Increased hydrolysis by Thermomyces lanuginosus lipase for omega-3 fatty acids in the presence of a protic ionic liquid

Abstract

We report that the hydrolytic performance of Thermomyces lanuginosus lipase, TLL, and its selectivity towards concentrating clinically important omega 3 fatty acids was increased by the addition of a protic ionic liquid, pIL, Triethylammonium mesylate, TeaMs. We show that TeaMs has a structure altering effect on TLL, changing both the secondary and tertiary structure of TLL. The thermal activity of TLL was also significantly enhanced by the addition of TeaMs.

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The clinical benefits of the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) for the treatment and prevention of disorders such as cardiovascular, Alzheimer's and Parkinson's diseases have been extensively studied.¹ Concentrated forms of EPA and DHA from fish oil are used as nutritional supplements, functional foods and pharmaceutical products.² Current techniques such as fractional distillation and urea complexation are used for the production of these concentrates.³ These methods are expensive, environmentally unfriendly and damaging to these chemically sensitive compounds often resulting in oxidation.⁴ The use of enzymes, in particular lipases,⁵ for the production of omega 3 fatty acids has many advantages over the above mentioned techniques, including preventing oxidation, use of mild temperatures and is relatively inexpensive.² However, there is a need to enhance the selectivity of commercially available lipases towards concentrating greater amounts of omega 3 in particular EPA and DHA from fish and algal oils.

“Solvent engineering” refers to the activity, enantioselectivity, regioselectivity, or chain length selectivity of an enzyme being significantly influenced by the nature of the solvent and the hydrolysis conditions.⁶ Ionic liquids, ILs, are solvents, which comprise entirely of ions. Ionic liquids, ILs are widely used and are often referred to as designer solvents. ILs are capable of being tailored to suit a range of applications. ILs as solvents for biomolecules has attracted attention due largely to the beneficial outcomes that result from their use, including increased thermal stability⁷ and shelf life⁸ of proteins, enhanced refolding of proteins,⁹ and as additives for protein crystallization¹⁰ and in particular for the enhancement of biocatalytic reactions.¹¹

In our laboratory, we are interested in protic ionic liquids (pILs) and their impact on chemical and enzymatic reactions when used as a solvent. PILs are a sub class of the ionic liquid family formed by the neutralization of a Bronsted base with a Bronsted acid. PILs have an additional tuneable feature that results due to the proton transfer, known as the proton activity. The proton activity has been used as a relative scale to select appropriate pILs for the solvation of biomolecules.¹²

In this communication, we report the impact of using the pIL Triethylammonium mesylate, TeaMs on the structure and activity of Thermomyces lanuginosus lipase, TLL. TLL is used commercially in both the immobilized and mobilized forms for producing or re-esterifying EPA and DHA.¹³ We selected TeaMs as previously the use of secondary and tertiary amines have been shown to enhance lipase activity.¹⁴ Additionally, we recently reported on the ability of TeaMs to alter the structure of the Aβ_1–40 peptide.¹⁵ We found that TeaMs induces an increased helical structure. Given the relationship between structure and activity for enzyme selectivity, and the fact that lipase activity is known to vary with solvent, we sought to use TeaMs as a novel solvent to apply a solvent engineering approach aimed at enhancing the rate of hydrolysis by TLL for use in the concentration of omega-3 fats, in particular DHA and EPA from anchovy oil.

Fig. 1a shows the percentage hydrolysis of anchovy oil by TLL, as a function of TeaMs concentration (see ESI† for experimental, all experiments reproduced in triplicate). TLL was used as received in solution form. It can be seen that enhanced hydrolysis was achieved with increasing concentration of TeaMs, with the maximum hydrolysis found at the highest achievable TeaMs concentration of 48 wt% TeaMs. At this TeaMs concentration both the degree of hydrolysis and the hydrolysis time is enhanced. Greater information regarding the nature of the lipid classes obtained from the TLL hydrolysed anchovy oil can be determined using lipid class determination. As such, we have determine the various lipids obtained in the absence and at 48 wt% TeaMs, these are presented in Fig. 1b and c respectively. Typically, partial hydrolysis using lipases will yield low amounts of monoacylglycerol (MAG) and high amounts of diacylglycerol (DAG) and free fatty acid (FFA), as clearly observed in Fig. 1b in the absence of TeaMs, where the % MAG is low at 1% after 36 hours of hydrolysis. Fig. 1c shows the same classification for the various lipid classes obtained from TLL hydrolysed anchovy oil in the presence of 48 wt% TeaMs. A clear increase in the MAG percentage is observed, with the MAG percentage being 37% at 36 hours. The increase in the MAG percentage due to the addition of TeaMs is likely due to the further hydrolysis of DAG into MAG since the DAG percentage is reduced to 8% (with TeaMs Fig. 1c), compared to 20% (without TeaMs Fig. 1b). This was a promising outcome as previous studies have shown that significant amounts of EPA and DHA are often deposited in the MAG phase due to partial selectivity of hydrolysis for shorter-chain fatty acids.¹⁶ From here, we investigated the relative percentage of EPA and DHA concentrated by TLL from anchovy oil in the absence and presence of TeaMs using gas chromatography as shown in Table 1. It can be seen that in the absence of TeaMs the DHA concentrated in the glyceride portion is 22.94%. In the presence of 48 wt% TeaMs the concentration of DHA in the glyceride portion is considerably higher and calculated to be 34.03%. This is a significant improvement, most likely driven by the ability of the TeaMs dissolved TLL to cleave a higher percentage of the shorter chain fatty acids including C14 : 0, C16 : 0, C16 : 1n7 and C18 : 1n9, from the triglyceride backbone. As a result, these shorter chain fatty acids are concentrated in the FFA portion. Table 1 also shows that the TLL dissolved in 48 wt% TeaMs was significantly more selective towards the enrichment of both EPA and DHA when considering the percentage increase. The total increase in DHA and EPA, from both the FFA and glyceride portions, was 25% and 34% respectively. This compares with a 15% increase in the shorter chain fatty acids in the same reaction. These results indicate that the presence of TeaMs has not only enhanced overall hydrolysis but has specifically enhanced DHA and EPA concentration.

Fig. 1 (a) Percentage hydrolysis of anchovy oil using TLL at various TeaMs concentrations, (b) TLC profile of lipid classes from hydrolysed anchovy oil using TLL at 0 wt% TeaMs and (c) TLC profile of lipid classes from hydrolysed anchovy oil using TLL at 48 wt% TeaMs.

Table 1 Fatty acid composition of fish oil before and after hydrolysis

Fatty acid (%)	Anchovy oil	Hydrolyzed oil (0% TeaMs)		Hydrolyzed oil (48 wt% TeaMs)
		FFA	Glyceride	FFA	Glyceride
Myristic acid (C14 : 0)	8.40	8.77	3.42	11.21	2.58
Palmitic acid (C16 : 0)	16.70	20.66	4.82	27.23	3.49
Palmitoleic acid (C16 : 1n7)	9.80	11.91	3.44	15.66	2.42
Oleic acid (C18 : 1n9 − cis + trans)	7.10	9.82	2.96	13.41	2.06
EPA (C20 : 5n3)	17.00	10.62	12.27	17.00	18.03
DHA (C22 : 6n3)	13.00	4.88	22.94	3.24	34.03
Hydrolysis degree (%)	—	63.68		79.76

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The immobilization of lipases can often alter the structure and this can directly impact the performance of the lipase. To determine if the enhanced activity observed in the presence of TeaMs was due to a change in structure, we determined the structure of TLL using Circular Dichroism. Fig. 2a and b show the secondary and tertiary structure of TLL at TeaMs concentrations of 0, 24 and 48 wt% and a TLL concentration of 215 μg mL⁻¹ and 645 μg mL⁻¹ respectively. At TeaMs concentrations above 9 wt% a change in the secondary structure similar to that represent by 24 wt% (red curve) in Fig. 2a is observed, which is consistent with a shift from α-helical to β-sheet structure, while the tertiary structure remains the same, Fig. 2b red curve. (ESI† for all wt% spectra) At TeaMs concentrations of 46 wt% and above, Fig. 3a green curve, another shift in the secondary structure is observed. This structure is considered to be predominately helical in nature as indicated by the minimum at 207 nm. Interestingly the tertiary structure also shows a change at 46 wt% TeaMs, Fig. 2b green curve. The change in structure observed with increasing TeaMs concentration is likely driven by the decrease in “available” water. It has previously been shown that lipases in low water environments adopt a firmer and more compact structure.¹⁷ The change in tertiary structure may also account for the specific enhancement of DHA and EPA shown in Table 1. Previously, aprotic ILs have been used to investigate the activity of TLL and other lipases.¹⁷ The impact of aprotic ILs on TLL was varied and depended largely on the aprotic IL and the lipase.

Fig. 2 CD spectrum for TLL dissolved in 0 wt% TeaMs (black), 24 wt% (red), 46 wt% (green) where (a) secondary structure and (b) tertiary structure.

Fig. 3 Log % remaining activity as a function of temperature over a two hour period for TLL dissolved in (a) phosphate buffer and (b) 48 wt% TeaMs.

Finally, we explored the thermal stability of TLL in the presence of TeaMs. Fig. 3a and b show the activity of TLL at various temperatures over a two hour period in the absence and presence of 48 wt% TeaMs respectively. It can be seen that the presence of TeaMs enhances the thermal stability of TLL at 80 °C. TLL retains 50% of its original activity in the presence of 48 wt% TeaMs at 80 °C. In contrast, all activity is lost in the TLL dissolved in phosphate buffer at 80 °C after two hours.

In this study, we showed that the pIL, TeaMs has the ability to alter the structure and enhance the activity of the Thermomyces lanuginosus lipase, TLL. The hydrolysis activity was enhanced from 64% to 80% over a 36 hour period. We also showed an enhancement in the thermal stability of TLL in the presence of TeaMs. Future work is aimed at exploring the structure inducing effects of other pILs and determining the influence this has on hydrolytic performance and selectivity of the lipase.

N. B. acknowledges the generous financial support of the Australian Research Council through the award of an Australian Postdoctoral Fellowship.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Experimental procedure supplied and additional CD data. See DOI: 10.1039/c2cy20392h

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