Elsevier

Process Biochemistry

Volume 53, February 2017, Pages 30-35
Process Biochemistry

Tween 80 influences the production of intracellular lipase by Schizochytrium S31 in a stirred tank reactor

https://doi.org/10.1016/j.procbio.2016.11.026Get rights and content

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 were the optimum condition 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.

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 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 come 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 enzymatic methods are faster than other methods and can be carried out under milder conditions than methods involving chromatographic separation, molecular distillation or urea complexation [11]. Commercially available lipases have been exploited for omega-3 fatty acid concentration [12], [13], [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 source 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.

Section snippets

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), α-linolenic acid (C18:3), eicosapentaenoic acid (C20:5) and

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

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

Acknowledgement

AB acknowledges the PhD scholarship award by Deakin University, Australia.

References (38)

Cited by (12)

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    The extracellular lipase activity with p-NPP confirmed that thraustochytrids could hydrolyze hydrophobic substrates and assimilate for growth and lipid synthesis. Byreddy et al. (2017) has previously reported the intracellular lipase synthesis for Schizochytrium S31 using tween-80 as substrate. The “de novo'' mode of fermentation led to higher biomass, lipid and DHA content but lower TUFA and TUFA/TSFA ratio.

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    The fatty acid components observed in this study were similar to those detected in previous studies (Chen et al., 2016; Ethier et al., 2011; Lung et al., 2016; Byreddy et al., 2017). The DHA content of cells cultured in TWW ranged from 22.36 to 23.20% of the total fatty acids, higher than that obtained in a study by Byreddy et al. where a DHA content of only approximately 20% of total fatty acids was obtained using the same Schizochytrium species evaluated in this study (Byreddy et al., 2017). However, compared to other Schizochytrium species, the DHA content obtained in this study was lower.

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