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.

Lipases are considered the most widely used industrial biocatalysts, and play a significant role in diverse industrial and biotechnological applications such as in biodiesel. Biodiesel is a renewable and ecologically friendly biofuel, and has received significantly increased attention in recent years. The reaction media of biodiesel synthesis via the transesterification process of fats and oils could be considered cascade reactions. The use of a mixture of immobilized lipases as biocatalysts in biodiesel reactions has received much consideration since it is a ‘greener technique’ and has several advantages over free lipases. The selection of appropriate carrier materials and immobilization techniques fundamentally affects the immobilized lipases’ catalytic performance for biodiesel production. Hence, this review discusses the recent developments for carrier materials and techniques for lipase immobilization as well as the current status of immobilized lipases mixtures and their application for biodiesel synthesis. The initial part of this review presents a brief introduction of lipases and their application for biodiesel preparation. New carrier materials and different basic and recent lipase immobilization techniques are also evaluated. Most significantly, studies carried out on mixtures of immobilized lipase‐catalyzed biodiesel for the last 15 years (2006–2021) are summarized. The economic and environmental evaluation of enzymatic biodiesel production is also presented. In the future, the application of mixed and co‐immobilized lipases may be enlarged to other enzymes and processes. © 2022 Society of Chemical Industry and John Wiley & Sons, Ltd

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