Processes involving lipases in obtaining active pharmaceutical ingredients (APIs) are crucial to increase the sustainability of the industry. Despite their lower production cost, microbial lipases are striking for their versatile catalyzing reactions beyond their physiological role. In the context of taking advantage of microbial lipases in reactions for the synthesis of API building blocks, this review focuses on: (i) the structural origins of the catalytic properties of microbial lipases, including the results of techniques such as single particle monitoring (SPT) and the description of its selectivity beyond the Kazlauskas rule as the “Mirror-Image Packing” or the “Key Region(s) rule influencing enantioselectivity” (KRIE); (ii) immobilization methods given the conferred operative advantages in industrial applications and their modulating capacity of lipase properties; and (iii) a comprehensive description of microbial lipases use as a conventional or promiscuous catalyst in key reactions in the organic synthesis (Knoevenagel condensation, Morita–Baylis–Hillman (MBH) reactions, Markovnikov additions, Baeyer–Villiger oxidation, racemization, among others). Finally, this review will also focus on a research perspective necessary to increase microbial lipases application development towards a greener industry.

Ionic additives affect the structure, activity and stability of lipases, which allow for solving common application challenges, such as preventing the formation of protein aggregates or strengthening enzyme–support binding, preventing their desorption in organic media. This work aimed to design a biocatalyst, based on lipase improved by the addition of ionic additives, applicable in the production of ethyl esters of fatty acids (EE). Industrial enzymes from Thermomyces lanuginosus (TLL), Rhizomucor miehei (RML), Candida antárctica B (CALB) and Lecitase^®, immobilized in commercial supports like Lewatit^®, Purolite^® and Q-Sepharose^®, were tested. The best combination was achieved by immobilizing lipase TLL onto Q-Sepharose^® as it surpassed, in terms of %EE (70.1%), the commercial biocatalyst Novozyme^® 435 (52.7%) and was similar to that of Lipozyme TL IM (71.3%). Hence, the impact of ionic additives like polymers and surfactants on both free and immobilized TLL on Q-Sepharose^® was assessed. It was observed that, when immobilized, in the presence of sodium dodecyl sulfate (SDS), the TLL derivative exhibited a significantly higher activity, with a 93-fold increase (1.02 IU), compared to the free enzyme under identical conditions (0.011 IU). In fatty acids ethyl esters synthesis, Q-SDS-TLL novel derivatives achieved results similar to commercial biocatalysts using up to ~82 times less enzyme (1 mg/g). This creates an opportunity to develop biocatalysts with reduced enzyme consumption, a factor often associated with higher production costs. Such advancements would ease their integration into the biodiesel industry, fostering a greener production approach compared to conventional methods.

Chemical modification is a valuable strategy for tuning enzyme functionality by introducing new reactive groups without disrupting the overall fold. Conventional amination using ethylenediamine (EDA) is effective, but the resulting modified proteins show limited reactivity for conjugation at neutral pH, and the modifier itself poses safety concerns due to its volatility and corrosive nature. Dihydrazides, in contrast, offer a safer and more versatile alternative: they operate through the same carboxyl-activation mechanism while enabling systematic investigation of chain-length effects. In this study, Thermomyces lanuginosus lipase (TLL) and Myceliophthora thermophila laccase (MTL) were modified using dihydrazides with different alkyl chain lengths (carbonyl (CZ), oxalyl (OX), succinyl (SC), and adipic (AA)), and compared to EDA-modified and unmodified enzymes to evaluate their effects on catalytic performance. Hydrazide-modified variants exhibited enhanced catalytic performance, reaching up to 2.5-fold (TLL-CZ) and 4.2-fold (MTL-AA and MTL-OX) higher efficiencies than unmodified and EDA-modified enzymes. Notably, AA provided the most consistent improvement across both enzymes (1.3-fold in TLL and the best in MTL). Molecular dynamics and docking analyses supported these findings, linking increased flexibility (higher RoG and RMSF) with higher k_cat, and changes in substrate binding with lower k_m. Overall, hydrazide-based modification broadens the spectrum of enzyme variants attainable through amination, while offering safer procedures, thus representing an alternative that overcomes the limitations of using EDA as a conventional aminating agent.