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.