Naringinase (E.C. 3.2.1.40) is an enzyme complex that's recently garnered significant attention due to its enzymatic activity and wide industrial utility [1] This complex comprises two enzymatic components: α-L-rhamnosidase (E.C. 3.2.1.40) and β-D-glucosidase (E.C. 3.2.1.21) [2], [3]. Naringinase catalyzes the sequential hydrolysis of the α-L-rhamnosidic and β-D-glycosidic linkages in naringin (C₂₇H₃₂O₁₄). The enzyme first converts naringin to prunin (C₂₁H₂₂O₁₀) by removing the rhamnose moiety, then further hydrolyzes prunin to release the aglycone naringenin and glucose (Fig. 1). This enzymatic process is particularly valuable because naringin is a naturally occurring bitter flavonoid glycoside found predominantly in citrus fruits like grapefruits (Citrus paradisi) and oranges (Citrus sinensis Fig. 1). It is responsible for the characteristic bitterness of these fruits [4], [5], [6] It was first discovered in 1938 in grapefruit leaves and celery seeds [7]. Structurally, naringin is a flavanone backbone, and naringenin is linked to two sugar moieties - rhamnose and glucose [8]. The reaction involves the hydrolysis of naringin, a flavanone glycoside, into naringenin through intermediate steps, reducing bitterness and improving the sensory quality of citrus-based juices. Since the enzyme can interact with various glycosides, it has been proven essential for flavour enhancement and several other transformative biochemical mechanisms. This broad specificity enables naringinase to hydrolyse different glycosidic bonds in multiple substrates, thus helping convert complex glycosides into more readily utilisable forms [9].

Naringinase is a glycoprotein with a quaternary structure, mainly from fungal and bacterial origin, such as A. niger, Penicillium decumbens, and Bacillus amyloliquefaciens [10], [11], [12]. The enzyme consists of specific catalytic domains—α-L-rhamnosidase and β-D-glucosidase—that catalyse the release of rhamnose and glucose, respectively [13]. The enzyme has broad substrate specificity to catalyse reactions on various flavonoid glycosides, including naringin, rutin, and hesperidin, as the low Michaelis constant (Km) and high maximum velocity (Vmax) values indicate its effectiveness. Optimal activity occurs within a pH range of 4.0–6.0 and temperatures of 40°C–60°C, although thermostable variants have a broader tolerance. Several substrates are used to immobilise the enzyme, making it more stable and reusable, enabling scalable, continuous processing systems and expanding its potential for diverse industrial uses [14], [15], [16].

Naringinase is an enzyme that has applications in several industries. The global enzyme market size was approximately USD 10 billion in 2022. The global naringinase market size was around USD 5.2 million in 2023 and is estimated to reach USD 9.8 million by 2032 with a CAGR of 7.2 % [17]. The steady growth is primarily due to the rising demand for organic products to reduce the negative impact on the environment. In the food sector, it is used principally to reduce bitterness in citrus juices, such as grapefruit, by hydrolysing naringin into non-bitter compounds, thus enhancing flavour and consumer acceptance. Also, it plays a vital role in developing functional foods and increases the aroma and taste of wine and beer. In the pharmaceutical field, naringinase is involved in the biotransformation reactions of glycosidic drugs that enhance their bioavailability and therapeutic value, as well as in the synthesis of antibiotics and antiviral drugs [18], [19], [20]. Its biotechnological applications include the production of rhamnose, prunin, and diosgenin, use in debittering juices and enhancement of the aroma in wine [21], [22], [23]. Immobilized naringinase boosts the efficiency and scalability of industrial operations [24], [25].

The review encompasses structural and molecular characteristics, microbial and fungal sources, production of naringinase through solid-state and submerged fermentation, optimisation techniques, purification of the enzyme, enzymatic characteristics, advances in naringinase immobilization and applications of naringinase in various fields. This review further pushes the improvement in enzyme engineering and new microbial sources relevant to scalable and sustainable practices to impact future studies and promote industrial optimization [26].