Effect of Triton X-100 on the Activity and Selectivity of Lipase Immobilized on Chemically Reduced Graphene Oxides.

The effect of support hydrophobicity on lipase activity and substrate selectivity was investigated with and without Triton X-100 (TX-100). Lipases from Thermomyces lanuginosa (TL) and Alcaligenes sp. (QLM) were immobilized on graphene oxide (GO) and a range of chemically reduced graphene oxides (CRGOs) with different levels of surface hydrophobicity. Activity assays using 4-hydroxy-N-propyl-1,8-naphthalimide (NAP) esters of varying chain lengths (NAP-butyrate (NAP-B), NAP-octanoate (NAP-O), and NAP-palmitate (NAP-P)) showed that the activity of immobilized QLM and TL decreased by more than 60% on GO and 80% on CRGO (2 h), with activity decreasing further as surface hydrophobicity of the CRGOs increased. Across the hydrophobicity range of GO/CRGOs, the substrate selectivity of QLM shifted from more readily hydrolyzing NAP-P to NAP-B, while TL retained its substrate selectivity for NAP-O. Lipase TL was also shown to desorb from GO and 2 h CRGO when mixed with NAP-O and NAP-P, whereas QLM did not. Circular dichroism analyses of the lipase α-helix content correlate to the observed activity data, with decreases in the α-helical content (40% in TL and 20% in QLM relative to free lipase) consistent with decreases in activity after immobilization on GO. α-Helical content decreased even further as the surface hydrophobicity of CRGOs increased. Attenuated total reflectance-Fourier transform infrared spectroscopy also showed significant changes to the lipase secondary structure upon immobilization. The addition of TX-100 into the activity assay modified the substrate selectivity of immobilized QLM, improving the activity against NAP-O (90%) and NAP-P (67%) compared to the activity measured without TX-100. It was shown that TX-100 primarily affected the activity of QLM by interacting with the ester substrate and the lipase itself. This study provides an improved understanding of how support hydrophobicity and the presence of TX-100 can affect activity/selectivity of lipases immobilized on hydrophobic supports.

Lipase-catalysed synthesis of mono- and di-acyl esters of glyceryl caffeate in propylene carbonate and their antioxidant properties in tuna oil.

Development of new non-toxic antioxidants with diverse hydrophobic properties is important due to growing concerns about the toxicity of artificial oil-soluble antioxidants, the comparatively low effectiveness of natural options, and the complex role hydrophobicity plays in antioxidant effectiveness. Using caffeic acid, a naturally occurring phenolic acid with potent antioxidant activity, a range of glyceryl caffeate esters (decanoate and palmitate) were prepared using lipase-catalysed esterification reactions. Glyceryl-1-caffeate (GC) was prepared from ethyl caffeate and glycerol (acting as both the solvent and the substrate), catalysed by immobilised Candida Antarctica lipase B (Novozym-435) at 80 °C under vacuum. Esterification of GC with decanoic acid using immobilised Thermomyces lanuginosus lipase (TLIM) or Novozym-435 was found to be selective towards mono-acylated or di-acylated products, respectively. The reaction was performed in an unconventional solvent, propylene carbonate (PC), which has many of the attributes of a green solvent. Product conversions in PC were comparable to the best performing conventional solvents. In contrast to conventional volatile solvents, the low volatility of PC allowed the reaction to be performed under vacuum, without the need for molecular sieves for removal of water produced during the reaction. Diisopropyl ether was effective at extracting the more lipophilic products from PC. Both the lipase (Novozym-435) and PC were reused four times with only a small loss in conversion efficiency. Glyceryl caffeate esters performed much better than α-tocopherol at protecting bulk tuna oil from oxidation (analysed using Rancimat). A comparison of glyceryl caffeate esters (decanoate/palmitate and mono-/di-acylated) showed that their antioxidant effectiveness in bulk tuna oil was not affected by chain-length, but compounds containing only one fatty ester were slightly more effective than those containing two fatty esters.

Immobilisation of Candida rugosa lipase on a highly hydrophobic support: A stable immobilised lipase suitable for non-aqueous synthesis.

Lipase from Candida rugosa (CrL) was immobilised on highly hydrophobic, octadecyl methacrylate resin (Lifetech™ ECR8806M) via interfacial adsorption. The aim was to produce a stable biocatalyst suitable for use in a range of lipid-modifying reactions. Immobilisation was carried out in 10 mM phosphate buffer (pH 6.0) over 24 h at 21 °C. High protein binding of 58.7 ±â€¯4.9 mg/g dry support accounted for ∼53 % of the applied protein. The activity recovery against tributyrin was 74.0 ±â€¯1.1 %. The specific activity of immobilised CrL against tributyrin was considerably higher than that of Novozym® 435, at 1.79 ±â€¯0.05 and 1.08 ±â€¯0.04 U/mg bound protein, respectively. Incubation with high concentrations (10 % w/v) of both Triton X-100 and SDS resulted in only a small reduction in immobilised lipase activity. Solvent-free synthesis of glycerides by the FFA-saturated immobilised CrL was successful over 6 reaction cycles, with no apparent loss of activity.

Polydatin-fatty acid conjugates are effective antioxidants for stabilizing omega 3-containing bulk fish oil and fish oil emulsions.

Candida antarctica lipase B-catalysed synthesis of lipophilic esters of polydatin was investigated along with their antioxidant activities. The effects of synthesis parameters such as solvent, substrate molar ratio, enzyme concentration, addition of molecular sieves, reaction temperature and time on the production of ester were studied and optimised. The highest production of esters was obtained with acetone as the reaction solvent. The antioxidant activities of the esters were compared with those of commercial butylated hydroxytoluene (BHT) and α-tocopherol. All polydatin esters inhibited the oxidative destruction of ß-carotene more effectively than did BHT and α-tocopherol. Results of thiobarbituric acid tests showed that in bulk fish oil, all esters were more effective than α-tocopherol at 2 mmol/kg concentration but were not as effective as BHT. In fish oil-emulsions, all esters were more effective than both BHT and α-tocopherol at 2 mmol/kg concentration. The synthesized polydatin esters are promising antioxidants for oil/fat-based foods.

A simplified method for active-site titration of lipases immobilised on hydrophobic supports.

The aim of this work was to develop a simple and accurate protocol to measure the functional active site concentration of lipases immobilised on highly hydrophobic supports. We used the potent lipase inhibitor methyl 4-methylumbelliferyl hexylphosphonate to titrate the active sites of Candida rugosa lipase (CrL) bound to three highly hydrophobic supports: octadecyl methacrylate (C18), divinylbenzene crosslinked methacrylate (DVB) and styrene. The method uses correction curves to take into account the binding of the fluorophore (4-methylumbelliferone, 4-MU) by the support materials. We showed that the uptake of the detection agent by the three supports is not linear relative to the weight of the resin, and that the uptake occurs in an equilibrium that is independent of the total fluorophore concentration. Furthermore, the percentage of bound fluorophore varied among the supports, with 50 mg of C18 and styrene resins binding approximately 64 and 94%, respectively. When the uptake of 4-MU was calculated and corrected for, the total 4-MU released via inhibition (i.e. the concentration of functional lipase active sites) could be determined via a linear relationship between immobilised lipase weight and total inhibition. It was found that the functional active site concentration of immobilised CrL varied greatly among different hydrophobic supports, with 56% for C18, compared with 14% for DVB. The described method is a simple and robust approach to measuring functional active site concentration in immobilised lipase samples.

Controlling enzyme function through immobilisation on graphene, graphene derivatives and other two dimensional nanomaterials.

Robust enzyme immobilisation methods that preserve enzyme activity while enabling enzymes to be recovered and reused multiple times have important applications in biocatalysis. However, immobilisation can change the functionality of enzymes, both in terms of their level of activity and their selectivity. These changes in activity are unpredictable and at present cannot be controlled, but if fully understood at a fundamental level could offer the opportunity to create highly targetted enzyme systems for specific applications. In this review, we will highlight the use of two dimensional nanomaterials (2D NMs), particularly graphene and its derivatives, as immobilisation materials to modify and control the selectivity and activity of various enzymes. The fundamental information obtained from immobilising enzymes on 2D NMs allows for the implementation of improved immobilisation strategies and assists in the design of next generation nano- and macro-materials for enzyme immobilisation. We hope that this review will highlight the potential for tailoring enzyme activity and selectivity through immobilisation.

Raman Spectroscopy of Fish Oil Capsules: Polyunsaturated Fatty Acid Quantitation Plus Detection of Ethyl Esters and Oxidation.

Fish oils are the primary dietary source of ω-3 polyunsaturated fatty acids (PUFA), but these compounds are prone to oxidation, and commercial fish oil supplements sometimes contain less PUFA than claimed. These supplements are predominantly sold in softgel capsules. In this work, we show that Fourier transform (FT)-Raman spectra of fish oils (n = 5) and ω-3 PUFA concentrates (n = 6) can be acquired directly through intact softgel (gelatin) capsules. These spectra could be used to rapidly distinguish supplements containing ethyl esters from those containing triacylglyceride oils. Raman spectroscopy calibrated with partial least-squares regression against traditional fatty acid methyl ester analyses by gas chromatography-mass spectrometry could be used to rapidly and nondestructively quantitate PUFA and other fatty acid classes directly though capsules. We also show that FT-Raman spectroscopy can noninvasively detect oxidation with high sensitivity. Oils with peroxide values of as low as 10 mequiv kg-1, which are on the cusp of falling outside of specification, could be readily distinguished from oils that were within specification (7 mequiv kg-1).

4-Hydroxy-N-propyl-1,8-naphthalimide esters: New fluorescence-based assay for analysing lipase and esterase activity.

Research using 1,8-naphthalimide derivatives has expanded rapidly in recent years owing to their cell-permeable nature, ability to target certain cellular locations and fluorescent properties. Here we describe the synthesis of three new esters of 4-hydroxy-N-propyl-1,8-naphthalimide (NAP) and the development of a simple and sensitive assay protocol to measure the activity of carboxylester hydrolases. The NAP fluorophore was esterified with short (butyrate), medium (octanoate) and long (palmitate) chain fatty acids. The esters were spectroscopically characterised and their properties investigated for their suitability as assay substrates. The esters were found to be relatively stable under the conditions of the assay and levels of spontaneous hydrolysis were negligible. Non-specific hydrolysis by proteins such as bovine serum albumin was also minimal. A simple and rapid assay methodology was developed and used to analyse a range of commercially available enzymes that included enzymes defined as lipases, esterases and phospholipases. Clear differences were observed between the enzyme classes with respect to the hydrolysis of the various chain length esters, with lipases preferentially hydrolysing the medium chain ester, whereas esterases reacted more favourably with the short ester. The assay was found to be highly sensitive with the fluorophore detectable to the low nM range. These esters provide alternate substrates from established coumarin-based fluorophores, possessing distinctly different excitation (447 nm) and emission (555 nm) optima. Absorbing at 440-450 nm also offers the flexibility of analysis by UV-visible spectrophotometry. This represents the first instance of a naphthalimide-derived compound being used to analyse these enzymes.

The use of immobilised digestive lipase from Chinook salmon (Oncorhynchus tshawytscha) to generate flavour compounds in milk.

The aim of this research was to determine the potential of immobilised digestive lipase from Chinook salmon (Oncorhynchus tshawytscha) to generate flavour compounds in milk. The lipase was immobilised on hydrophobic resin (Toyopearl® Butyl) and used to hydrolyse milk lipids in a batch reactor. The lipase was stable when immobilised and there was no significant resin fouling or enzyme inhibition between cycles. Eight cycles were achieved before the hydrolysis rate dropped significantly because of physical losses of the immobilised lipase. The immobilised lipase showed the highest specificity towards short-chain fatty acids butanoic and hexanoic acids, the main dairy product flavour and odour compounds. Based on the performance of the reactor, and the ability of the lipase to alter free fatty acid composition and sensory characteristics of milk, the immobilised salmon lipase has potential applications in developing dairy products with unique flavours.

Rapid Quantitative Determination of Squalene in Shark Liver Oils by Raman and IR Spectroscopy.

Squalene is sourced predominantly from shark liver oils and to a lesser extent from plants such as olives. It is used for the production of surfactants, dyes, sunscreen, and cosmetics. The economic value of shark liver oil is directly related to the squalene content, which in turn is highly variable and species-dependent. Presented here is a validated gas chromatography-mass spectrometry analysis method for the quantitation of squalene in shark liver oils, with an accuracy of 99.0 %, precision of 0.23 % (standard deviation), and linearity of >0.999. The method has been used to measure the squalene concentration of 16 commercial shark liver oils. These reference squalene concentrations were related to infrared (IR) and Raman spectra of the same oils using partial least squares regression. The resultant models were suitable for the rapid quantitation of squalene in shark liver oils, with cross-validation r (2) values of >0.98 and root mean square errors of validation of ≤4.3 % w/w. Independent test set validation of these models found mean absolute deviations of the 4.9 and 1.0 % w/w for the IR and Raman models, respectively. Both techniques were more accurate than results obtained by an industrial refractive index analysis method, which is used for rapid, cheap quantitation of squalene in shark liver oils. In particular, the Raman partial least squares regression was suited to quantitative squalene analysis. The intense and highly characteristic Raman bands of squalene made quantitative analysis possible irrespective of the lipid matrix.

Purification and properties of digestive lipases from Chinook salmon (Oncorhynchus tshawytscha) and New Zealand hoki (Macruronus novaezelandiae).

Lipases were purified from delipidated pyloric ceca powder of two New Zealand-sourced fish, Chinook salmon (Oncorhynchus tshawytscha) and hoki (Macruronus novaezelandiae), by fractional precipitation with polyethylene glycol 1000, followed by affinity chromatography using cholate-Affi-Gel 102, and gel filtration on Sephacryl S-300 HR. For the first time, in-polyacrylamide gel activity of purified fish lipases against 4-methylumbelliferyl butyrate has been demonstrated. Calcium ions and sodium cholate were absolutely necessary both for lipase stability in the gel and for optimum activity against caprate and palmitate esters of p-nitrophenol. A single protein band was present in native polyacrylamide gels for both salmon and hoki final enzyme preparations. Under denaturing conditions, electrophoretic analysis revealed two bands of 79.6 and 54.9 kDa for salmon lipase. It is proposed that these bands correspond to an uncleaved and a final form of the enzyme. One band of 44.6 kDa was seen for hoki lipase. pI values of 5.8±0.1 and 5.7±0.1 were obtained for the two salmon lipase forms. The hoki lipase had a pI of 5.8±0.1. Both lipases had the highest activity at 35°C, were thermally labile, had a pH optimum of 8-8.5, and were more acid stable compared to other fish lipases studied to date. Both enzymes were inhibited by the organophosphate paraoxon. Chinook salmon and hoki lipases showed good stability in several water-immiscible solvents. The enzymes had very similar amino acid composition to mammalian carboxyl ester lipases and one other fish digestive lipase. The salmon enzyme was an overall better catalyst based on its higher turnover number (3.7±0.3 vs. 0.71±0.05 s(-1) for the hoki enzyme) and lower activation energy (2.0±0.4 vs. 7.6±0.8 kcal/mol for the hoki enzyme) for the hydrolysis of p-nitrophenyl caprate. The salmon and hoki enzymes are homologous with mammalian carboxyl ester lipases.