Red algae are a renewable bioresource, with a high annual output. Sulfated galactans are the main component of cell walls in some red algae. These compounds can be divided into agar and carrageenan according to their different monomer compositions. Carrageenans are linear sulfated polysaccharides that consist of D-β-galactose (D-Gal, G-unit) and 3,6-anhydro-α-D-galactose (D-AHG, DA-unit). The formation of ether bonds between the C-3 and C-6 hydroxyl groups in D-AHG makes it difficult for most microorganisms to utilize, and it has been found to have anti-inflammatory properties that could be useful in the pharmaceutical industry (Yun et al., 2012). Based on the degree and position of sulfate groups on the monosaccharide units, carrageenans are usually divided into κ-carrageenan (KC), ι-carrageenan (IC), and λ-carrageenan (LC). On its disaccharide units, KC contains only a C-4 hydroxyl sulfated D-Gal residue (G4S), IC contains both G4S and C-2 hydroxyl sulfated D-AHG residues (DA2S), and LC contains C-2 hydroxyl sulfated D-Gal residues (G2S) and C-2 and C-6 hydroxyl sulfated D-galactopyranosyl residues (D2,6S) (Fig. 1) (Usov, 2011). In addition, the hybrid carrageenan, mainly composed of β/κ-carrageenan (B/KC), extracted from Furcellaria lumbricalis has attracted widespread attention due to its excellent film-forming ability; this carrageenan consists of two kinds of disaccharide units, G-DA and G4S-DA (Correc et al., 2012; Marangoni Junior et al., 2021). Another hybrid carrageenan (κ/ι-carrageenan, K/IC) is the main biopolymer structure extracted from Mastocarpus stellatus, and has potential for edible active film application (Hilliou et al., 2006). Although carrageenan has already been explored as a gelling or thickening agent for application in the food industry, its further high-value application is limited due to its low bioavailability and lack of obvious bioactivity (Guo et al., 2022). By comparison, carrageenan oligosaccharides exhibit good bioavailability, and studies have demonstrated that these compounds exhibit various physiological activities, including antioxidant, antitumour, anti-inflammatory, antiviral, and antibacterial activities (Zia et al., 2017). Therefore, the transformation of carrageenan into carrageenan oligosaccharides should offer an ideal route for achieving high-value applications. Based on the nonreducing ends, carrageenan oligosaccharides can be divided into neocarrageenan-oligosaccharides (NCOSs) and carrageenan-oligosaccharides (COSs), the nonreducing ends of which are D-AHG and D-Gal, respectively. Corresponding to different types of carrageenans, NCOSs and COSs can be further divided into κ-, ι-, λ-, and hybrid types (Fig. 1).

Among the methods for preparing carrageenan oligosaccharides, the use of carrageenolytic enzymes is the preferred green method for preparing carrageenan oligosaccharides with specific structures (Guo et al., 2022; Li et al., 2021). The most reported carrageenolytic enzymes are carrageenases, they can directly hydrolyse carrageenan to produce even-numbered NCOSs (ENCOSs) usually achieved through the endolytic action. According to differences in their substrate specificity, they can generally be classified into κ-, ι-, λ-, and β-carrageenases. The most reported of these enzymes is κ-carrageenase, which belongs to glycoside hydrolase family 16 subfamily 17 (GH16_17) (Drula et al., 2022; Viborg et al., 2019). Until now, the hydrolysis products of the characterized κ-carrageenases mostly contained both κ-neocarrabiose (Nκ2) and κ-neocarratetrose (Nκ4). The currently characterized ι-carrageenases that belong to GH82 tend to produce ι-neocarratetrose (Nι4) as the main hydrolysate. The least reported was λ-carrageenase, which belongs to the GH150 family, in which only five members have been identified and characterized. The products were distributed with degrees of polymerization (DP) ranging from 2 to 6 (Guibet et al., 2007; Li et al., 2014; Ohta and Hatada, 2006). The last known type of carrageenase is β-carrageenase, which belongs to GH16_13. Schultz-Johansen et al. (2018) discovered β-carrageenases Ph1656 and Ph1663 in the carrageenan-degrading bacterium Paraglaciecola hydrolytica S66T. Ph1656 and Ph1663 showed no significant hydrolytic activity on KC, but were able to hydrolyze B/KC to produce Nβ/κCOSs.

In addition to the abovementioned carrageenases, other types of carrageenolytic enzymes, including sulfatases, exo-α-3,6-anhydro-D-galactosidases (D-ADAGase), and exo-β-galactosidases (BGase), have been discovered via the analysis of metabolic pathways in marine microorganisms. There was one novel sulfatase type described in the carrageenan polysaccharide utilization loci (PUL) from Zobellia galactanivorans (Ficko-Blean et al., 2017), and there were two others that were previously described (Prechoux et al., 2013; Prechoux et al., 2016). These compounds act on the different types of NCOSs to remove their sulfate groups and produce hybrid NβCOSs. Then, D-ADAGase subsequently hydrolyses the first α-1,3-glycosidic linkage at the β-neocarrabiose (Nβ2) nonreducing end of ENCOSs to release D-AHG and generate corresponding odd-numbered COSs (OCOSs) (Ficko-Blean et al., 2017; Jiang et al., 2022a). These novel carrageenolytic enzymes from the carrageenan metabolic pathway show significant potential for obtaining carrageenan oligosaccharides with different structures, expanding the preparation scope of enzymatic methods.

A systematic review by Zhu et al. (2018b) provided a detailed introduction to the progress of the sources, categories, and enzyme properties of κ-, ι-, and λ-carrageenases. In the past five years, a certain number of carrageenases have been cloned and characterized. In particular, novel types of carrageenolytic enzymes involved in carrageenan metabolism have been identified and show potential for the preparation of carrageenan oligosaccharides with different structures. These novel carrageenolytic enzymes have been reviewed in several related studies (Bäumgen et al., 2021; Hettle et al., 2022; Michel et al., 2006; Rhein-Knudsen and Meyer, 2021), which provide important references for the use of these enzymes. However, a systematic and detailed review article that specifically focuses on this subject and includes an update on the previously characterized κ-, ι-, and λ-carrageenases, as well as information about new types of carrageenolytic enzymes, is still lacking. Therefore, in the present review, we introduce carrageenolytic enzymes in detail and summarize the characteristics of recently reported carrageenolytic enzymes, which are sorted by family and function to provide an overall understanding of carrageenolytic enzymes. Moreover, the different carrageenan metabolic pathways associated with these carrageenolytic enzymes are also described. Furthermore, the potential applications of these enzymes are highlighted, as well as the challenges and future perspectives for the mining and application of carrageenolytic enzymes.