## 1 Introduction  
The formation of flavor compounds in cheese is a highly complex and versatile process. Different catabolic reactions, mainly lipolysis and proteolysis, lead to the formation of piquant cheesy flavors [1]. For certain types of cheese, especially for white cheeses, so-called brine cheeses, such as Feta and its variants created from cow's milk, for kashkaval and many other Italian cheeses, such as _Parmigiano Regsiano_ or Provolone, the majority of character impact compounds are short- to medium-chain free fatty acids such as butanoic acid (C4:0), hexanoic acid (C6:0), octanoic acid (C8:0), or decanoic acid (C10:0) [2]. Milk fat contains more than 400 different fatty acids, of which only 15 are present in amounts above 1% of the total fatty acids, chemically bound in triglycerides [3]. However, only volatile free fatty acids (vFFA) contribute the flavor. These vFFA possess different olfactory properties depending on their chain lengths, ranging from goats, chees (e.g., C6:0, C8:0, C10:0) to soapy, waxy (e.g., C12:0, C14:0) flavors [4]. The formation of these vFFA is catalyzed by lipolytic enzymes during the ripening process. Endogenous lipases in milk were originally used to produce this special type of flavor, but due to the pasteurization of the raw milk, which is the industrial standard to inactivate pathogens and other microorganisms to increase the shelf life of the products, those enzymes are inactivated. To compensate for this heat damage, enzyme preparations may be added. Most commonly used enzymes are derived from goats, sheep, or calves in the form of extracts of sublingual tissue, so-called pregastric esterases (PGE), or paste rennet from the stomachs of the aforementionedanimals [5; 6; 7]. PGEs show a high specificity towards short-chain fatty acids (C4:0 to C10:0), which enables them to create goats, cheesy flavors, without adding a soapy note caused by the hydrolytic release of long-chain fatty acids [8]. However, these animal-derived enzyme preparations are neither suitable for a vegetarian, kosher, or halal diet, nor for the complete value chain of the side stream product whey. Therefore, alternative enzyme preparations are highly sought-after. To overcome these limitations, the market offers numerous lipidic enzymes from different origins such as a lipase from _Rhizomucor miehei_, which is sold under the tradename Piccantase(r). Nevertheless, these enzymes do not meet all requirements by either lacking the functionality in terms of producing a flavor that is typical for cheese prepared with PGE or tending to release lipids non-specifically, which may lead to soapy off-flavors. Therefore, finding suitable replacements for PGEs is challenging [8; 9]. Despite these challenges, a suitable candidate was found in a previous study, as described by [PERSON] et al. [10]. Application tests with different culture supernatants of basidiomycetous fungi revealed that these kinds of enzyme preparations may represent suitable replacements for PGEs. With the purified lipase of the golden oyster mushroom, _Pleutrotus citrinopileatus_ (PCI_Lip), the sensory properties of the produced cheese were close to those of a cheese prepared with a PGE reference enzyme (optizym z10 uc). Unfortunately, these cheeses still had a slight soapy aftertaste [10]. Therefore, a semi-rational design was used as a powerful tool in the present study to engineer the PCI_Lip chain length specificity, in particular, to reduce the hydrolysis of milk triglycerides with long-chain fatty acids. The semi-rational design combines the advantages of both, directed evolution and rational design, by the usage of partial knowledge of the protein structure without the need for a crystal structure. Smart libraries can be created and analyzed in silico, providing smaller and easier-to-screen libraries compared to directed evolution approaches [11]. Most lipases belong to the group of \(\alpha/\beta\)-hydrolase fold proteins.