Co-Immobilization of Rhizopus oryzae and Candida rugosa Lipases onto mMWCNTs@4-arm-PEG-NH2—A Novel Magnetic Nanotube–Polyethylene Glycol Amine Composite—And Its Applications for Biodiesel Production

Abstract

This article describes the successful synthesis of a novel nanocomposite of superparamagnetic multi-walled nanotubes with a four-arm polyethylene glycol amine polymer (mMWCNTs@4-arm-PEG-NH2). This composite was then employed as a support for the covalent co-immobilization of Rhizopus oryzae and Candida rugosa lipases under appropriate conditions. The co-immobilized lipases (CIL-mMWCNTs@4-arm-PEG-NH2) exhibited maximum specific activity of 99.626U/mg protein, which was 34.5-fold superior to that of free ROL, and its thermal stability was greatly improved. Most significantly, CIL-mMWCNTs@4-arm-PEG-NH2 was used to prepare biodiesel from waste cooking oil under ultrasound conditions, and within 120 min, the biodiesel conversion rate reached 97.64%. This was due to the synergy effect between ROL and CRL and the ultrasound-assisted enzymatic process, resulting in an increased biodiesel yield in a short reaction time. Moreover, after ten reuse cycles, the co-immobilized lipases still retained a biodiesel yield of over 78.55%, exhibiting excellent operational stability that is attractive for practical applications. Consequently, the combined use of a novel designed carrier, the co-immobilized lipases with synergy effect, and the ultrasound-assisted enzymatic reaction exhibited potential prospects for future applications in biodiesel production and various industrial applications.

Recent developments of lipase immobilization technology and application of immobilized lipase mixtures for biodiesel production

“…It is the most efficient immobilization technique and it has more consideration because of the very strong interactions between the carrier materials and the enzyme, preventing the enzyme from leaking, reducing the diffusional limitations of the products or substrate and improving enzyme activity and stability. 1,10 This approach also improves enzymes' half-lives and thermal stability, 66 but the main weakness is the high cost of the carrier and the loss of enzyme activity after chemical treatment. 1,79 In addition, the process is complex, the reaction conditions are severe and the conditions are strictly monitored compared with the physical method.…”

“…81,82 In this regard, glutaraldehyde treatment was employed as a cross-linking agent for covalently immobilized different enzymes on the surface of fabricated magnetic multiwall carbon nanotubes (mMWCNTs) and applied for biodiesel production. 10,52,61 Additionally, covalent bonds could be also multipoint, in which the enzyme is connected to the carrier materials via multiple covalent chains, making the enzyme structure rigid and fixed. 67 These advantages give the immobilized lipase more stability, and protect it from extreme pH, heat, and organic solvent denaturation.…”

“…107 Another recent study was performed by our workgroup, in 2021, in which we designed a novel carrier comprising magnetic MWCNTs modified by the hyperbranched polymer of four-arm polyethylene glycol amine (4-arm-PEG-NH 2 ). 10 Four-arm-PEG-NH 2 has a large number of amino groups and can, thus, make an immobilized lipase once a cross-linking agent is linked with protein, improving the enzyme's stability and loading capacity. In this research, for easy separation purposes, mMWCNTs were first synthesized via a simple method that encapsulated Fe 2 O 4 nanoparticles within their interiors.…”

“…On the other hand, the industrialization of immobilized lipase-catalyzed biodiesel synthesis has been restricted by long reaction times, inefficient yields and expensive manufacturing methods. 9,10 Researchers in this field have sought to find the best methods to overcome these problems. In this respect, during the last 15 years, some research groups, for example Lee and co-workers (Korea), Yan and co-workers (China), Poppe and his associates (Brazil), Shahedi and his colleagues (Iran) and other researchers, have focused on lipase specificity and employed mixtures of immobilized lipases with different site specificities as green biocatalysts for biodiesel production.…”

“…4,13,14 Most importantly, different site-specificity lipases will work synergistically, target different triglyceride sites in the oil composition, and produce high biodiesel yields in a short reaction time. 10,15 Hence, this review shows a path forward for addressing biodiesel production using immobilized lipase, including recent developments of lipase immobilization technology and a mixture of immobilized lipases strategies that have shown great potential in recent studies. In reality, it looks to be a straightforward solution for boosting the efficiency of biodiesel production operations.…”