Seaweed Phenolics as Natural Antioxidants, Aquafeed Additives, Veterinary Treatments and Cross-Linkers for Microencapsulation.

Driven by consumer demand and government policies, synthetic additives in aquafeed require substitution with sustainable and natural alternatives. Seaweeds have been shown to be a sustainable marine source of novel bioactive phenolic compounds that can be used in food, animal and aqua feeds, or microencapsulation applications. For example, phlorotannins are a structurally unique polymeric phenolic group exclusively found in brown seaweed that act through multiple antioxidant mechanisms. Seaweed phenolics show high affinities for binding proteins via covalent and non-covalent bonds and can have specific bioactivities due to their structures and associated physicochemical properties. Their ability to act as protein cross-linkers means they can be used to enhance the rheological and mechanical properties of food-grade delivery systems, such as microencapsulation, which is a new area of investigation illustrating the versatility of seaweed phenolics. Here we review how seaweed phenolics can be used in a range of applications, with reference to their bioactivity and structural properties.

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.

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.

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.

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.