Graphical abstract
Introduction
Sterols are lipids that play a crucial role in all multicellular eukaryotes. The distribution and synthesis of sterols varies among the eukaryotic community, with animals primarily producing cholesterol (C27), whereas fungi and plants synthesis sterols with 28 to 29 carbon atoms (C28 and C29), known as ergosterols and phytosterols[1, 2]. These differences reflect the complex evolutionary history of sterol synthesis[3]. Phytosterols, which include phytostanols and sterols, are similar in structure and biological function to cholesterol[4, 5]. They are primarily found in vegetable oils, nuts, fruits, grains, and other plant products[6]. Phytosterols have been recognized for their various pharmacological properties, including the potential to lower total and low-density lipoprotein (LDL) cholesterol levels, thereby reducing the risk of cardiovascular disease[7-9]. Other health-promoting effects of phytosterols include anti-obesity[10], anti-diabetic[9], anti-microbial[11], anti-inflammatory[12], and immunomodulatory effects[13]. Additionally, it has been strongly suggested that phytosterols possess anti-cancer properties, as phytosterol-rich diets may reduce the risk of cancer by 20%[14, 15]. However, the high melting point and low solubility in both water and oils have greatly limited their further application in food, medicine, and other fields. To overcome this problem, phytosterol esters are considered suitable alternatives because they maintain all the excellent properties of original phytosterol[16, 17].
The chemical synthesis of phytosterol esters is industrially feasible, but the high energy consumption, low selectivity of the reaction, and the unavoidable by-products of dehydrated sterols limit its further applications[18]. Enzymatic catalysis which is performed under mild operating conditions, has high selectivity and fewer by-products and is therefore attractive in this field[19]. In recent years, several lipases, including Novozym@435[20], Candida rugosa lipase[21], and others, have demonstrated their ability in the synthesis of phytosterol esters. However, these reactions usually were performed in organic solvents, which limited the space-time yield and were harmful to the enzymes. To overcome the damage of organic solvent, some environmentally friendly mediums, such as ionic liquids[22], and supercritical carbon dioxide[23] were applied in biocatalysis. In this work, the esterification process is performed in a solvent-free system. However, we just found that the solvent-free system’s high viscosity limited the substrates’ mass-transfer effect, which made the reaction time extremely long. It indicated that the reaction can only be performed at a relatively higher temperature (50℃), which may not be beneficial to the lipase.
In response to this challenge, the immobilization of enzymes that aimed to improve stability and recyclability has attracted more and more interest, and various immobilization methods and supports with low cost, large specific surface area, and low diffusion limits of substrates, have been developed in past decades. For instance, diatomite, which is mainly composed of silicon dioxide (SiO2), was widely used in the immobilization of various enzymes due to its low cost, porous structure, low density, and chemical inertness. Chen et al.[24] suggested that the half-lives of diatomite immobilized of D-allulose 3-epimerase can be improved to 109 and 124 times than that of free enzyme at 55℃ and 60℃, respectively. Polyaniline-coated magnetic diatomite can efficiently immobilize invertase, β-galactosidase, and trypsin[25]; Candida sp. 99-125 immobilized on diatomite can be used to produce biodiesel efficiently[26]; Burkholderialipase immobilized on diatomite can be applied to a fixed-bed bioreactor and continuous biodiesel conversion[27]. However, single immobilization support does not meet the needs of all enzymes, therefore, for the immobilization of different enzymes, different modifiers need to be designed to confer a more suitable microenvironment to the support as well as the strength of interaction with the enzyme, e.g., basso’s study indicated that the octadecyl functional group can change the hydrophobic microenvironment of lipase, which can have a significant effect on the enzyme activity[28], and Singh modified the surface of silica nanoparticles with carboxylic acids with different numbers of alkyl chains, and found that silica-immobilized lipase modified by stearic acid with more hydrophobicity has a higher activity[29]. Moreover, the production of water during the esterification reactions will drive the reaction toward hydrolysis and lead to enzyme inactivation via promoting the aggregation of enzymes[30, 31]. Additionally, it will also accelerate the leakage of lipase from the support during reutilization.
Therefore, to develop a low-cost, sustainable, and high-efficiency enzymatic synthesis strategy for phytosterol esters, the solvent-free system was applied in the present study. To overcome the challenge of the low stability of lipase, a cheap inorganic porous material, namely diatomite was applied as a support for the immobilization ofCandida rugosa lipase (CRL). To further enhance its efficiency, the magnetic nano-Fe3O4 was first attached to the surface of diatomite via a co-precipitation method. Moreover, the octadecyl and sulfonyl groups were applied as modifiers of the diatomite for regulating its hydrophobicity, flexibility, and interaction and thus improve the efficiency of diatomite. Additionally, the immobilized enzyme prepared by this method was used in the enzymatic synthesis of phytosterol esters to test its stability and recyclability. This study is anticipated to provide a green and efficient innovative method for the enzymatic synthesis of phytosterol esters.