Lipases are a class of serine hydrolases that catalyze the hydrolysis of triglycerides as well as a variety of other reactions [1]. These enzymes are widely distributed in nature, with sources including animal tissues, plant seeds, and various microorganisms. Among them, microbial lipases have become the predominant source for industrial applications owing to their wide diversity, ease of large-scale fermentation, and generally high stability [2]. Owing to their versatile catalytic properties, lipases are widely employed in various industries, including food processing, biodiesel production, chiral drug resolution, and detergent formulation, etc. [3], [4], [5], [6].

Among the various microbial lipases, Rhizopus oryzae lipase (ROL) has attracted considerable attention because of its strict sn-1,3 positional specificity [7]. This unique selectivity provides a distinct advantage in the synthesis of structurally tailored lipids with defined nutritional properties, such as human milk fat substitutes and cocoa butter analogues [8], [9]. Furthermore, its excellent enantioselectivity renders it an efficient biocatalyst for resolution of chiral alcohols, acids, and their derivatives [10], [11].

Although ROL has been successfully heterologously expressed and is available as a commercial enzyme, its intrinsic thermal instability remains a major bottleneck hindering its broader industrial application [12], [13]. Thermal instability represents a fundamental challenge for many industrial enzymes. As protein catalysts, enzymes depend on their precisely organized yet inherently fragile three-dimensional structures refined through extensive evolutionary optimization to perform catalytic functions [14]. This structural integrity is primarily maintained by noncovalent interactions, including hydrogen bonding, hydrophobic interactions, ionic bonds, and van der Waals forces [15]. When the environmental temperature exceeds the enzyme’s optimal range, these weak interactions will be disrupted, leading to marked alterations in molecular conformation [16]. Such conformational changes distort the geometry of the active site, ultimately resulting in loss of catalytic activity. In case of ROL, previous studies have reported that rapid inactivation occurs above 50 °C, with severe and irreversible denaturation and precipitation observed above 60 °C [17]. In addition to thermal stress, ROL is also susceptible to interfacial and chemical inactivation [18], [19]. However, existing studies primarily report macroscopic inactivation phenomena and provide limited insights into the underlying mechanisms: the kinetic pathways governing thermal inactivation remain unresolved, and the relationship between activity loss and structural perturbations at multiple levels needs to be clarified. Therefore, systematic investigation of the thermal inactivation mechanism of ROL, along with the development of effective stabilization strategies, is essential for expanding its industrial applicability.

Currently, strategies for improving enzyme stability can be broadly classified into four main categories: protein engineering, immobilization, chemical modification, and addition of stabilizing agents [20]. Protein engineering enhances enzyme stability by introducing targeted mutations at the genetic level through rational design or directed evolution, thereby modifying the enzyme’s molecular structure [21], [22]. Although this approach offers great potential, it generally requires an in-depth understanding of the enzyme’s three-dimensional structure and involves complex and costly experimental techniques [23], [24]. Immobilization technology anchors enzymes onto specific carrier materials, facilitating their separation and recovery from reaction systems for reuse, while occasionally conferring moderate improvements in stability [25], [26]. However, immobilization may lead to partial loss of activity, and factors such as carrier cost and mass transfer resistance must be carefully considered. Chemical modification aims to alter the surface properties of enzymes by covalently attaching specific chemical groups [27]. Nevertheless, this method carries inherent risks, including non-specific modification sites and potential impairment of catalytic activity. In contrast, addition of stabilizing agents represents the simplest, most cost-effective, and most easily scalable strategy. This approach involves incorporating substances such as sugars [16], [28], [29], polyols [30], certain salts [31], or nonionic surfactants [32] into enzyme formulations. These additives modulate microenvironment surrounding enzyme molecules through mechanisms such as preferential exclusion and hydrogen-bond network formation, thereby helping to maintain native conformation of the enzyme under stress conditions and preventing denaturation and aggregation. Importantly, this strategy does not require any modification of the enzyme molecule itself, thus preserving its intrinsic properties while being readily applicable to large-scale production processes. However, current research on ROL primarily focuses on gene cloning, expression system optimization, fermentation control, and enzyme immobilization, while systematic studies on the rational screening of stabilizers grounded in a profound understanding of the inactivation mechanisms remain limited.

Therefore, in this study, we systematically investigated the thermal stability of ROL from three perspectives: kinetics, structure, and formulation optimization. Thermal inactivation kinetics were analyzed to elucidate the mechanism and key parameters. High-purity ROL was subjected to spectroscopic analysis, which revealed heat-induced alterations in secondary and tertiary structures. Guided by these insights, stabilizers including sugars, polyols, metal ions, and surfactants were screened and further optimized using Plackett-Burman and response surface methodology (RSM). Overall, the aim of this work is to elucidate the molecular basis of ROL thermal inactivation and then correspondingly develop an effective strategy to enhance its stability for industrial applications.