Recent Advances in Alginate Lyase Engineering for Efficient Conversion of Alginate to Value‐Added Products

ABSTRACT

Alginate lyases depolymerize alginate and generate alginate oligosaccharides (AOS) and eventually 4‐deoxy‐L‐erythro‐5‐hexoseulose uronate (DEH), a monosaccharide. Recently, alginate lyases have garnered significant attention due to the increasing demand for AOS, which exhibit bioactivities beneficial to human health, livestock productivity, and agricultural efficiency. Additionally, these enzymes play a crucial role in producing DEH, essential in alginate catabolism in bacteria. This review explains the industrial value of AOS and DEH, which contribute broadly to industries ranging from the food industry to biorefinery processes. This review also highlights recent advances in alginate lyase applications and engineering, including domain truncation, chimeric enzyme design, rational mutagenesis, and directed evolution. These approaches have enhanced enzyme performance for efficient AOS and DEH production. We also discuss current challenges and future directions toward industrial‐scale bioconversion of alginate‐rich biomass.

This review covers alginate lyases that depolymerise alginate into AOS (alginate oligosaccharides) and DEH (4‐deoxy‐L‐erythro‐5‐hexoseulose uronate). It discusses the industrial applications of AOS and DEH in food, agriculture, and biorefinery processes. Additionally, it explores recent advancements in enzyme engineering, including chimeric enzyme construction, truncation, computer‐aided design, and directed evolution, to enhance alginate lyase efficiency.

1. Introduction

Alginate is a co‐polymer of D‐mannuronic acid (M)/L‐guluronic acid (G) and is a major structural polysaccharide in brown macroalgae (Gundewadi et al. 2018; Arroyo et al. 2020; Sahoo and Biswal 2021). Its degraded products, such as alginate oligosaccharides (AOS) and 4‐deoxy‐L‐erythro‐5‐hexoseulose uronate (DEH), also demonstrate potential in the medical, pharmaceutical, human nutrition, agricultural, and biorefinery industries (Liu et al. 2019; Kawai and Hashimoto 2022). Specifically, AOS refer to oligosaccharides with a degree of polymerisation (DP) ranging from 2 to 25 and exhibit various clinical activities, including anti‐tumour, anti‐obesity, anti‐inflammatory, and antimicrobial effects, making them promising ingredients for functional supplements in the food industry (Liu et al. 2019). Beyond these clinical applications, AOS can enhance productivity in agriculture and the livestock industry, positioning them as eco‐friendly agents for sustainable food security (Yamasaki et al. 2016; Zhang, Wang, et al. 2020). DEH, as a metabolizable sugar, can serve as a sustainable nutrient source for microbial production of food additives (Hobbs et al. 2016). This versatility highlights its potential utility in food chemistry, particularly in the production of functional ingredients. Moreover, it can be converted into value‐added chemicals, including precursors for polymer synthesis and bioethanol, through microbial bioconversion (Kawai et al. 2014; Lim et al. 2019; Lee et al. 2024; Moon et al. 2024).

Alginate lyases can break down alginate in a mild and specific manner, making it a promising tool for producing AOS and DEH (Garron and Cygler 2010; Costa et al. 2021). The enzymes can be classified by the carbohydrate‐active enzyme database (CAZy database), the polysaccharide lyase (PL) families containing alginate lyases and the differences in activities across families (Garron and Cygler 2010). Their biological activities are known to vary depending on the DP and M/G ratios (Liu et al. 2019; Vasudevan, Mrudulakumari, et al. 2021). Chemical or physical degradation of alginate is harsh and uncontrolled, also leading to the production of saturated oligosaccharides (Wang et al. 2016; Ravanal et al. 2017; Nguyen et al. 2020). However, alginate lyases specifically cleave the glycosidic bonds of alginate via β‐elimination and generate unsaturated oligosaccharides, which are known to have superior bioactivities compared to saturated oligosaccharides (Wong et al. 2000). Endo‐type alginate lyases exhibit random cleavage sites, producing unsaturated AOS with double bonds between C4 and C5. In contrast, exo‐type alginate lyases cleave alginate from the terminal residues, producing DEH directly, and occasionally also produce dimers (Suzuki et al. 2006; Takeda et al. 2011; Park et al. 2012; Gimpel et al. 2018; Narsico et al. 2020; Jiang et al. 2023). For bacterial metabolism, DEH is quite unstable and will spontaneously hydrolyse and ring‐open, followed by a new 5‐carbon ring formation (DHF) (Arntzen et al. 2021).

Hence, recent efforts have focused on exploiting alginate lyases to effectively obtain AOS and DEH. Heterologous expression and culture condition optimization were performed for scaled production of alginate lyases (Chen et al. 2018; Guo et al. 2023). Utilisation of crude enzymes from alginate‐metabolising microorganisms was cost‐effective by omitting purification steps (Zhang et al. 2024). Additionally, immobilising alginate lyases could enhance their reusability and stability, further contributing to cost‐effective bioprocessing (Chen et al. 2018; Guo et al. 2023). However, thermal instability and low catalytic activity are significant limitations to the industrial production and application of AOS and DEH (Cui et al. 2024). With the development of protein engineering technologies, it is possible to enhance their properties even more efficiently and effectively (Li, wang, et al. 2019).

This review offers a comprehensive overview of recent advancements in alginate lyases, emphasising the industrial value of AOS and DEH, the applications of alginate lyases, and further engineering improvements for enhanced AOS and DEH production. By summarising current knowledge and recent developments in the field, this review can provide a valuable resource for researchers and practitioners aiming to harness the full potential of alginate lyases (Figure 1).

FIGURE 1.

Enzymatic degradation of alginate by alginate lyases: characterisation, industrial applications, and advanced engineering strategies. (A) The enzymatic degradation of cell walls using alginate lyase and the industrial applications of AOS and DEH are discussed. Endolytic and exolytic alginate lyases cleave alginate polysaccharide chains, releasing AOS and DEH, which have significant industrial applications. (B) The discovery and characterisation of alginate lyases are detailed in grey boxes. Conventional and novel alginate lyases are depicted in yellow and red, respectively. For efficient AOS and DEH production, novel alginate lyases with resistance to metal ions, dual activity, broad substrate specificity, and high thermal stability have been identified and studied. (C) Advancements in the production and application of alginate lyases are presented in green boxes. To enhance alginate lyase yields, heterologous expression systems have been developed. The use of crude enzymes from alginate‐degrading microorganisms allows the omission of purification steps. Immobilisation on solid matrices enhances the activity and durability of alginate lyases. (D) Enzyme engineering strategies for alginate lyase are described in orange boxes. Creating chimeric and truncated enzymes through fusion and truncation of alginate lyase modules, respectively, can improve enzyme performance. Computer‐aided rational design, including point mutations and mechanism analysis, can also produce mutant alginate lyases with enhanced properties. Directed evolution using error‐prone PCR induces simultaneous mutations in the coding sequence of alginate lyase. AOS, Alginate oligosaccharide; CBM, Carbohydrate‐binding module; CD, Catalytic domain; DEH, 4‐deoxy‐L‐erythro‐5‐hexoseulose uronate; O.E, Overexpression; PolyG, Poly‐guluronate; PolyM, Poly‐mannuronate; SP, Signal peptide; WT AL, Wild‐type alginate lyase.

2. Applications of Alginate‐Degraded Products

Numerous studies have demonstrated that AOS obtained by alginate lyase‐mediated degradation exhibit beneficial effects (Table 1), particularly on human health (Zhang et al. 2023). Firstly, AOS are recognised for their antitumour activities; treating osteosarcoma patients with AOS of various DP (DP 2–5) revealed that only AOS with DP 5 exhibited anticancer effects against osteosarcoma cells (Chen et al. 2017). Unsaturated AOS from alginate lyase showed higher antitumour activity than acid‐hydrolyzed AOS, inducing greater TNF‐α secretion in RAW264.7 cells (mouse macrophage cell line), highlighting the importance of the unsaturated terminal structure (Iwamoto et al. 2005). Using the alginate lyase Aly30 from the marine psychrophilic bacterium Cobetia marina HQZ08, alginate was primarily degraded into trisaccharides. Low‐molecular‐weight AOS exhibited superior antitumour activity compared to high‐molecular‐weight AOS (Qiu et al. 2023). Moreover, AOS can act as immunostimulatory agents. AOS with DP 3–9 derived from sodium alginate acted as immunostimulatory agents on RAW264.7 cells, promoting the production of nitric oxide and reactive oxygen species (ROS). Both are cytotoxic molecules secreted by macrophages to kill pathogens and tumours. This promoting effect was most pronounced when AOS were produced using alginate lyase (Xu et al. 2014, 2015).

TABLE 1.

Applications of AOS.

Alginate source	Degradation method	Size	Effect of alginate oligosaccharides	Reference
Commercial sodium alginate (from Qingdao Qingya Chemical Co.)	Enzymatic degradation (alginate lyase from Agarivorans sp. L11)	DP 2–5	Antitumor effect	Chen et al. (2017)
Commercial sodium alginate (from Nacalai Tesque Inc.)	Acid hydrolysis/Enzymatic degradation (alginate lyase from Pseudoalteromonas sp.)	DP 3–9	Antitumor effect	Iwamoto et al. (2005)
Saccharina japonica	Enzymatic degradation (alginate lyase from Cobetia marina HQZ08)	Mainly DP 3	Antitumor effect	Qiu et al. (2023)
Commercial sodium alginate (from Sigma‐Aldrich)	Enzymatic degradation (Pseudoalteromonas sp.)	DP 3–9	Immunostimulatory effect	Xu et al. (2015)
Commercial sodium alginate (from Nuotai)	Acid hydrolysis/Enzymatic degradation (Pseudoalteromonas sp.)	Average DP 5	Immunostimulatory effect	Xu et al. (2014)
Commercial AOS (from BZ Oligo Biotech Co. Ltd.)	—	—	Antimicrobial effect	Asadpoor et al. (2021)
Commercial AOS (from DuPont Nutrition and Health)	Acid hydrolysis	Average DP 20.135	Antimicrobial effect	Bouillon et al. (2019)
Commercial AOS (from AlgiPharma AS)	—	—	Antimicrobial effect	Hengzhuang et al. (2016)
Commercial AOS (from AlgiPharma AS)	—	—	Antimicrobial effect	Pritchard, Powell, et al. (2017)
Commercial AOS (from AlgiPharma AS)	—	—	Antimicrobial effect	Powell et al. (2023)
Commercial sodium alginate (from Sigma‐Aldrich)	Enzymatic degradation (alginate lyase from Sigma‐Aldrich)	—	Antimicrobial effect	Park et al. (2016)
Commercial AOS (from AlgiPharma AS)	—	—	Antimicrobial effect	Pritchard, Jack, et al. (2017)
Commercial AOS (from BZ Oligo Biotech Co. Ltd.)	—	—	Antioxidant effect	Pan et al. (2021)
Commercial sodium alginate (from DuPont)	Enzymatic degradation (alginate lyase from Nagase Enzymes)	DP 2–4	Antioxidant effect	Falkeborg et al. (2014)
Commercial sodium alginate	Enzymatic degradation (alginate lyase from Microbulbifer sp. ALW1)	DP 2–3	Antioxidant effect	Zhu et al. (2016)
Commercial AOS (from BZ Oligo Biotech Co. Ltd.)	—	—	Antioxidant effect	Feng et al. (2021)
Oligomannuronate prepared by school of Medicine and Pharmacy, Ocean University of China	—	—	Antidiabetic effect	Hao et al. (2015)
Commercial AOS (from Nanjing Junlan Biotechnology Company)	—	DP 1–4	Antidiabetic effect	Wang et al. (2020)
Saccharina japonica	Acid hydrolysis/Enzymatic degradation (alginate lyase from Vibrio sp. SY01)	DP 2–3	Anti‐obesity effect	Li, He, and Wang (2019)
Saccharina japonica	Enzymatic degradation (alginate lyase from Vibrio sp. SY01)	DP 2–3	Anti‐obesity effect	Li, Wang, et al. (2020)
Commercial sodium alginate (from KIMICA Co.)	Enzymatic degradation (alginate lyase from Pseudoalteromonas sp.)	DP 1–3	Productivity increase in the livestock and fishery industry	Yamasaki et al. (2016)
AOS prepared by Chinese Academy of Agricultural Sciences	Enzymatic degradation	—	Productivity increase in the livestock and fishery industry	Yan et al. (2011)
Commercial sodium alginate (from Kimika Co.)	Enzymatic degradation (alginate lyase from Pseudoalteromonas sp. Strain no. 272)	DP 2–4	Productivity increase in the livestock and fishery industry	Hu, Zhang, et al. (2021)
Commercial sodium alginate (from Cargill)	Enzymatic degradation	—	Prebiotic effect	Gupta et al. (2019)
Commercial AOS (from Sinopharm Chemical Reagent Co. Ltd)	—	—	Extension of food shelf life	Ma et al. (2015)
Commercial AOS (from Qingdao Oligo Biotech Co. Ltd)	—	Mw ≈ 2800 Da	Extension of food shelf life	Han et al. (2023)
Commercial sodium alginate	Enzymatic degradation (alginate lyase from Flavobacterium sp. S20)	DP 2–8	Extension of food shelf life	Liu et al. (2020)
Commercial sodium alginate (from Sigma‐Aldrich)	Enzymatic degradation (alginate lyase from Vibrio sp. C42)	DP 3	Productivity increase in agriculture	Wang et al. (2024)
AOS prepared by the Dalian Institute of Chemical Physics	—	—	Productivity increase in agriculture	Du et al. (2023)
Commercial sodium alginate	Enzymatic degradation (alginate lyase from Flavobacterium sp. LXA)	Average DP 6.8	Productivity increase in agriculture	An et al. (2009)
Bifurcaria bifurcata	Radical hydrolysis (H2O2)	Mw ≈ 3700 Da	Productivity increase in agriculture	Aitouguinane et al. (2020)

Abbreviations: DP, degree of polymerisation; Mw, molecular weight.

Additionally, AOS possess antimicrobial properties. AOS have shown capability of inhibiting the growth of pathogenic bacteria such as Pseudomonas aeruginosa , Streptococcus agalactiae , and Candida species by suppressing biofilm formation (Hengzhuang et al. 2016; Pritchard, Powell, et al. 2017; Bouillon et al. 2019; Asadpoor et al. 2021; Powell et al. 2023). Park et al. (2016) improved oral drug delivery against Escherichia coli by incorporating alginate and its oligosaccharides into artificial gastric juice, achieving antimicrobial activity comparable to pure lysozyme while maintaining enzyme stability and immobilisation efficiency in gastric juice (Park et al. 2016). Moreover, AOS have been shown to effectively reduce the expression of virulence factors in fungal species such as Candida albicans (Pritchard, Jack et al. 2017).

AOS have demonstrated beneficial effects on human health in various aspects, particularly highlighting that unsaturated AOS produced by alginate lyases show higher bioactivities. AOS have strong radical scavenging effects, making them excellent antioxidants. They may help prevent oxidative stress‐related degenerative diseases, such as neurodegenerative and cardiovascular disorders (Li et al. 2023). AOS reduced ROS levels and oxidative stress, protecting mitochondrial function and preventing D‐galactose‐induced cardiac ageing (Feng et al. 2021). Their protective role extended to kidney ageing, where AOS upregulated key antioxidant enzymes such as heme oxygenase‐1 (HO‐1) and NADPH quinone oxidoreductase 1 (NQO1) (Pan et al. 2021). Moreover, AOS produced using various alginate lyases have shown strong radical scavenging activity against 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH), 2,2′‐azino‐bis‐3‐ethylbenzthiazoline‐6‐sulphonic acid (ABTS), and hydroxyl radicals (Falkeborg et al. 2014; Zhu et al. 2016). AOS have been shown to increase insulin sensitivity in skeletal muscle cell lines, and their addition has been shown to increase the concentration of beneficial microbiota that can enhance glucose tolerance and insulin secretion, thereby proving the antidiabetic effects (Hao et al. 2015; Wang et al. 2020). Unsaturated AOS generated through enzymatic degradation have been reported to help prevent obesity by modulating gut microbiota and reducing total cholesterol (Li, He, et al. 2019; Li, Wang, et al. 2020).

The bioactivities of AOS have been actively utilised not only for enhancing human health but also for increasing productivity in the livestock and fishery industry. AOS have been reported to enhance the immune response of broiler chickens and promote the growth of Ruditapes philippinarum (Manila clam) (Yan et al. 2011; Yamasaki et al. 2016). Additionally, their prebiotic effects suggest ample potential for application in the feed industry (Gupta et al. 2019; Zhang et al. 2023). A recent study by Hu, Zhang, et al. (2021) found that supplementing grass carp ( Ctenopharyngodon idella ) feed with AOS improved both growth and non‐specific immunity, contributing to increased fishery production. The AOS‐fed carp exhibited higher levels of lysozyme, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, total protein, suggesting enhanced immunity (Hu, Zhang, et al. 2021).

AOS can be utilised to extend the shelf life of food products. Few studies have reported that AOS can slow down the quality deterioration of frozen meat. When added to frozen Litopenaeus vannamei shrimp, AOS prevented texture and colour deterioration and inhibited the denaturation of functional proteins (Ma et al. 2015; Zhang, Yao, et al. 2020). Ultrasound‐assisted soaking with AOS improved water retention, reduced ice crystal size, and preserved protein structure in crayfish during freeze–thaw cycles, enhancing physicochemical properties (Han et al. 2023). AOS can also extend the shelf life of fruits. AOS treatment on harvested kiwifruit inhibited moulds and rot, suppressed pectin degradation, and boosted antioxidant enzyme activity and phenol levels. These findings suggest that AOS can extend postharvest fruit quality by preventing decay and promoting antioxidants (Liu et al. 2020).

AOS have exhibited significant potential in agricultural applications, including enhancing plant growth, alleviating growth inhibition caused by environmental stress, and activating plant defence responses (El‐Mohdy 2017; Bose et al. 2019; Zhang, Wang, et al. 2020). In particular, Wang et al. (2024) demonstrated the agricultural applicability of AOS (DP 3) produced by AlyC7, a PL7 family alginate lyase from Vibrio sp. C42. The prepared AOS increased root lengths in various plants from 9.7 up to 27.5%. This approach provides a solid foundation for the industrial and agricultural utilisation of AlyC7 (Wang et al. 2024). In another approach, AOS enhanced salt tolerance in rice by increasing antioxidant enzyme activity and improving stem diameter, root number, and biomass under salt stress. Transcriptomic and metabolomic analyses revealed that AOS regulated genes involved in antioxidant activity, photosynthesis, and cell wall synthesis, while increasing metabolites associated with antioxidant properties. This study highlighted AOS's role in alleviating salt stress‐induced damage in rice (Du et al. 2023). AOS can also enhance plant resistance against pathogenic attacks. Plants identify pathogens through molecules or molecular fragments that have structures or chemical patterns similar to those of the pathogens. These molecules are known as pathogen‐associated molecular patterns (PAMPs). Oligosaccharides from external sources, which possess structures resembling pathogens, act as PAMPs to trigger the immune system (Zhang, Wang, et al. 2020). When AOS (average DP 6.8), produced by alginate lyase from Flavobacterium sp. LXA, were applied to soybean cotyledons, they induced the production of phytoalexins. This indicates that AOS acted as PAMPs on soybean cotyledons (An et al. 2009). In an experiment conducted on tomato seedlings, AOS stimulated the plant's natural defence system by inducing phenylalanine ammonia‐lyase (PAL) activity and promoting the accumulation of phenolic compounds. These phenolic compounds possess antioxidant properties, playing a key role in inhibiting pathogen invasion and enhancing the plant's immune response. The results showed that AOS treatment increased PAL activity starting at 12 h, with a significant rise in polyphenol levels after 24 h, contributing to enhanced disease resistance in tomatoes (Aitouguinane et al. 2020).

Alginate and its degraded products can be converted into various value‐added chemicals through microbial fermentation (Table 2) (Wargacki et al. 2012; Hobbs et al. 2016). This application involves providing DEH, the final monomeric compound degraded by alginate lyase, as a substrate (Hobbs et al. 2016) and using microbial hosts that express alginate lyase either natively or heterologously to directly degrade and convert alginate into value‐added compounds (Takeda et al. 2011; Wargacki et al. 2012; Enquist‐Newman et al. 2014; Takagi et al. 2017; Lim et al. 2019; Lee et al. 2024; Moon et al. 2024). While early studies focused on bioethanol production from alginate and its degraded products (Takeda et al. 2011; Wargacki et al. 2012; Enquist‐Newman et al. 2014; Takagi et al. 2017), recent studies have diversified the target value‐added chemicals to include 2,3‐butanediol, citramalate, itaconate, and 3‐hydroxypropionic acid. Additionally, the successful synthesis of lycopene, renowned for its antioxidant properties, underscores the potential of alginate‐derived pathways in the dietary supplements market and food industry (Kang et al. 2022; Lee et al. 2024; Moon et al. 2024). Collectively, given the high industrial value of alginate‐degraded products, which find applications in areas ranging from agriculture to the biorefinery industry, efficient alginate degradation using alginate lyase holds great promise.

TABLE 2.

Bioconversion of alginate and DEH into value‐added chemicals through microbial fermentation.

Alginate source	Microorganisms	Product	Titre	Yield	Maximum productivity	Reference
Commercial sodium alginate (from Nacalai Tesque)	Metabolically modified Sphingomonas sp. A1	Ethanol	13.0 g/L	—	343 mg/L/h	Takeda et al. (2011)
Saccharina japonica	Metabolically modified Escherichia coli	Ethanol	4.7% (v/v)	0.281 weight ethanol/weight dry macroalgae	—	Wargacki et al. (2012)
Commercial sodium alginate (from Sigma‐Aldrich)	Metabolically modified Saccharomyces cerevisiae	Ethanol	4.6% (v/v) (36.2 g/L)	83% of the maximum theoretical yield	1.9 g/L/h	Enquist‐Newman et al. (2014)
Commercial sodium alginate	Metabolically modified Saccharomyces cerevisiae	Ethanol	8.8 g/L	32% of the maximum theoretical yield	—	Takagi et al. (2017)
Commercial sodium alginate	Co‐culutre of Vibrio sp. dhg and Metabolically modified E. coli	3‐hydroxypropionic acid	293.55 m g/L	—	—	Kang et al. (2022)
Commercial sodium alginate (from Junsei Chemical Co.)	Metabolically modified Vibrio sp. dhg	Citramalate	9.8 g/L	0.47 g/g (57% of maximum theoretical yield)	0.20 g/L/h	Lee et al. (2024)
Saccharina japonica			8.7 g/L	—	—
Commercial sodium alginate (from Junsei Chemical Co.)	Metabolically modified Vibrio sp. dhg	Itaconate	2.5 g/L	0.13 g/g	—	Moon et al. (2024)
Saccharina japonica			1.5 g/L	0.08 g/g	—
Commercial sodium alginate (from Sigma‐Aldrich)	Metabolically modified Vibrio sp. dhg	Ethanol	25.7 g/L	64% of maximum theoretical yield	1.8 g/L/h	Lim et al. (2019)
Kelp powder			19.2 g/L	63% of maximum theoretical yield	0.80 g/L/h
Commercial sodium alginate (from Sigma‐Aldrich)		2,3‐butanediol (including acetoin)	31.3 g/L	81% of maximum theoretical yield	1.3 g/L/h
Commercial sodium alginate (from Sigma‐Aldrich)		lycopene	6.2 mg/L	—	—

3. Discovery and Characterisation of Alginate Lyases

Given the numerous benefits of enzymatic alginate degradation, there is a sustained focus on producing AOS and DEH using alginate lyases and advancing this process for industrial applications. This section covers the efforts to discover novel alginate lyases and their applications.

3.1. Alginate Lyases With Metal Ion Resistance

Metal ions interact with amine or carboxylic acid groups of amino acids within the alginate lyases, which can either inhibit or enhance the activity of the enzymes (Ferdhous et al. 2022). Since marine biomass processing often occurs in environments where various metal ions are present, stability in such conditions is important for industrial applications of alginate lyase (Gao et al. 2018; Ferdhous et al. 2022). For instance, Alyw202 from Vibrio sp. W2, which belongs to PL family 7 and alginate lyase superfamily 2, demonstrated a significant increase in activity, with around 200% and 180% boost in the presence of 1 mM Mn²⁺ and Co²⁺, respectively, and also showed slight activation with Fe²⁺, Cu²⁺, and Zn²⁺. Moreover, Alyw202 maintained its activity even at high metal ion concentrations, such as 10 mM, without complete inhibition (Ma et al. 2020). Similarly, a PL family 6 alginate lyase, TsAly6A from Thalassomonas sp. LD5, exhibited a 177% and 136% increase in enzyme activity with Ca²⁺ and Mg²⁺, respectively, while other ions such as Li⁺, Ba²⁺, and K+ exerted minimal inhibitory effects. The observed activation by Ca²⁺ and Mg²⁺ is likely attributable to their coordination with specific amino acid residues within the enzyme (Gao et al. 2018). Chen et al. (2023) reported TAPL7B, identified as a PL7 family alginate lyase from Thalassotalea algicola, which was activated by a wide range of metal ions, including Ca²⁺, K⁺, Na⁺, Ni⁺, Cu²⁺, Mg²⁺, Zn²⁺, Co²⁺, and Fe³⁺. Notably, TAPL7B exhibited a remarkable increase in activity under 1.2 mM Ca²⁺ conditions (Chen et al. 2023). The pronounced metal ion tolerance of these alginate lyases makes them highly suitable for industrial applications, as they can efficiently degrade alginate in ion‐rich environments.

3.2. Alginate Lyases With Dual Endolytic and Exolytic Activities

Several studies have identified alginate lyases with dual endolytic and exolytic activities, enabling the complete breakdown of alginate into products ranging from oligosaccharides to monosaccharides, thus facilitating efficient saccharification (Chu et al. 2020). A novel alginate lyase, Alg17B, which was isolated from a marine bacterium inhabiting decomposing Sargassum, exhibited dual activity, degrading alginate into DEH or AOS with a DP of 2–6 (Huang et al. 2019). Chu et al. (2020) identified a novel alginate lyase with dual activity, KJ‐2, from Stenotrophomonas maltophilia KJ‐2 (Chu et al. 2020). Furthermore, by comparing the crystal structure with the previously characterised exolytic alginate lyase Alg17c (Kim et al. 2012), they predicted that four amino acid residues in KJ‐2 (Tyr238, Arg241, Arg418, and Glu644) were crucial in forming the active site and participating in hydrogen bonding with the substrate. Introducing site‐specific mutations to Arg241 and Glu644 decreased exolytic activity, thereby confirming the key residues for the enzyme's exolytic function. Dual active alginate lyases are highly advantageous for industrial applications. They are crucial for processes like biofuel production from seaweed biomass, where thorough depolymerisation is necessary to produce fermentable sugars and valuable biochemicals.

3.3. Alginate Lyases with Broad Substrate Specificity

Since brown macroalgae, the primary source of alginate, have varying compositions of M and G depending on the harvest season and species, alginate lyases with broad substrate specificity are essential to efficiently extract alginate from brown macroalgae (Schiener et al. 2016; Zheng et al. 2022). These enzymes enable proficient and complete alginate breakdown, maximising substrate utilisation (Yagi et al. 2016). Gu et al. (2024) identified Aly448, an alginate lyase with broad substrate specificity, from a metagenomic library of macroalgae collected in Indonesia (Gu et al. 2024). They figured out that Aly448 has binding sites for alginate, polyG, and polyM substrates. Lu et al. (2019) characterised a novel endo‐lyase AlyPB1 and a novel exo‐lyase AlyPB2 from a marine bacterium, Photobacterium sp. FC61 (Lu et al. 2019). Particularly, AlyPB2 showed activity towards both polyG and polyM, while AlyPB1 only had activity towards polyG. Although having a broad substrate activity could bring about less specificity, it could reduce the need for multiple alginate lyases and individual optimisation, thereby enhancing the efficiency and cost‐effectiveness of industrial alginate degradation.

3.4. Thermophilic Alginate Lyases

The discovery of thermophilic alginate lyases, which exhibit greater durability and stability at high temperatures (above approximately 50°C), is particularly noteworthy. Alginate degradation at high temperatures offers several industrial advantages, including lower viscosity of alginate, prevention of bacterial contamination, and compatibility with commercially available cellulase that has optimal activity at high temperatures (Rhein‐Knudsen et al. 2021; Zhang et al. 2021). Recently, two thermophilic alginate lyases of the PL6 family, AlyRmA and AlyRmB from Rhodothermus marinus , which exhibit optimal activity at 70°C, were characterised (Zhu et al. 2024). Their primary protein structures suggest that their thermal resistance may be attributed to the high content of proline and arginine (Watanabe et al. 1994). Due to its superior metal ion tolerance and stability compared to AlyRmA, AlyRmB was selected for AOS production in this study. As a result, AlyRmB produced 0.42 g/L of AOS with a DP 2–6, degrading 0.5% (w/v) alginate over 10 h (Zhu et al. 2024). Another thermostable alginate lyase identified from a metagenomic data set using the dbCAN data server were employed for one‐step saccharification of brown macroalga in combination with commercial cellulase. The enzyme mixture of endolytic AMOR_PL7 (optimal at 65°C), exolytic AMOR_PL17 (optimal at 50°C), and commercial cellulase mixture Cellic CTec2 (optimal at 45°C ~ 50°C) effectively yielded glucose, mannitol, and DEH from Saccharina latissima at 55°C (Rhein‐Knudsen et al. 2021).

4. Advancements in Production and Application of Alginate Lyases

4.1. Heterologous Expression Systems for Enhancing Production of Alginate Lyase

Since alginate lyases are naturally produced by non‐platform strains, their low yield limits their harvest and industrial exploitation. To maximise the diverse properties and advantages of alginate lyases from various species, platform strains can be utilised as expression hosts. Exploiting the characteristics of these strains could enhance alginate lyase yield, thereby increasing the production of AOS and DEH. For instance, E. coli offers significant benefits, including robust growth, cost‐effective large‐scale fermentation, and high protein synthesis efficiency, enhancing alginate lyase productivity (Zhang et al. 2015; Sun et al. 2019; Depping et al. 2022; Falak et al. 2022). In addition, Bacillus subtilis and Yarrowia lipolytica are highly valued in industrial applications for their strong secretion of heterologous enzymes and proteins, ease of genetic manipulation, and well‐characterised genetic systems, making them ideal workhorses for industrial and pharmaceutical uses (Madzak et al. 2004; Madzak 2015; Yagi et al. 2016; Cui et al. 2018; Zheng et al. 2023). However, most studies have focused on heterologous alginate lyase expression, primarily assessing the performance of the recombinant enzymes (Zheng et al. 2023). At best, the efforts were limited to optimising the culture conditions to enhance the catalytic activity. Meng et al. (2021) characterised a new PL7 family alginate lyase, Aly01, derived from Vibrio natriegens SK42.001, and enhanced its yield through heterologous expression in Terrific Broth (TB) medium supplemented with 120 mM glycine and 10 mM calcium (Meng et al. 2021). Similarly, Zheng et al. (2023) focused on overexpressing Alg62, classified as PL7 family, from Vibrio alginolyticus in B. subtilis WB600, determining that the optimal medium for maximum activity included 15 g/L glycerol, 25 g/L yeast extract, and 1.5 mM K⁺ (Zheng et al. 2023). Therefore, maximising the potential of platform strains with diverse advantages could further increase the yield of alginate lyases.

In this regard, incorporating host‐compatible signal peptides is beneficial, as it is crucial for protein secretion and localisation (Owji et al. 2018). The alginate lyase derived from Vibrio sp. QY102 was expressed in E. coli by replacing its native signal peptide with the signal peptide sequence from the ompA gene in E. coli (Sun et al. 2019). The extracellular enzyme activity of alginate lyase with the ompA signal peptide averaged 350 U/mL, which is approximately 1.5 to 6 times higher than that with the native signal peptide. A similar strategy was reported using alginate lyase AlyC7, classified within the PL7 family, from Vibrio sp. C42, which exhibited high activity, broad substrate specificity, and high yields of trimers (Wang et al. 2024). The authors integrated five different signal peptides in the type II secretion pathway of E. coli (the pelB, malE, ompA, phoA, and ompT genes) to efficiently produce AlyC in E. coli . Among them, AlyC7 combined with the pelB signal peptide achieved the highest yield of 1122.8 U/mL after 27 h of cultivation in LB medium.

Collectively, it has been confirmed that using the appropriate expression host and signal peptide can improve protein expression and secretion efficiency, resulting in significant technical contributions to large‐scale production of alginate lyases. Additionally, engineering signal peptides to enhance secretion efficiency would further accelerate the economic feasibility of alginate lyase production and alginate degradation process.

4.2. Utilisation of Crude Enzyme From Alginate‐Degrading Microorganisms

To reduce enzyme purification costs (Zhang et al. 2024), recent efforts have focused on using crude enzymes from alginate lyase‐producing microorganisms for alginate degradation. Using crude enzymes also offers the advantage of simultaneously utilising multiple alginate lyases with diverse substrate specificities and cleavage modes. Gomaa et al. (2018) utilised crude enzymes from the culture supernatant of the marine fungus Dendryphiella arenaria to degrade crude alginate extracted from Sargassum latifolium for biofuel production. The crude enzyme converted 1 g of crude alginate into around 440 mg of reduced sugars (Gomaa et al. 2018). Similarly, crude enzymes from Pseudoalteromonas sp. Alg6B were used to produce AOS from Saccharina japonica. Combined with commercial cellulase, this study achieved a degradation rate of 97% for Saccharina japonica, producing AOS with a DP of 2 and 4 (Sun et al. 2020). In addition, crude enzymes from Vibrio sp. B1Z05, containing 17 different alginate lyases, were used to produce AOS with DP 2–6 from Saccharina japonica (Zhang et al. 2024).

4.3. Immobilisation of Alginate Lyases

Immobilising the enzyme on a solid matrix has shown advantages over using the free enzyme, including improved stability and reusability of the enzymes (Maghraby et al. 2023). A study has explored the use of mesoporous titanium oxide particles (MTOPs) to enhance the thermal stability and reusability of alginate lyase (Li, Hu, et al. 2020). The authors reported that immobilising AlyPL6 from Pedobacter hainanensis NJ‐02 on MTOPs maintained 84% of its initial activity after being heated at 45°C for 1 h, compared to only 9.6% for the free enzyme, demonstrating a significant improvement in thermal stability. Furthermore, the immobilised enzyme retained 75% and 54% of its initial activity after 5 and 10 cycles of reuse, respectively (Li, Hu, et al. 2020). In another study, Tanaka et al. (2022) immobilised the endolytic AlyFRA (PL7 family) and the exolytic AlyFRB (PL15 family) from Falsirhodobacter sp. alg1 onto κ‐carrageenan, which was used as a support material, to produce DEH. These lyases degrade alginate into unsaturated uronate monomers, which are then converted to DEH. The immobilised enzymes achieved an 80% DEH yield after seven cycles of reuse, highlighting the potential for a cost‐effective production process Tanaka et al. 2022.

5. Protein Engineering of Alginate Lyases

5.1. Domain Truncation of Alginate Lyase

The truncation of the specific domain is guided by the enzyme's resolved structure. Most alginate lyases consist of catalytic domains (CD) and non‐catalytic domains (NCD), which include the carbohydrate‐binding module (CBM). Although CBM does not directly catalyse reactions, it crucially determines enzyme functionality (Várnai et al. 2013). Thus, truncation of the domain could result in changes in the enzyme's performance, including alterations in substrate affinity, catalytic activity, and thermal stability of the alginate lyases (Hu et al. 2019; Yan et al. 2019; Yang Li, et al. 2019; Zhang et al. 2019) (Table 3). Yang, Li, et al. (2019) analysed domains of AlyM, classified as PL7 family, from Microbulbifer sp. CGMCC 14061 by comparing its sequence within its genomic database. Subsequently, they generated various truncated AlyM variants by reconstructing its domains. Among them, cAlyM, containing only the CD, showed the highest enzymatic activity of approximately 390 U/mg, representing a 6.3‐fold increase compared to the full‐length AlyM. The mutant also exhibited significantly enhanced thermal stability, retaining nearly 70% of the enzymatic activity at 45°C. Interestingly, AlyMΔ58C, with the F5/8 type C domain—known for its roles in protein–protein interactions and one of CBM (Ficko‐Blean and Boraston 2006)—removed, demonstrated a higher affinity for polyM, whereas the intact AlyM preferentially bound to polyG (Yang, Li, et al. 2019). This mutant can be utilised to produce AOS with mannuronate at the reducing end, such as pentaM and pentaG.

TABLE 3.

Engineering strategies for alginate lyases.

Enzyme	Source of the enzyme	Strategy	Result of engineering	Reference
AlyM	Microbulbifer sp. CGMCC 14061	Reconstructing CD	Improved thermal stability, substrate affinity, and catalytic efficiency	Yang, Li, et al. (2019)
AlgH‐I	Marinimicrobium sp. H1	Truncating NCD	Improved thermal stability, pH stability, and salt tolerance	Yan et al. (2019)
TsAly7B	Thalassomonas sp. LD5	Reconstructing CD	Improved thermal stability, catalytic efficiency, and regulating the products distribution	Zhang et al. (2019)
Aly7C	Vibrio sp. W13	Constructing a hybrid enzyme by recombining modules	Improved catalytic efficiency and catalytic efficiency	Hu, Cao et al. (2021)
AlyRm6A‐Zu7	AlyRM6A from Rhodothermus marinus 4252 & AlyZu7 from Zobellia uliginosa	Constructing a hybrid enzyme by recombining modules	Improved thermal stability and achieving bifunctional catalytic capability	Guo et al. (2024)
FlAlyA	Flavobacterium sp. UMI‐01	Prediction of mutation sites by B‐factor and free energy change	Improved thermal stability and expression efficiency	Zhang et al. (2022)
AlyRm6A	Rhodothermus marinus 4252	Prediction of mutation sites by free energy change	Improved thermal stability	Guo et al. (2023)
AlyMc	Microbulbifer sp. Q7	Prediction of mutation sites by B‐factor and free energy change	Improved thermal stability	Cui et al. (2024)
AlgL‐CD	Pseudoalteromonas sp. zb‐7	Substituting residues near the active site to alkaline amino acid	Improved substrate affinity and catalytic efficiency	Xu et al. (2021)
AlgL‐CD	Pseudoalteromonas sp. aly – SJ02	Targeting the lid loop and sites at the predicted substrate entrance	Improved thermal stability and catalytic efficiency	Su et al. (2021)
AlyG2	Seonamhaeicola algicola	Substitution of residues with bulky steric hindrance close to the active pocket and prediction of mutation sites by free energy change	Improved thermal stability and catalytic efficiency	Huang et al. (2024)
cAlyM	Microbulbifer sp. Q7	Prediction of potential sites of disulfide bond formation using dynamic simlations	Improved thermal stability	Yang, Yang, et al. (2019)
Alg‐2	Tamlana sp. S12	Error‐prone PCR	Improved substrate affinity	Shu et al. (2019)
FsAly7	Flammeovirga sp.	Error‐prone PCR	Improved thermal stabilit	Jiang et al. (2025)

Yan et al. (2019) identified the protein domains of AlgH, classified as PL7 family, from Marinimicrobium sp. H1 using a similar strategy, and investigated the impacts of removing NCDs in AlgH (Yan et al. 2019). AlgH‐I containing only the CD exhibited significantly improved thermal stability, preserving 80% and 75% of its activity after 2 h of incubation at 40°C and 50°C, respectively, while the full‐length AlgH retained only 10% of its activity under the same conditions. Moreover, AlgH‐I showed 2.1 times higher catalytic activity than AlgH, without any change in substrate preference (Yan et al. 2019).

Furthermore, elucidating the function of modules can provide valuable insights for engineering and developing alginate lyases. The PL7 family alginate lyase TsAly7B from the marine bacterium Thalassomonas sp. LD5 was identified, and its CBM and CD were characterised using sequence analysis tools (Zhang et al. 2019). The enzyme was predicted to contain two types of CBMs (CBM9 and CBM32) and a CD. In alginate lyase, CBM32s are key in recognising alginate and influencing the composition of degradation products. In contrast, the specific role of CBM9 in alginate lyases remains unclear and is yet to be fully understood. To figure out the functionality of CBM9, truncated mutants TM1 (containing CBM32 and CD), TM2 (containing CBM9 and CD), and TM3 (containing only CD) were constructed. Experimental results demonstrated that both CBM9 and CBM32 facilitated alginate degradation, with TM2 exhibiting a specific activity 7.6 times higher than TM3. The team successfully demonstrated the functionality of CBM9 for the first time (Zhang et al. 2019).

While these approaches highlight the potential of studying this domain to develop more efficient and robust alginate lyases for industrial applications, truncation may cause undesirable conformational changes that affect functionality (Kittur et al. 2003; Hu et al. 2019). Moreover, deleting the conserved NCDs could be detrimental, reducing substrate activity and potentially leading to loss of function (Hu et al. 2019). As truncating NCDs relies on the resolved enzyme structure, it is essential to consider structural information and stability when constructing mutant enzymes.

5.2. Chimeric Alginate Lyase Construction

Enzymes are composed of several modules, each presenting distinct functionalities. By recombining these modules, particularly from different alginate lyases, it is possible to create hybrid enzymes with improved properties such as enhanced catalytic efficiency, substrate specificity, or stability (Yan et al. 2019; Yang, Li, et al. 2019; Zhang et al. 2019; Hu, Cao, et al. 2021; Guo et al. 2024). Such engineered enzymes could offer significant advantages in industrial applications, where tailored enzymatic activity is crucial (Abascal and Valencia 2003).

Recombination of conserved domains could improve functionality by integrating superior structural components (Hu, Cao et al. 2021; Guo et al. 2024). Hu et al. (2021) aimed to prepare AOS by hybridising the domains of the PL7 family alginate lyases Aly7A and Aly7B (Hu, Cao, et al. 2021), previously identified from Vibrio sp. W13 (Zhu et al. 2015, 2019; Hu et al. 2019). Sequence alignment within the PL7 family revealed that Aly7A possessed an N‐terminal CBM and a C‐terminal catalytic module. Aly7B contained two unique catalytic modules absent in any other lyases in the family. The researchers observed a significant decrease in Aly7A's activity upon the removal of its CBM, indicating that the CBM plays a pivotal role in enzymatic function. Among the possible combinations, the hybrid enzyme Aly7C, which incorporates Aly7A's CBM and Aly7B's catalytic module, demonstrated higher thermal stability and maximal activity at 40°C and pH 9.0 (Hu, Cao, et al. 2021). In contrast, Aly7B‐CDII, a previously developed truncated enzyme of Aly7B, exhibited maximal activity at 35°C and pH 9.0 (Hu et al. 2019). Additionally, Aly7C could degrade sodium alginate, polyM, and polyG into oligosaccharides with DP of 2–5, demonstrating a broad range of product specificity. Despite the novel hybrid enzyme's enhanced performance, the underlying cause remains unexplained. Developing a universal rationale and strategy could facilitate advancements in other alginate lyases, thereby improving bioprocessing efficiency.

Furthermore, Guo et al. (2024) successfully constructed a multifunctional enzyme by recombining the endoactive and thermostable PL6 family AlyRm6A from Rhodothermus marinus 4252 with the exoactive PL7 family AlyZu7 from Zobellia uliginosa and elucidated the cause by structural analysis (Guo et al. 2024). The team identified conserved domains of AlyZu7 and selected key residues for fusion with AlyRm6A via the linker. The resulting hybrid enzyme, AlyRm6A‐Zu7, maintained approximately 90% of its activity after incubation at temperatures ranging from 30°C to 50°C. Product analysis showed that AlyRm6A‐Zu7 produced more products (DP 1–4 and DEH) than the mixture of AlyZu7 and AlyRm6A, and significantly higher amounts of DP 1 and DEH compared to AlyZu7 at 25°C. Structural analysis using AlphaFold2 suggested that the enhanced protein–protein interactions between AlyRm6A‐Zu7 and its substrate potentially increased protein stability. This mutant exhibited excellent thermostability and dual endo‐ and exo‐activity, making it a valuable biocatalyst for industrial applications (Guo et al. 2024). These studies demonstrate that combining domains from different alginate lyases can induce a synergistic effect, offering promising strategies for enzyme engineering and application.

5.3. Computer‐Aided Rational Design

When the structure and function of the target protein are understood, rational structure‐based protein design becomes a promising approach (Narad et al. 2023). Site‐directed mutagenesis is frequently used to investigate the role of a specific amino acid in the context of the entire protein (Siloto and Weselake 2012). Traditionally, it enabled the evaluation of a specific residue's role in the catalytic activity of alginate lyases (Yamamoto et al. 2008; Kim et al. 2015). With advancements in computer‐aided rational design, more precise site‐directed mutagenesis considering structural information can now be applied to engineer alginate lyases.

When introducing a mutation to an enzyme, calculating changes in free energy (ΔΔG) upon mutation and predicting the dynamic changes caused by the mutation helps decide the alteration in the enzyme's stability and functionality (Musil et al. 2017; Zhang et al. 2022; Guo et al. 2023; Cui et al. 2024). Zhang et al. (2022) aimed to enhance the thermal stability and expression yield of FlAlyA, a PL7 family alginate lyase from Flavobacterium sp. UMI‐01 characterised in 2014 (Inoue et al. 2014). They calculated the B‐factor values, which indicate the dynamic flexibility of each atom in the protein (Mlynek et al. 2024), to assess protein stability and selected potential mutation sites through calculation of ΔΔG (Zhang et al. 2022). Among the mutants, H176D and H71K showed improvements in thermal stability, with melting point increased by 1.2°C and 0.3°C, respectively. At 50°C, the half‐lives of these mutants were extended by 7.6 min and 1.7 min, respectively. The H71K mutation particularly exhibited comprehensive performance enhancements over the wild‐type, including better expression levels, thermal stability, and specific activity. To investigate the molecular mechanisms behind the increased thermostability of the mutants, molecular dynamic simulations were conducted. The results indicated that the enhanced stability of H71K was due to the formation of new hydrogen bonds and a reduction in the solvent‐accessible surface area (Zhang et al. 2022).

Similarly, Guo et al. (2023) focused on engineering AlyRm6A, a novel PL6 family alginate lyase from Rhodothermus marinus , which naturally exhibits high thermostability (Guo et al. 2023). To enhance the thermostability of AlyRm6A while preserving its catalytic activity, the researchers calculated the ΔΔG values to compare protein stability after mutagenesis, identifying optimal mutation sites for improved thermal stability. Their rational design predicted and confirmed that single‐point mutants T43I and Q216I displayed enhanced thermostability, with respective half‐life increases of 20% and 25% (Guo et al. 2023). Structural analysis of the WT and mutants revealed that T43I and Q216I formed new and strong hydrophobic interactions with nearby amino acid residues, explaining the enhanced thermostability of these mutants. Unfortunately, to alleviate workload, the team eliminated most mutation points due to the lack of accurate computer‐aided verification of beneficial mutations, potentially discarding more advantageous mutants.

Moreover, Cui et al. (2024) focused on enhancing the thermal resilience of the PL7 family alginate lyase AlyMc from Microbulbifer sp. Q7. They calculated B‐factor and ΔΔG values to assess protein stability and introduced site‐specific mutations to generate and evaluate mutants for thermal stability (Cui et al. 2024). The resulting mutants, Q246V and K249V, exhibited significant improvements, with thermal half‐lives of 3.9 h and 3.7 h, respectively, compared to that of 2.4 h of the wild‐type AlyMc. The team argued that favourable changes in electrostatic charges on the surface of the mutants enhanced the thermal stability of AlyMc, as charged amino acids on the protein surface can form a protective network through electrostatic interactions.

Another approach focuses on the reaction mechanism to select desirable mutations (Yang, Yang, et al. 2019; Su et al. 2021; Xu et al. 2021; Huang et al. 2024). Xu et al. (2021) applied this approach to the endo‐type PL18 family alginate lyase from Pseudoalteromonas sp. aly‐SJ02, identified in 2011 (Li et al. 2011; Dong et al. 2014). They aimed to enhance its substrate affinity and catalytic efficiency by introducing single‐point mutations near the active site to improve substrate affinity and study the enzyme's structure–function relationship (Xu et al. 2021). Specifically, they designed AlgL‐CD mutants by substituting acidic amino acid residues near the active site with basic ones. The E226K mutant exhibited a 10‐fold increase in substrate affinity compared to the wild‐type. Structural analysis of the E226K mutant revealed that it had more flexible lid loops, thereby increasing substrate binding affinity (Xu et al. 2021).

Subsequently, Su et al. (2021) further engineered the E226K mutant (Xu et al. 2021) to enhance its catalytic efficiency and thermal stability (Su et al. 2021). Focusing on the enzyme's mechanism, they targeted the lid loop and sites at the predicted substrate entrance. Given the high polarity of the alginate substrate, the researchers hypothesised that the lid loop's polarity would significantly impact functionality. They replaced hydrophobic residues with polar ones and substituted bulky residues at the entrance with smaller ones. The resultant triple mutant, E226K/I211T/R294V, showed a 4.8‐fold increase in catalytic efficiency and a substantial improvement in thermal stability, with the half‐life at 45°C increasing from 89 min to 560 min. Molecular dynamics simulations demonstrated that the mutant had a more flexible loop and a wider substrate entrance, which facilitated efficient proton transfer and enhanced enzymatic activity (Su et al. 2021).

A similar strategy was employed to engineer the alginate lyase AlyG2 from Seonamhaeicola algicola, which was characterised previously (Yun et al. 2024), to enhance its thermal stability and catalytic efficiency (Huang et al. 2024). The main strategy was to increase specific activity by substituting residues with bulky steric hindrance near the active pocket (T91S). The T91S mutant exhibited a 1.9‐fold increase in specific activity compared to the wild‐type (Huang et al. 2024). Moreover, to enhance the thermal stability of T91S, mutants with negative ΔΔG values in the thermal flexibility region were selected. Among them, the S72Y^a mutant demonstrated an 18% increase in specific activity and improved thermal stability compared to T91S, with a 31% increase in relative activity after a 1‐h incubation at 42°C. To understand the improvement, the team conducted molecular dynamics and found that S72Y^a showed reduced surface loop flexibility and more stable hydrogen bonds.

Moreover, Yang, Yang, et al. (2019) applied this approach to the previously modified alginate lyase cAlyM, derived from AlyM of Microbulbifer sp. Q7 (Ficko‐Blean and Boraston 2006). Using molecular dynamics simulations, they rationally assessed the structural stability of cAlyM and predicted potential sites for disulfide bond formation to effectively enhance protein thermal stability (Mansfeld et al. 1997; Yang, Yang, et al. 2019). Of the six mutants constructed, five successfully formed new disulfide bonds. Notably, the D102C‐A300C and G103C‐T113C mutants exhibited half‐lives of 4.2 h and 3.1 h at 45°C, respectively. Additionally, the D102C‐A300C mutant showed a specific activity 1.1 times higher than that of cAlyM and improved thermal stability, making it a promising candidate for meeting the industrial demand for the preparation of AOS (Yang, Yang, et al. 2019).

These studies demonstrate that targeted mutations, guided by thermodynamics and molecular dynamic predictions, can significantly enhance the performance of alginate lyases. Using structural stability assessments can improve specific activity and thermal stability, showing great potential for developing industrially valuable biocatalysts for AOS and DEH preparation. Integrating computer‐aided rational engineering and targeted mutations can induce beneficial structural changes, resulting in more robust and efficient enzymes for the exploitation of brown macroalgae.

5.4. Directed Evolution

While computer‐aided design has refined mutagenesis sites for alginate lyase engineering, it is limited to enzymes with known structural information. Directed evolution has been widely utilised to enhance the activity, stability, and industrial applicability of alginate lyases. Various strategies, including error‐prone PCR, iterative mutagenesis, and combinatorial mutagenesis, have been employed to optimise enzyme performance (Mansfeld et al. 1997; Yang, Yang et al. 2019).

Shu et al. (2019) applied error‐prone PCR to improve the PL7 family alginate lyase Alg‐2 from Tamlana sp. S12, identifying two mutants with 160% and 240% higher enzymatic activity than the wild‐type (Shu et al. 2019). The P2‐81 mutant exhibited a 50% reduction in K_m, indicating improved substrate affinity, with key amino acid substitutions (Glu, Thr, Ser, Asp, and Tyr) contributing to enhanced activity. Similarly, iterative rounds of mutagenesis and selection were performed on PL7 family FsAly7 from Flammeovirga sp., leading to a 10% increase in catalytic efficiency, a shift in optimal temperature from 40°C to 50°C, and enhanced thermal stability, making it more suitable for industrial applications (Jiang et al. 2025). Additionally, error‐prone PCR identified mutants with 20% lower K_m and a 1.5‐fold increase in specific activity, further improving substrate affinity and enzyme efficiency.

These studies collectively demonstrate the power of directed evolution in improving alginate lyases, enabling more efficient bioconversion of alginate‐rich biomass and expanding their industrial potential in bioprocessing, biofuel production, and marine biomass utilisation.

6. Future Perspectives and Challenges

Alginate lyases show significant potential for producing AOS and DEH, with promising applications in agriculture, pharmaceuticals, and value‐added chemical production. Recent advancements in the discovery and characterisation of alginate lyases have brought us closer to enhancing AOS and DEH productivity. However, challenges such as instability at high temperatures and low catalytic activity must be addressed to optimise production. Identifying new enzymes with superior performance and advancing protein engineering techniques are crucial steps in overcoming these hurdles. These technological improvements have not only increased production efficiency but also expanded the application scope of alginate lyases.

Optimising industrial processes involving alginate lyases requires a thorough evaluation of analytical techniques and alginate properties. Current methods for separating and characterising AOS and DEH, including chromatography, mass spectrometry, and nuclear magnetic resonance, are time‐consuming and costly, posing challenges for the industrialisation of alginate lyase‐based processes (Cheng et al. 2020). Furthermore, the properties of alginate, primarily derived from brown macroalgae, vary depending on the species, harvest time, and growth location, necessitating standardised enzymatic degradation processes tailored to specific alginate characteristics.

By addressing these challenges and fully leveraging the potential of alginate lyases, we can successfully achieve improvements in the efficiency and sustainability of industrial processes. In the biorefinery sector, alginate lyases can play a pivotal role in converting brown macroalgae into valuable biofuels and biochemicals, contributing to a more sustainable and circular economy. Continued investment in research and development will unlock new applications and drive innovation, positioning alginate lyases as key biocatalysts for a sustainable future.

Author Contributions

Conceptualisation, H.J.S., J.H.M., S.W., C.W.L., G.Y.J., H.G.L.; investigation, H.J.S., J.H.M., C.W.L.; supervision and validation, G.Y.J., H.G.L.; writing – original draft, H.J.S., J.H.M., S.W., C.W.L., G.Y.J., H.G.L.; H.J.S. and J.H.M. contributed equally to this work. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

Data sharing is not applicable to this article as no new data were created or analyzed in this study.