHz, 40% amplitude and the pulse was 40 s on and 20 s off with total working time of 20 min (Sonics, Newtown, CT, USA). Sample tubes were kept on ice during the sonication process to prevent overheating. Sonication conditions such as time and acoustic power were optimized. The sonicator used in this study was a horn type with 500 W power output and 20 KHz. 2.5. Lipase assay and protein estimation Lipase activity was measured in the supernatant obtained after cell disruption using pnitrophenol palmitate (pNPP) as a substrate, following a previously described method with some modifications [21]. The lipase substrate solution was prepared by adding solution A consisting of 30 mg of pNPP in 10 ml of isopropanol and the solution B consisting of 0.4% Triton X-100, 0.1% Gum Arabic and 50 mM Tris-HCl buffer, pH 7.0. Solution A (10 ml) was added to solution B (90 ml) drop by drop with stirring. The substrate solution (2.9 ml) and 0.1 ml of enzyme solution was incubated at 37 oC for 10 min and absorbance was measured at 410 nm. One unit (IU) of lipase activity was defined as one nmol of p-
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nitrophenol released per minute under the standard assay conditions. The relative activity of crude enzyme is calculated as a percentage activity divided by the most hydrolysed substrate (designated 100% for each substrate). Protein concentration was estimated from the supernatant using the Bradford method with bovine serum albumin as the standard [22]. All enzyme and protein assays were done in four replicates and the standard error (SE) calculated using Microsoft excel software. 2.6. Lipid extraction Lipid was extracted and quantified from freeze-dried biomass every 48 h according to a previously published protocol [23] with some modifications. Freeze-dried biomass (10 mg) was added to 600 μL solvent (chloroform and methanol in 2:1 ratio) and the sample vortexed for 2 min, followed by centrifugation at 13,000 rpm for 15 min. This extraction process was repeated three times. The supernatants were collected in a pre-weighed vials and dried in an oven at 50 °C. The lipid percentage was measured gravimetrically. All the experiments were performed in triplicate. 3. Results and discussion 3.1. Optimisation of culture conditions for lipase production Lipase production is influenced by the type of carbon source used, temperature and initial pH of the culture medium [24]. Tween 80 has been successfully used as a carbon source for the production of lipases from various microbes [25, 26]. In this study, Tween 80 was used as the sole carbon source and its concentration was optimised to maximise biomass and lipase productivity (Fig. 1). Tween 80 (1%) resulted in the highest level of biomass (0.9 g L-1) and lipase (39 IU/g) productivity by Schizochytrium S31. Similar results were found previously for Acinetobacter radioresistens [25].Another study reported that the addition of tween 80 stimulated the lipase production from Pseudozyma hubeiensis HB85A [27]. Lipase production did not improve when higher tween 80 concentration (1.5%) was used in the
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medium, although the reason for this is unclear. Remaining experiments were performed with 1% tween 80. Initial pH and temperature are known to influence lipase production [26]. In Schizochytrium S31 maximal lipase activity was observed at pH 6 (38 U/g) for the pH range of 5 to 8. Similar results were reported for lipase production from Thraustochytrium sp. when the medium pH was preadjusted to 6 [17]. The effect of incubation temperature on lipase production was investigated for the four temperatures 15 oC, 20 oC, 25 oC and 30 oC at pH 6 and agitation 150 rpm, with lipase levels being highest at 25 oC (data not shown). Out results are comparable with lipase production from Colletotrichum gloeosporioides where maximum lipase activity at 25 oC [29]. 3.2. Production of lipase in stirred tank reactor Growth kinetics of Schizochytrium S31 was studied in terms of biomass production, lipid accumulation in cells, and lipase production. After 48 h acclimatisation in the medium, dry cell weight went from 0.16 g L-1 to 0.2 g L-1, with the log phase beginning after this time, up to 120 h when growth slows and the stationary phase begins (Fig 2). Schizochytrium S31 cells grew well in a fermentation medium containing Tween 80 (1%) as sole carbon source. Fig. 2 shows the time course of the investigated parameters at different growth phases and also showed that lipase and lipid production were growth associated. Biomass increased with incubation time and was maximal (0.9 g) after 5 days of incubation. Lipase production reached a maximum of 39 U/g in the late exponential phase, then the lipase activity declined (10.7 U/g) up until 9 days of incubation, the possible reason might be the final product inhibition by the accumulation of fatty acids. Our results are in agreement with other researchers who observed the maximum lipase activity of Yarrowia lipolytica KKP 379 in late exponential phase (after 72 h) [30]. Another study conducted by Kanchana et al, (2011), found that the maximum lipase activity was observed in thraustochytrids between 4-7 days of
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incubation [17]. The pH and substrate concentration of the medium decreased as the incubation time increasing, which may be due to the hydrolytic activity of lipase on Tween 80 releasing oleic acid for utilisation. In the bioreactor, lipase production increased with increasing biomass concentration as a result of increasing Tween 80 concentration from 0.5 to 1 %. In addition, improved control of environmental factors in the vessel resulted in increased enzyme production, compared with that achieved in flasks. To investigate lipid content and fatty acid composition, biomass was collected and analysed at different time intervals up to 9 days. DHA content increased with total lipid content and reached a maximum of 5.4% on day 5. Biomass, lipid and DHA all decrease after day 5, although DHA decreases more rapidly than does total lipid (Fig. 3). Lipid is used for energy and membrane synthesis during starvation and DHA is associated with many cellular functions. DHA appears to be used preferentially in the late stages, which also indicates that lipase is acting on DHA during this stage (Fig. 2 and 3). The fatty acid composition of oil from Schizochytrium S31 grown in Tween 80 medium is given in Fig. 4, with growth in glucose medium as a control. The oleate in Tween 80 was directly accumulated into cells (Fig. 4). A similar pattern was observed in Thraustochytrium aureum ATCC 34304, where Tween 80 in the medium increased the oleic acid content in lipid [29]. A corresponding decrease in the percentages of other fatty acids was observed in Tween 80 medium. There was also changes in the ratio of fatty acids with Tween 80 medium, with relatively decrease in C16 (26%), C18 (70%) and traces of C14, EPA and DHA, relative to DHA. 3.3. Disruption of Schizochytrium S31 using sonication In order to investigate the fatty acid selectivity of lipase from Schizochytrium S31, sonication was used to release the intracellular lipase. The effect of sonication on Schizochytrium S31 was evaluated by microscopic observation and cells were shown to be fully broken at high acoustic power. Table 1 shows lipase activity extracted from
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thraustochytrid cells at an acoustic power of 20 kHz and 40 % amplitude for intervals of 5 to 25 mins. Lipase activity increased to 15 minutes but plateaued after that, indicating partial lipase inactivation with increased time over 15 minutes. Since sonication releases intracellular enzymes but also degrades, however, optimising time and power are important for maximising lipase extraction [32, 33]. Fig. 5 (a) and (b) show the effect of sonication on lipase activity from Schizochytrium S31 at different levels of sonication time and power. As the sonicator power increased from 30% to 70%, the total proteins released increased from 53 to 122 µg/ml and the most lipase activity was observed at 50% power (64 IU/ml). Total proteins released after sonication treatment was 3 folds higher at 50 % (0.75 IU/µg) sonication power than 70% (0.25 IU/µg) (Fig. 5 a). Our results are in agreement with previous studies showing that the amount of protein release from microbial cells increased linearly with increasing sonication time and power [34]. The highest protein extraction levels from Schizochytrium was observed after 15 min of sonication time at 70% of sonication power. However, both total lipase activity and specific lipase activity decreased at 70% power, with extended time causing additional lipase activity degradation. A recent study by Kapturowska et al, 2013, demonstrated the negative effects of extended sonication time on lipase activity from Yarrowia lipolytica KKP 379 [35]. 3.4. Hydrolysis of p-Nitrophenyl esters Understanding and controlling lipase fatty acid selectivity is important in the modification of fats and oils in the production of structured lipids [36]. Most of the commercially available lipases catalyse the hydrolysis of long chain fatty acids poorly compared to short chain due to their complex structure. Marine microbes offer a potential source of enzymes which are stable at very high and low temperatures and active at broad pH range with unique fatty acids selectivity [37]. The substrate selectivity of S31 crude lipase activity was determined using the standard assay using the different p-NP esters of various
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chain lengths: p-NP acetate (C2); p-NP butyrate (C4); p-NP caprylate (C8); p-NP decanoate (C10), p-NP laurate (C12), p-NP myristate (C14), p-NP palmitate (C16), p-NP stearate (C18), p-NP-EPA and p-NP-DHA. Lipase extracted from Schizochytrium S31 showed a broad range of substrate activity (Fig. 6). Maximum activity was observed towards short chain length fatty acids, followed by medium chain length fatty acids. Schizochytrium lipase preferred pNP-DHA with relative activity 20.14 ± 1.7 compared to other PUFA esters (Fig 6). The pNP-EPA was poorly hydrolyzed with relative activity 12.8 ± 1.4, compared to the other PUFA esters. An unusual fatty acid hydrolysis was observed towards p-NP-DHA, when compared with p-NP-EPA. Similar lipase selectivity towards DHA has been reported from Pseudomonas sp. In contrast a fish digestive lipase salmon intestines, hydrolyzed DHA at a faster rate than EPA, although this lipase was never characterised [38]. However, a study investigated the fatty acid selectivity of five commercial lipases using p-NP esters found that all lipase showed preference to EPA over DHA [18]. This selectivity might be due to the requirement of the Schizochytrium to process DHA more than EPA, since EPA is present in only trace amounts in the oil from this strain, whereas DHA levels are relatively high in Schizochytrium sp. Confirmation of DHA to EPA selectively will require additional work to purify the S31 lipase and confirm that observed activity is due to a single lipase. 4. Conclusion Schizochytrium S31 was grown in a bioreactor using Tween 80 as the carbon source, which resulted in high biomass and oleic acid levels. Lipase extraction by sonication was optimised and the fatty acid selectivity of the extracted lipase investigated using p-NP fatty acid esters. The lipase extract preferentially hydrolysed shorter chain fatty acids, however, DHA was preferentially hydrolysed over EPA. This preferential hydrolysis of DHA is unusual and may be industrially useful. This selectivity may be a result of this
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Schizochytrium species having much higher levels of DHA then EPA, and so DHA processing is more important for the organism growth and function. Acknowledgement AB acknowledges the PhD scholarship award by Deakin University, Australia.
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Figure and table captions
Fig. 1. Effect of Tween 80 concentration on lipase production by Schizochytrium S31. Fig. 2. Fermentation profile of lipase production by Schizochytrium S31 in a stirred tank reactor. Fig. 3. DHA production as a function of incubation. Fig. 4. Comparison of fatty acid composition of Schizochytrium S31 lipid in Tween 80 vs glucose medium. Fig. 5. Cell disruption optimisation of Schizochytrium S31 a) as a function of sonication power, and b) time duration. Fig. 6. Substrate specificity of lipase from Schizochytrium S31 Footnote: p-nitrophenyl esters of different chain lengths were incubated with crude enzyme.
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Fig. 1.
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Lipid pH
100 7.0
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Substrate depletion (%)
350
Biomass Lipase Substrate depletion
20
10
20 4.0
0.2 0 0.1 0
2
4
6
8
3.5 10
10
0
0
Incubation time (day)
Fig. 2.
21
Fig.
3.
22
Fig. 4
23
a
b
Fig. 5.
24
Fig. 6.
25
Table 1. Effect of sonication on protein release and lipase activity from Schizochytrium S31. Table 1.
Sonication (min)
period Lipase (IU/ml)
activity Protein concentration
Specific activity (IU/µg)
(µg/ml) 5
22.8±1.4
43±1.5
0.512
10
32.5±1.6
60±1.8
0.533
15
37.8±1.5
82±2.1
0.451
20
26.3±1.3
105±9
0.247
26
S1359-5113(16)30992-8 http://dx.doi.org/doi:10.1016/j.procbio.2016.11.026 PRBI 10871
To appear in:
Process Biochemistry
Received date: Revised date: Accepted date:
26-2-2016 17-11-2016 30-11-2016
Please cite this article as: Byreddy Avinesh R, Rao Nalam Madhusudhana, Barrow Colin J, Puri Munish.Tween 80 influences the production of intracellular lipase by Schizochytrium S31 in a stirred tank reactor.Process Biochemistry http://dx.doi.org/10.1016/j.procbio.2016.11.026 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Manuscript for Process Biochemistry
Tween 80 influences the production of intracellular lipase by Schizochytrium S31 in a stirred tank reactor
Avinesh R. Byreddya, Nalam Madhusudhana Raob, Colin J. Barrowa,*, Munish Puria,* a
Centre for Chemistry and Biotechnology, Deakin University, Waurn Ponds, Geelong,
Australia. 3217 b
Centre for Cellular and Molecular Biology, Council of Scientific and Industrial Research,
Uppal road, Hyderabad, Telangana 500007, India
*Corresponding authors Dr Munish Puri CCB, Deakin University, Victoria 3217, Australia. E-mail addresses: [email protected] (M. Puri).
1
Graphical abstract
Lipid pH
100 7.0
0.9
300
6.5
0.8 250
150
100
6.0
5.5
0.5
5.0 0.4 4.5
50
0.3
40
80 70 60
0.6
pH
200
Biomass (g/L)
Lipid (mg/g)
0.7
90
50 40 30
30 Lipase Activity (IU/g)
Biomass Lipase Substrate depletion
1.0
Substrate depletion (%)
350
20
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20 4.0
0.2 0 0.1 0
2
4
6
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3.5 10
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Incubation time (day)
Biomass, lipase and lipid production
Lipase substrate selectivity Lipase extraction
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Research Highlights
Tween 80 influenced lipase productivity in Schizochytrium S31
Sonication was optimised for the extraction of intracellular lipase from Schizochytrium S31
Fatty acid chain length selectivity of crude lipase was evaluated using p-NP esters
The extracted lipase showed preferential hydrolysis of DHA over EPA
Abstract Marine microorganisms are a potential source of enzymes with structural stability, high activity at low temperature and unique substrate selectivity. Thraustochytrids are marine heterotrophic microbes, well known for the production of omega-3 fatty acids. In this study the effect of Tween 80 as a carbon source was investigated with regard to biomass, lipase and lipid productivity in Schizochytrium sp. S31. Tween 80 (1%) and 120 h of incubation was the optimum period for biomass, lipid and lipase productivity in a stirred tank reactor. The yields obtained were 0.9 g L-1 of biomass, 300 mg g-1 of lipid and 39 U/g of lipase activity. Sonication was optimised in terms of time and acoustic power to maximise the yield of extracted lipase. The extracted lipase from Schizochytrium S31 was observed to hydrolyse long chain polyunsaturated fatty acids DHA and EPA.
Key words: lipase, DHA, Omega-3 fatty acids, marine, bioactives
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1. Introduction Lipases (triacylglycerol lipases, EC 3.1.1.3) belongs to the hydrolase family of enzymes that catalyse the hydrolysis of triglycerides into free fatty acids and glycerol. However, under certain conditions, they are also able to catalyse synthetic reactions [1]. In addition, lipases have some important functional properties such as substrate selectivity, regio-selectivity and high enantio-selectivity. These properties make them potential catalyst in numerous industrial processes, such as food, detergent, chemical, pharmaceutical industries and lipid modification [2, 3]. Lipases are ubiquitous enzymes widely distributed in plants, animals and microorganisms. Among them, microbial lipases have displayed a broad range of substrate specificities. This property might have evolved through the access of microbial lipases to different carbon sources [4]. There is a growing demand for the novel lipases from a microbial sources, because of their ease in production and access to genetic manipulations. Also because they work under mild reaction conditions without requirement of any co-factors [5]. The health benefits of omega-3 fatty acids have been evaluated by a number of clinical studies [6]. There is strong evidence for the efficacy of omega-3 fatty acids in the prevention of sudden death from cardiovascular disease and rheumatologic conditions [7]. The anti-cancer activity of omega-3 fatty acids, particularly against colorectal cancer, has recently been shown [8]. Infant brain and eye development is influenced by DHA, which has led to the addition of DHA to the infant formulae. Learning capacity of school going children has been shown to increase by taking DHA enriched food supplements [9]. Oily marine fish are the primary source for the industrial production of omega-3 fatty acids. These fish derived omega-3 fatty acids from their diet, primarily from phytoplankton. Researchers have implemented different methods for concentrating omega-3 fatty acids [10]. A promising green approach for concentration of omega-3 fatty acids is the use of lipases. Generally the
4
enzymatic methods are faster than other methods and can be carried out under milder conditions then methods involving chromatographic separation, molecular distillation or urea complexation [11]. Commercially available lipases have been exploited for omega-3 fatty acid concentration [12-14]. However, selectivity of fatty acid hydrolysis is poor and no lipases were detected which selectively hydrolyse DHA and EPA, a property that would be useful in selective concentration of these fatty acids from fish or microbial oils. Thraustochytrids are marine heterotrophic microbes, well known for the production of omega-3 fatty acids, carotenoids and enzymes [15]. Thraustochytrids produce lipases and because of their high productivity of omega-3 fatty acids, particularly DHA, they are potential sources of omega-3 selective lipases, although none have yet been characterised [16, 17]. In this work, we optimised culture conditions to enhance lipase production from Schizochytrium sp. S31 in a bioreactor and optimised the extraction of this lipase using sonication. Using a recently developed p-nitrophenyl esters (p-NP) assay we evaluated the substrate selectivity of lipase extracted from Schizochytrium S31. This is an initial attempt to understand the shift in lipid change during lipase production and optimization of lipase extraction from Schizochytrium sp. 2. Materials and methods 2.1. Microorganism and maintenance of culture Schizochytrium S31 (ATCC 20888) was procured from the American type culture collection (ATCC). The stock culture was maintained on GYP medium consisting of glucose 5, yeast extract 2, mycological peptone 2, agar 10 g L-1 and artificial seawater 50% at 25 °C and sub-cultured after 15 days. All medium components and chemicals including p-NP were obtained from Sigma, Australia. The p-NP fatty acid esters of linoleic acid (C18:2), αlinolenic acid (C18:3), eicosapentaenoic acid (C20:5) and docosahexaenoic acid (C22:6)
5
were synthesised based on a recently published protocol. The p-NP-EPA and p-NP-DHA yields were 90% and remained stable for 6 months [18].
2.2. Schizochytrium S31 growth and lipase production Schizochytrium S31 was grown in a medium containing peptone 0.5, yeast extract 0.5 g L-1 and Tween 80 (1% w/v; as sole carbon source). 50 ml of medium in a 250 ml Erlenmeyer flask was inoculated with a loop full of culture from the plates. This culture flask was incubated for 48 h at 20 oC and shaken at 150 rpm. A 48 h old 5% seed culture (v/v) was used to inoculate sterilised production medium (100 ml in 500 ml Erlenmeyer flask) containing peptone 0.5, yeast extract 0.5 g L-1 and Tween 80 for the production of lipase. The flasks were incubated in a rotary shaker for up to 9 days. Samples were withdrawn aseptically at regular time intervals and then dried and weighted and lipase activity determined. For dry weight determination, the entire content of the liquid culture was transferred to a pre-weighed centrifuge tube and harvested by centrifugation at 3500 rpm for 10 min and the supernatant was discarded. Harvested cells were washed thoroughly with phosphate buffer (pH 7.0) and then freeze-dried for 24 h. The biomass weight was determined gravimetrically. Tween 80 concentrations were estimated based on a colorimetric method [19]. A standard curve was prepared by varying the Tween 80 concentration and the unknown sample concentration was determined using the standard curve. Temperature (15 to 30 oC) and initial pH (5-8) of the medium were optimised to maximise lipase activity. All experiments were carried out in triplicates. Fermentation of thraustochytrids was carried out in a 2 L bioreactor (New Brunswick, USA) with a working volume of 1.4 L at 25 oC with aeration of 0.5 vvm and agitation of 150 rpm. Varying concentration of Tween 80 (0.5 to 1.5%, v/v) was used to optimise biomass, lipase and lipid production in the bioreactor. Samples were withdrawn at regular time 6
intervals and analysed for residual substrate. Changes in pH were also monitored during the course of fermentation.
2.3. Estimation of Schizochytrium S31 growth The growth of the organism was estimated by taking 1-ml aliquot periodically and measuring the optical density (OD) at 600 nm in a UV-visible spectrophotometer (Shimadzu1601, Japan) with medium as a blank. The OD value was converted into cell mass concentration (mg L−1) using a standard curve. 2.4. Optimization of cell disruption through sonication Cell disruption of Schizochytrium S31 biomass was performed as previously described [20]. Thraustochytrid cell biomass (50 mg) was suspended in 3 mL of solvent in a 15-mL centrifuge tube. Sample was sonicated at 20 kHz, 40% amplitude and the pulse was 40 s on and 20 s off with total working time of 20 min (Sonics, Newtown, CT, USA). Sample tubes were kept on ice during the sonication process to prevent overheating. Sonication conditions such as time and acoustic power were optimized. The sonicator used in this study was a horn type with 500 W power output and 20 KHz. 2.5. Lipase assay and protein estimation Lipase activity was measured in the supernatant obtained after cell disruption using pnitrophenol palmitate (pNPP) as a substrate, following a previously described method with some modifications [21]. The lipase substrate solution was prepared by adding solution A consisting of 30 mg of pNPP in 10 ml of isopropanol and the solution B consisting of 0.4% Triton X-100, 0.1% Gum Arabic and 50 mM Tris-HCl buffer, pH 7.0. Solution A (10 ml) was added to solution B (90 ml) drop by drop with stirring. The substrate solution (2.9 ml) and 0.1 ml of enzyme solution was incubated at 37 oC for 10 min and absorbance was measured at 410 nm. One unit (IU) of lipase activity was defined as one nmol of p-
7
nitrophenol released per minute under the standard assay conditions. The relative activity of crude enzyme is calculated as a percentage activity divided by the most hydrolysed substrate (designated 100% for each substrate). Protein concentration was estimated from the supernatant using the Bradford method with bovine serum albumin as the standard [22]. All enzyme and protein assays were done in four replicates and the standard error (SE) calculated using Microsoft excel software. 2.6. Lipid extraction Lipid was extracted and quantified from freeze-dried biomass every 48 h according to a previously published protocol [23] with some modifications. Freeze-dried biomass (10 mg) was added to 600 μL solvent (chloroform and methanol in 2:1 ratio) and the sample vortexed for 2 min, followed by centrifugation at 13,000 rpm for 15 min. This extraction process was repeated three times. The supernatants were collected in a pre-weighed vials and dried in an oven at 50 °C. The lipid percentage was measured gravimetrically. All the experiments were performed in triplicate. 3. Results and discussion 3.1. Optimisation of culture conditions for lipase production Lipase production is influenced by the type of carbon source used, temperature and initial pH of the culture medium [24]. Tween 80 has been successfully used as a carbon source for the production of lipases from various microbes [25, 26]. In this study, Tween 80 was used as the sole carbon source and its concentration was optimised to maximise biomass and lipase productivity (Fig. 1). Tween 80 (1%) resulted in the highest level of biomass (0.9 g L-1) and lipase (39 IU/g) productivity by Schizochytrium S31. Similar results were found previously for Acinetobacter radioresistens [25].Another study reported that the addition of tween 80 stimulated the lipase production from Pseudozyma hubeiensis HB85A [27]. Lipase production did not improve when higher tween 80 concentration (1.5%) was used in the
8
medium, although the reason for this is unclear. Remaining experiments were performed with 1% tween 80. Initial pH and temperature are known to influence lipase production [26]. In Schizochytrium S31 maximal lipase activity was observed at pH 6 (38 U/g) for the pH range of 5 to 8. Similar results were reported for lipase production from Thraustochytrium sp. when the medium pH was preadjusted to 6 [17]. The effect of incubation temperature on lipase production was investigated for the four temperatures 15 oC, 20 oC, 25 oC and 30 oC at pH 6 and agitation 150 rpm, with lipase levels being highest at 25 oC (data not shown). Out results are comparable with lipase production from Colletotrichum gloeosporioides where maximum lipase activity at 25 oC [29]. 3.2. Production of lipase in stirred tank reactor Growth kinetics of Schizochytrium S31 was studied in terms of biomass production, lipid accumulation in cells, and lipase production. After 48 h acclimatisation in the medium, dry cell weight went from 0.16 g L-1 to 0.2 g L-1, with the log phase beginning after this time, up to 120 h when growth slows and the stationary phase begins (Fig 2). Schizochytrium S31 cells grew well in a fermentation medium containing Tween 80 (1%) as sole carbon source. Fig. 2 shows the time course of the investigated parameters at different growth phases and also showed that lipase and lipid production were growth associated. Biomass increased with incubation time and was maximal (0.9 g) after 5 days of incubation. Lipase production reached a maximum of 39 U/g in the late exponential phase, then the lipase activity declined (10.7 U/g) up until 9 days of incubation, the possible reason might be the final product inhibition by the accumulation of fatty acids. Our results are in agreement with other researchers who observed the maximum lipase activity of Yarrowia lipolytica KKP 379 in late exponential phase (after 72 h) [30]. Another study conducted by Kanchana et al, (2011), found that the maximum lipase activity was observed in thraustochytrids between 4-7 days of
9
incubation [17]. The pH and substrate concentration of the medium decreased as the incubation time increasing, which may be due to the hydrolytic activity of lipase on Tween 80 releasing oleic acid for utilisation. In the bioreactor, lipase production increased with increasing biomass concentration as a result of increasing Tween 80 concentration from 0.5 to 1 %. In addition, improved control of environmental factors in the vessel resulted in increased enzyme production, compared with that achieved in flasks. To investigate lipid content and fatty acid composition, biomass was collected and analysed at different time intervals up to 9 days. DHA content increased with total lipid content and reached a maximum of 5.4% on day 5. Biomass, lipid and DHA all decrease after day 5, although DHA decreases more rapidly than does total lipid (Fig. 3). Lipid is used for energy and membrane synthesis during starvation and DHA is associated with many cellular functions. DHA appears to be used preferentially in the late stages, which also indicates that lipase is acting on DHA during this stage (Fig. 2 and 3). The fatty acid composition of oil from Schizochytrium S31 grown in Tween 80 medium is given in Fig. 4, with growth in glucose medium as a control. The oleate in Tween 80 was directly accumulated into cells (Fig. 4). A similar pattern was observed in Thraustochytrium aureum ATCC 34304, where Tween 80 in the medium increased the oleic acid content in lipid [29]. A corresponding decrease in the percentages of other fatty acids was observed in Tween 80 medium. There was also changes in the ratio of fatty acids with Tween 80 medium, with relatively decrease in C16 (26%), C18 (70%) and traces of C14, EPA and DHA, relative to DHA. 3.3. Disruption of Schizochytrium S31 using sonication In order to investigate the fatty acid selectivity of lipase from Schizochytrium S31, sonication was used to release the intracellular lipase. The effect of sonication on Schizochytrium S31 was evaluated by microscopic observation and cells were shown to be fully broken at high acoustic power. Table 1 shows lipase activity extracted from
10
thraustochytrid cells at an acoustic power of 20 kHz and 40 % amplitude for intervals of 5 to 25 mins. Lipase activity increased to 15 minutes but plateaued after that, indicating partial lipase inactivation with increased time over 15 minutes. Since sonication releases intracellular enzymes but also degrades, however, optimising time and power are important for maximising lipase extraction [32, 33]. Fig. 5 (a) and (b) show the effect of sonication on lipase activity from Schizochytrium S31 at different levels of sonication time and power. As the sonicator power increased from 30% to 70%, the total proteins released increased from 53 to 122 µg/ml and the most lipase activity was observed at 50% power (64 IU/ml). Total proteins released after sonication treatment was 3 folds higher at 50 % (0.75 IU/µg) sonication power than 70% (0.25 IU/µg) (Fig. 5 a). Our results are in agreement with previous studies showing that the amount of protein release from microbial cells increased linearly with increasing sonication time and power [34]. The highest protein extraction levels from Schizochytrium was observed after 15 min of sonication time at 70% of sonication power. However, both total lipase activity and specific lipase activity decreased at 70% power, with extended time causing additional lipase activity degradation. A recent study by Kapturowska et al, 2013, demonstrated the negative effects of extended sonication time on lipase activity from Yarrowia lipolytica KKP 379 [35]. 3.4. Hydrolysis of p-Nitrophenyl esters Understanding and controlling lipase fatty acid selectivity is important in the modification of fats and oils in the production of structured lipids [36]. Most of the commercially available lipases catalyse the hydrolysis of long chain fatty acids poorly compared to short chain due to their complex structure. Marine microbes offer a potential source of enzymes which are stable at very high and low temperatures and active at broad pH range with unique fatty acids selectivity [37]. The substrate selectivity of S31 crude lipase activity was determined using the standard assay using the different p-NP esters of various
11
chain lengths: p-NP acetate (C2); p-NP butyrate (C4); p-NP caprylate (C8); p-NP decanoate (C10), p-NP laurate (C12), p-NP myristate (C14), p-NP palmitate (C16), p-NP stearate (C18), p-NP-EPA and p-NP-DHA. Lipase extracted from Schizochytrium S31 showed a broad range of substrate activity (Fig. 6). Maximum activity was observed towards short chain length fatty acids, followed by medium chain length fatty acids. Schizochytrium lipase preferred pNP-DHA with relative activity 20.14 ± 1.7 compared to other PUFA esters (Fig 6). The pNP-EPA was poorly hydrolyzed with relative activity 12.8 ± 1.4, compared to the other PUFA esters. An unusual fatty acid hydrolysis was observed towards p-NP-DHA, when compared with p-NP-EPA. Similar lipase selectivity towards DHA has been reported from Pseudomonas sp. In contrast a fish digestive lipase salmon intestines, hydrolyzed DHA at a faster rate than EPA, although this lipase was never characterised [38]. However, a study investigated the fatty acid selectivity of five commercial lipases using p-NP esters found that all lipase showed preference to EPA over DHA [18]. This selectivity might be due to the requirement of the Schizochytrium to process DHA more than EPA, since EPA is present in only trace amounts in the oil from this strain, whereas DHA levels are relatively high in Schizochytrium sp. Confirmation of DHA to EPA selectively will require additional work to purify the S31 lipase and confirm that observed activity is due to a single lipase. 4. Conclusion Schizochytrium S31 was grown in a bioreactor using Tween 80 as the carbon source, which resulted in high biomass and oleic acid levels. Lipase extraction by sonication was optimised and the fatty acid selectivity of the extracted lipase investigated using p-NP fatty acid esters. The lipase extract preferentially hydrolysed shorter chain fatty acids, however, DHA was preferentially hydrolysed over EPA. This preferential hydrolysis of DHA is unusual and may be industrially useful. This selectivity may be a result of this
12
Schizochytrium species having much higher levels of DHA then EPA, and so DHA processing is more important for the organism growth and function. Acknowledgement AB acknowledges the PhD scholarship award by Deakin University, Australia.
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Figure and table captions
Fig. 1. Effect of Tween 80 concentration on lipase production by Schizochytrium S31. Fig. 2. Fermentation profile of lipase production by Schizochytrium S31 in a stirred tank reactor. Fig. 3. DHA production as a function of incubation. Fig. 4. Comparison of fatty acid composition of Schizochytrium S31 lipid in Tween 80 vs glucose medium. Fig. 5. Cell disruption optimisation of Schizochytrium S31 a) as a function of sonication power, and b) time duration. Fig. 6. Substrate specificity of lipase from Schizochytrium S31 Footnote: p-nitrophenyl esters of different chain lengths were incubated with crude enzyme.
19
Fig. 1.
20
Lipid pH
100 7.0
0.9
300
6.5
0.8 250
150
100
6.0
5.5
0.5
5.0 0.4 4.5
50
0.3
40
80 70 60
0.6
pH
200
Biomass (g/L)
Lipid (mg/g)
0.7
90
50 40 30
30
Lipase Activity (IU/g)
1.0
Substrate depletion (%)
350
Biomass Lipase Substrate depletion
20
10
20 4.0
0.2 0 0.1 0
2
4
6
8
3.5 10
10
0
0
Incubation time (day)
Fig. 2.
21
Fig.
3.
22
Fig. 4
23
a
b
Fig. 5.
24
Fig. 6.
25
Table 1. Effect of sonication on protein release and lipase activity from Schizochytrium S31. Table 1.
Sonication (min)
period Lipase (IU/ml)
activity Protein concentration
Specific activity (IU/µg)
(µg/ml) 5
22.8±1.4
43±1.5
0.512
10
32.5±1.6
60±1.8
0.533
15
37.8±1.5
82±2.1
0.451
20
26.3±1.3
105±9
0.247
26