Marine ecosystems host an exceptional variety of life forms, ranging from microorganisms to complex organisms, thriving in diverse habitats (Chandna et al., 2020; Griffin, 2020). This biodiversity is fundamental to sustaining ecosystem stability and functionality (Chandna et al., 2020). Among these organisms, algae are particularly significant as a rich source of bioactive compounds with substantial industrial importance (Tripathi et al., 2024). Of the numerous enzymes derived from these organisms, haloperoxidases have garnered increasing attention due to their unique ability to catalyze the halogenation of organic compounds (Höfler et al., 2019). This catalytic process, which involves the incorporation of halogens such as chlorine, bromine, or iodine into various substrates, plays a critical role in the synthesis of biologically active molecules and environmentally friendly chemical transformations (Franssen, 1994; Höfler et al., 2019). Consequently, haloperoxidases hold immense promise for applications in biocatalysis, pharmaceuticals, and environmental remediation due to their unique catalytic properties, such as regio- and stereoselectivity, as well as their ability to catalyze halogenation reactions (Ayala and Eduardo, 2015; Rebollar-Pérez et al., 2019). In biocatalysis, they can be used to synthesize complex organic compounds; in pharmaceuticals, they facilitate the production of halogenated drugs with enhanced efficacy and bioavailability; and in environmental remediation, they play a role in the degradation of persistent organic pollutants. Despite this potential, haloperoxidases from marine algae remain underutilized, partly due to challenges associated with their extraction and purification (Wever et al., 2018). Conventional purification methods often fall short in achieving the required yield, purity, and stability for industrial applications, highlighting the need for innovative approaches. Furthermore, large-scale utilization of these enzymes has been limited by difficulties in maintaining bioactivity and scalability. Therefore, addressing these knowledge and technical gaps is essential to fully exploit the potential of haloperoxidases and enable their efficient integration into industrial applications. The objective of this review is to provide an in-depth exploration of algal haloperoxidases, focusing on their diversity, purification methodologies, and the associated challenges. Special attention is given to underrepresented groups, such as green algae, and to innovative techniques that could overcome existing limitations. By identifying key knowledge gaps and proposing strategies for optimizing purification, this review aims to facilitate the broader application of haloperoxidases in industrial and biotechnological fields.

Haloperoxidases are enzymes that catalyze the incorporation of halogen atoms (such as chlorine, bromine, or iodine) into organic compounds using hydrogen peroxide or other peroxides as oxidants (Franssen, 1994; Gérard et al., 2023). This halogenation process is highly selective, facilitating the synthesis of halogenated compounds with significant biological and industrial value (Sharma et al., 2024). Haloperoxidases are typically classified based on the halogen they incorporate and their active site composition, with the main types being chloroperoxidases (CPOs), bromoperoxidases (BPOs), and iodoperoxidases (IPOs), with only BPOs and IPOs are found in marine algae (Butler and Baldwin, 1997; Pacios and Galvez, 2010; Fournier et al., 2014). In marine algae, these enzymes play a critical role in the biosynthesis of halogenated natural products that serve ecological functions such as chemical defense and signaling (Butler and Carter-Franklin, 2004; Baumgartner and Shaun, 2021).

Vanadium-dependent haloperoxidases, including bromoperoxidases (V-BPOs) and iodoperoxidases (V-IPOs), catalyze the oxidation of bromide (Br⁻) or iodide (I⁻) using hydrogen peroxide (H₂O₂), producing halogenated organic compounds and water (Figure 1). These enzymes activate hydrogen peroxide to form a highly reactive intermediate, facilitating regio- and stereoselective halogenation of organic substrates (Aboelnga, 2022; Chen et al., 2022; Zhang and Liu, 2023). V-BPOs, primarily found in red and brown algae, exhibit broad pH stability (4–10) and high thermal resistance, making them valuable for industrial biocatalysis. In contrast, V-IPOs, mainly from brown algae, function within a narrower pH range (5–7) and play key roles in marine iodine metabolism (Tarakhovskaya et al., 2015; Punitha et al., 2018; Ishikawa et al., 2022). In marine ecosystems, halogenation mediated by these enzymes contributes to the production of bioactive compounds involved in chemical defense, microbial inhibition, and ecological interactions. Both V-BPOs and V-IPOs have significant applications in green chemistry, particularly in selective halogenation processes (La Barre et al., 2010; Cabrita et al., 2010).

Marine algae, especially brown, red, and green algae, are notable sources of haloperoxidases. Brown algae, such as Laminaria spp., Macrocystis pyrifera, and Ascophyllum nodosum, are rich in vanadium-dependent haloperoxidases (V-HPOs), which are involved in bromination and iodination processes (Küpper and Kroneck, 2014; Tarakhovskaya et al., 2015; Punitha et al., 2018). These enzymes are responsible for the production of brominated and iodinated compounds for defense, tissue repair, and adhesion (La Barre et al., 2010). Red algae, such as Laurencia spp. and Gracilaria spp., produce haloperoxidases that generate halogenated phenols with antimicrobial and antifouling properties, contributing to their ecological success (Weinberger et al., 2007; Thapa et al., 2020; Ishikawa et al., 2022). Green algae, such as Ulva spp. and Codium spp., also harbor haloperoxidases (Rehder, 2014), although they are less extensively studied, and these enzymes catalyze the halogenation of fatty acids and terpenoids as part of the algae’s chemical defense strategies (Carter-Franklin et al., 2003; Weinberger et al., 2007; Thapa et al., 2020).

Haloperoxidases have garnered significant attention for their versatility in various industrial applications due to their unique ability to catalyze selective halogenation reactions (Höfler et al., 2019; Sharma et al., 2024). These enzymes are proving invaluable in biocatalysis, pharmaceuticals, environmental remediation, and emerging innovative applications (Littlechild, 1999; García-Zamora et al., 2018; Michail and Isupov, 2014; Höfler et al., 2019; Rebollar-Pérez et al., 2019).

In biocatalysis, haloperoxidases catalyze the selective halogenation of organic compounds using H₂O₂ as an oxidant, a process visually depicted in their catalytic mechanisms. These enzymes are broadly classified into heme-dependent and vanadium-dependent haloperoxidases. In heme-dependent haloperoxidases, the catalytic cycle begins with the activation of the heme iron to form a highly reactive intermediate known as Compound I, which facilitates the transfer of halide ions (X⁻) to the substrate, leading to halogenation (Figure 2). In vanadium-dependent haloperoxidases, the resting state involves a vanadate cofactor bound to the active site, which is oxidized by H₂O₂ to produce a peroxo vanadate species that mediates halogenation (Figure 3). These mechanisms are highly regio- and stereoselective, enabling precise chemical transformations that are challenging to achieve using conventional chemical halogenation. Additionally, the environmentally benign nature of this process, with water as the only byproduct, underscores its sustainability. Recent research by Sharma et al. (2024) highlighted the efficacy of V-HPOs as biocatalysts for selective halogenation, specifically facilitating the synthesis of 1,2,4-oxadiazole from substituted benzamidine hydrochlorides. This compound is a cornerstone in drug discovery, forming part of numerous experimental, investigational, and approved drugs (Boström et al., 2012; Dhameliya et al., 2022). Oxadiazoles, acting as bioisosteres for esters, hydroxamic acids, carboxamides, and carbamates, enhance the metabolic stability of drugs such as ataluren, naldemedine, amenamevir, ozanimod, azilsartan medoxomil, and opicapone, thereby improving their therapeutic efficacy and prolonging their action (Salahuddin et al., 2017; Baykov et al., 2023). Furthermore, studies have shown that V-HPOs exhibit broad substrate specificity and are capable of catalyzing complex reactions, including epoxidation, hydroxylation, and halogenation, which are often challenging to achieve with conventional chemical methods (Bhandari et al., 2023; Gérard et al., 2023). Their diversity and ability to construct greener synthesis routes match green chemistry concepts, decreasing waste and the requirement for hazardous chemicals. V-HPOs, have significant applications in green chemistry due to their ability to catalyze halogenation and oxidation reactions with high selectivity and stability. These enzymes facilitate the biosynthesis of halogenated organic compounds, widely used in pharmaceuticals and as synthetic intermediates in chemical industries. Their enzymatic approach offers an environmentally friendly alternative to using toxic and corrosive reagents like molecular bromine (Wischang and Hartung, 2011). For instance, V-HPOs from A. nodosum have been successfully applied to synthesize brominated pyrroles (Wischang et al., 2011a) and terpenes (Carter-Franklin et al., 2003), as well as optically pure sulfoxides, which are critical intermediates in asymmetric synthesis and pharmaceutical formulations (Ten Brink et al., 2001). Moreover, the immobilization of V-HPOs on supports such as magnetic beads has enabled efficient recycling of these biocatalysts (Wischang et al., 2011b). These applications demonstrate the potential of V-HPOs to drive more sustainable and selective chemical processes.

Despite extensive studies on V-HPOs, their application remains poorly exploited, particularly from marine algae. Notably, most research has focused exclusively on bromoperoxidases, neglecting the abundant iodoperoxidases in brown algae such as Laminaria digitata, A. nodosum, and Pelvetia canaliculate (Almeida et al., 2000; Colin et al., 2005; Verhaeghe et al., 2008). These iodoperoxidases hold significant potential for novel applications, yet their properties and industrial relevance remain largely unexplored. Expanding the study of V-HPOs to include these enzymes could open new avenues for biotechnological advancements and further harness the untapped potential of marine algae-derived biocatalysts.

Haloperoxidases were purified from algae using various purification techniques. Supplementary Table S1 provides an overview of haloperoxidases from various algal sources, focusing on their purification techniques, prosthetic groups, and optimal activity conditions. Algae are categorized into red, brown, and green types, with red and brown algae being the most extensively studied. Each algal group presents unique characteristics in terms of haloperoxidase stability and adaptability, reflecting their potential for diverse applications. In addition, the purification of haloperoxidases varies across different algal types, with distinct techniques influencing yield and purification fold (Table 1).

Red algae exhibit the highest yield (37.9%) but a moderate purification fold (68.87), primarily using ammonium sulfate precipitation, ion-exchange chromatography, and size-exclusion chromatography (Itoh et al., 1985; Sheffield et al., 1992; Kongkiattikajorn and Ruenwongsa, 2006). These algae are prominently represented by species including Corallina spp and Gracilaria spp (Itoh et al., 1985; Sheffield et al., 1992; Rush et al., 1995; Coupe et al., 2007). Vanadium-dependent bromoperoxidases (V-BPOs) exhibit impressive stability, functioning effectively across a wide pH range of 4–10. They are also highly temperature-resistant, with Gracilaria fisheri enzymes remaining stable up to 50°C (Kongkiattikajorn and Pongdam, 2006) and Corallina officinalis enzymes enduring temperatures as high as 80°C (Rush et al., 1995). This makes red algae a valuable source for enzymes suitable for industrial processes requiring robustness under varied conditions (Littlechild and Isupov, 2014).

Brown algae dominate the literature due to their rich diversity, including families like Laminariaceae, Fucaceae, and Phyllariaceae (Wever et al., 2018). Despite having the lowest yield (3.8%), brown algae achieve the highest purification fold (508), primarily using aqueous two-phase systems, hydrophobic interaction chromatography, and chromatofocusing chromatogramphy. These algae primarily produce vanadium-dependent iodoperoxidases (V-IPOs) and bromoperoxidases (V-BPOs), characterized by their moderate pH and temperature stability (pH 5–7, 20°C–60°C) (De Boer et al., 1986; Wever et al., 1985; Almeida et al., 1998; Hara and Sakurai, 1998; Almeida et al., 2000; Almeida et al., 2001; Colin et al., 2003; Verhaeghe et al., 2008; Tarakhovskaya et al., 2015). Brown algae are abundant in alginates, sulfated fucans, and polyphenolic compounds, which pose significant challenges during the extraction and purification of haloperoxidases (Wever et al., 2018). To address these complications, several studies have employed aqueous salt/polymer two-phase systems as an effective purification method (Almeida et al., 1998; Hara and Sakurai, 1998; Almeida et al., 2001; Colin et al., 2003; Verhaeghe et al., 2008; Tarakhovskaya et al., 2015). Additionally, hydrophobic interaction chromatography is frequently utilized, reflecting the complexity of haloperoxidases derived from brown algae (Verhaeghe et al., 2008; Almeida et al., 2001). While less commonly used, chromatofocusing chromatography has proven effective in a study involving Saccorhiza polyschides (Almeida et al., 1998).

Green algae remain underrepresented, with only Cladophora glomerata. This species contains a heme-based haloperoxidase, which is distinct from the vanadium prosthetic groups seen in red and brown algae. Green algae demonstrate an intermediate yield (20%) and purification fold (195), using ammonium sulfate precipitation, ion-exchange chromatography, and size-exclusion chromatography. The enzyme’s activity is optimal at an unusually low pH of 3.1, making it unique among algal haloperoxidases. This enzyme has been successfully purified from C. glomerata using ammonium sulfate precipitation, ion-exchange chromatography and size-exclusion chromatography. The limited exploration of green algae suggests a potential area for further investigation to uncover novel enzymes with unique properties (Verdel et al., 2000).

Haloperoxidases from marine algae represent a valuable yet underexplored class of enzymes with substantial potential in industrial biocatalysis, pharmaceutical applications, and environmental remediation. Despite the advancements in the purification of these enzymes, challenges remain, particularly in optimizing yield, purity, and bioactivity while scaling up the process for industrial use. The review highlights the underrepresentation of green algal species in haloperoxidase research, signaling a significant gap in our understanding of their enzymatic properties and applications. Additionally, while traditional chromatographic techniques remain effective, the introduction of novel methods, such as affinity chromatography, ATPS, and microfluidic technologies, offers promising avenues to improve purification efficiency and enzyme stability. Sustainable extraction methods that align with green chemistry principles are gaining attention, reflecting the increasing demand for environmentally friendly practices in enzyme production. Moving forward, developing robust, scalable purification techniques customized to the specific biochemical properties of algal species will be essential for maximizing the industrial potential of haloperoxidases. The growing body of research on these enzymes underscores their promise, but more comprehensive studies are needed to fully unlock their diverse applications. Future work should focus on expanding the range of algal species investigated, optimizing purification systems, and addressing scalability issues to ensure the successful commercialization of marine algal haloperoxidases.