High-efficiency and thermostable β-agarase Aga3 for sustainable production of neoagarooligosaccharides (NAOs) from algal biomass

Abstract

With growing concerns about the circular economy and carbon neutrality, attention has increasingly focused on the discovery and industrial application of powerful renewable resources. Agarose, a major structural component of red algae and classified as a third-generation biomass, has emerged as a promising renewable substrate for the production of high-value chemicals and bioactive compounds. In this study, we identified GH16 β-agarase, designated as Aga3, from the marine agarolytic bacterium, Cellulophaga omnivescoria MSK1 and conducted comprehensive biochemical characterization of the enzyme. Aga3 exhibited excellent agarose-degrading activity, with K_m and k_cat values of 0.93 mg/mL and 1300 s⁻¹, respectively. The enzyme also showed remarkable thermal stability above 50°C, and its activity increased more than six-fold in the presence of 5 mM cobalt ions. Under optimized conditions, Aga3 efficiently produced neoagarobiose (NA2) (6.61 ± 0.55 g/L) and neoagarotetraose (NA4) (10.4 ± 1.30 g/L) with maximum conversion rate of 88.8%. These results demonstrate that Aga3 is a robust and highly efficient biocatalyst with substantial potential for sustainable bioprocesses.

Keywords

GH16 β-agarase marine bacteria algal biomass neoagarooligosaccharides thermostable

Introduction

Agarose, a polysaccharide extracted from the cell walls of red algae, holds strong potential as a sustainable biorefinery feedstock due to its chemical composition, functional properties, and renewable nature. Its relatively simple structure, composed of D-galactose (G) and 3,6-anhydro-L-galactose (AHG), enables efficient processing and conversion into bio-based products such as pharmaceuticals, cosmetics, and other valuable biochemicals, making it well-suited for biorefinery applications.^1,2 Agarose degradation is catalyzed by β-agarases (EC 3.2.1.81), which hydrolyze the β-1,4-glycosidic bonds between the repeating units of G and AHG. These enzymes are classified into different glycoside hydrolase (GH) families based on their substrate specificities and modes of action. Enzymatic hydrolysis of agarose yields neoagarooligosaccharides (NAOs), including neoagarobiose (NA2), neoagarotetraose (NA4), neoagarohexaose (NA6), neoagarooctaose (NA8), and neoagarodecaose (NA10), depending on the number of G and AHG units in the oligosaccharides.³

Among these NAOs, NA2, and NA4 have gained significant attention as potent bio-compounds in various industrial sectors, including cosmetics, pharmaceuticals, and the food industry. Their diverse bioactivities including skin moisturization,⁴ whitening, antioxidant,⁵ anti-inflammatory, and ROS-scavenging effects⁶ contribute to their wide range of potential applications. Furthermore, their proven safety in animal models at doses up to 5000 mg/kg body weight,⁷ hepatoprotective effects via activation of nuclear factor (NF)-E2-related factor 2 (Nrf2),⁸ and potential to alleviate obesity-related metabolic defects have positioned them as promising candidates for pharmaceutical applications.⁹

Reflecting their expanding industrial relevance, the β-agarase market was valued at $200 million USD in 2025 and is projected to reach $350 million USD by 2033, representing a compound annual growth rate (CAGR) of 7% (Data Insights Market, 2024). In parallel, the marine oligosaccharides market, including NAOs, was valued at $3.56 billion USD in 2024 and is forecasted to reach $5.98 billion USD by 2034 (CAGR of 5.33%; Precedence Research, 2025).

Given this increasing demand and commercial potential, significant research efforts have focused on identifying highly efficient β-agarases capable of producing NA2 and NA4 from waste algal biomass. For example, the engineered GH16 β-agarase Aga0917originated from Pseudoalteromonas fulginea YTW1-15-1 capable of producing NA4 up to 1.38 mg/mg/h was developed through site-directed mutagenesis of two catalytic residues.¹⁰ Another study reported the isolation of GH16 β-agarase, Aga-ms-R from Microbulbifer sp. BN3, which predominantly produced NA4 and trace amounts of NA2 using red seaweeds Gracilaria sjoestedtii and Gelidium amansii as substrates.¹¹ In addition, the GH50 β-agarase GH50A, derived from Cellvibrio sp. KY-GH-1, produced 216 mg of NA2 under optimal conditions (pH 7.5, 35°C), with its enzymatic activity increasing 2.5-fold in the presence of 5 mM MnSO₄ and 10 mM tris(2-carboxyethyl)phosphine (TCEP).¹² Despite these advances, significant challenges remain in meeting the industrial requirements for β-agarase applications. Key limitations include insufficient substrate affinity and stability in vitro, which hinder the large-scale production of NA2 and NA4.¹³ Furthermore, enhancing thermal stability to withstand harsh industrial conditions and improving cofactor affinity to maintain enzymatic activity below the sol-gel transition temperature of agarose remain critical challenges.¹⁴ Therefore, the development of novel β-agarases with improved catalytic efficiency and enhanced resilience to extreme environments is essential to enable industrial utilization.

We previously isolated and reported Cellulophaga omnivescoria MSK1, a strain capable of utilizing agarose, a marine biomass, as a carbon source for xanthophyll carotenoid biosynthesis.¹⁵ Based on previous results, the strain exhibited high agarose-degrading activity and efficiently converted agarose into xanthophylls. However, the mechanism underlying the high agarose-degrading capacity of the MSK1 strain has not yet been clearly elucidated. Therefore, in this study, we identified several agarases from the MSK1 strain, including a newly discovered GH16 β-agarase, Aga3, which plays the most important role, and analyzed its catalytic activity, substrate specificity, and stability. This study elucidates the enzymatic basis for agarose utilization by the MSK1 strain and highlights the potential of this enzyme as a biocatalyst for the sustainable utilization of marine biomass.

Materials and methods

Whole genome sequencing of C. omnivescoria MSK1

The whole genome sequence of C. omnivescoria MSK1 strain was constructed de novo based on the result of Pacbio sequencing analysis. The preparation of a genome library and sequencing was carried out by CJ Bioscience (Seoul, Republic of Korea). In brief, genomic DNA (gDNA) was isolated and purified from 10 mL of cell cultures using a Wizard Genomic DNA Isolation kit (Promega, Madison, WI, USA). To generate libraries, 5 µg of genomic DNA was sheared and then purified to remove small fragments (< 3 kb) using AMpureXP bead purification kit (Beckman Coulter, Inc., CA, USA). The sequencing primer v4 was annealed to the SMRTbell template, and DNA polymerase was bound to the complex using Sequel Binding kit 3.0 (PacBio Bioscience, CA, USA). The SMRTbell library was sequenced using Sequel Sequencing Kit v3.0 and SMRT Cell 1 M v2, and the data was collected through 1 × 10 h movie per each SMRT Cell 1 M v2 using the sequel sequencing platform (Pacific Biosciences). The resulting contigs from PacBio sequencing data were circularized and rearranged at the start position of dnaA using Circlator 1.4.0 (Sanger institute). The gene annotation was analyzed using NCBI Prokaryotic Genome Annotation Pipeline (PGAP) version 6.9 with GeneMarkS2 + annotation method.¹⁶

Amino acid sequence analysis

The nucleotide and amino acid sequence of β-agarase, Aga3 (accession No. WP_271081340.1) were obtained from the genome information of C. omnivescoria MSK1.¹⁵ Basic Alignment Search Tool (BLASTp) (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to determine the identity of the Aga3 sequence. The phylogenetic tree was constructed based on amino acid sequences of Aga3 with various β-agarases derived from other bacterial species using Neighbor-Joining (NJ) method with 1000 bootstrap replications using Mega X program.¹⁷ The conserved catalytic residues of Aga3 were analyzed by multiple alignments with other β-agarases belonging to glycoside hydrolase family 16 (GH16) in Uniprot (https://www.uniprot.org/align).

Cloning, expression, and purification of the recombinant Aga3 protein

The genomic DNA of C. omnivescoria MSK1 was used for the original source of the β-agarase gene, aga3 gene, and as a PCR (Polymerase chain reaction) template.¹⁵ The aga3 gene without signal peptide sequence (1–51 bp) was amplified by PCR using Pfu DNA polymerase premix (Bioneer, South Korea), and Aga3F (5’-cgatggatccatgtgtagtgataatgaatctac-3’) and Aga3R (5’-agcactcgagatctgctttaaccggtttaa-3’) primers containing BamHI and XhoI sites (underlined), respectively. The resulting DNA fragments were digested with BamHI and XhoI restriction enzymes.¹⁸ After digestion, the DNA fragment of aga3 gene was ligated into pET21a (Invitrogen) to construct the aga3 expression plasmid.¹⁹ The recombinant plasmid was then transformed into BL21(DE3).²⁰ The transformant was inoculated in 3 mL of LB (Luria-Bertani) broth medium supplemented with ampicillin (100 μg/mL) and incubated overnight at 37 °C. Subsequently, 1% (v/v) of seed culture was transferred into 500 mL of LB broth supplemented with ampicillin (100 μg/mL). When cell density reached an OD₆₀₀ of 0.4 to 0.5 at 37 °C, IPTG (isopropyl β-D-1-thiogalactopyranoside) was added to a final concentration of 1 mM, and incubation continued for 16 h at 16 °C.²¹ After incubation, the cells were harvested by centrifugation at 13,000 × g for 15 min and washed twice with distilled water. The pellet was then resuspended in 5 mL of buffer A (10 mM NaH₂PO4, 1.8 mM KH₂PO₄, 500 mM NaCl, pH 7.4). The cell lysates were prepared by ultrasonication (90% power, pulse 30 s and 30 s cooling on ice for 5 min). The supernatant was recovered by centrifugation at 13,000 × g for 30 min and filtered using 0.45 μm syringe filter (Sartorius, Germany) to remove residual cell debris. The soluble fraction containing the 6xHis-tagged Aga3 protein was loaded onto Ni-NTA agarose (Qiagen, Germany) for affinity chromatography. The column was washed and eluted with buffer A containing 20 to 60 mM imidazole.²² Eluates were dialyzed against buffer B (50 mM Tris-Cl, pH 8.0) and concentrated with Amicon^® Ultra-30 K centrifugal filter (Merck, Millipore, Rahway, NJ, USA). The protein purity and concentration of the Aga3 recombinant protein were determined by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and bicinchoninic acid (BCA) assay (SMART^TM BCA protein assay kit, iNtRON, South Korea), respectively. The purification profile of Aga3 is summarized in Supplemental Table S1.

β-agarase activity assay

The β-agarase activity was determined by the 3,5-dinitrosalicyclic acid (DNS) method, as previously reported by Seo et al.,²³ with slight modification. Briefly, the DNS solution was prepared by mixing 6.5 g of DNS, 325 mL of 2 N NaOH, and 45 mL of glycerol in 1 L of deionized water. The purified Aga3 protein (50 μg) was added to 2 g/L low melting agarose solution and incubated at 50 °C for 15 min. After inactivating the enzyme reaction at 100 °C for 5 min, 300 μL of DNS reagent was added and heated for 5 min. The reducing sugar content in the reaction mixture was measured using UV-vis spectrophotometer at OD₅₄₀ (Biotek, USA).

Control reactions containing DNS solutions without enzyme were included to account for background absorbance. Enzyme activities (U/mL) were calculated by subtracting this background signal. D-galactose was used as the standard, and one unit (U) of Aga3 activity was defined as the amount of enzyme required to liberate 1 μmol of reducing sugar per minute under the assay conditions.²⁴

Biochemical properties of Aga3

The effect of pH on enzyme activity of Aga3 was determined in 50 mM Na₂HPO₄/citric acid buffer ranging from pH 3 to 9.^25,26 A single buffer system was used throughout the pH range to maintain consistent ionic strength and minimize potential buffer-specific effects on enzyme activity, thereby ensuring that observed differences in activity were attributable solely to pH variation.²⁷ To determine the effect of temperature, the reaction was performed in 50 mM Na₂HPO₄/citric acid buffer (pH 5) at 20 °C to 65 °C supplemented with 2 g/L low melting agarose for 15 min.

To determine the kinetic parameters of Aga3, the enzyme reactions were carried out with different concentrations of low melting agarose, ranging from 0.2 to 2 g/L (w/v). The reaction condition was 80 μg of the purified Aga3 in pH 5, 50 mM Na₂HPO₄/citric acid buffer (total volume, 500 μL) at 50 °C for 15 min. The values for kinetic parameters (K_m, V_max, k_cat) were calculated using a Lineweaver-Burk plot.²⁸

To evaluate the thermostability and thermal inactivation of Aga3, the enzyme solution was pre-incubated for 0 to 60 min at different temperatures (0 °C to 65°C). After thermal treatment, all samples were supplemented with 2 g/L agarose and equilibrated at pH 5 and 50 °C for 15 min, the optimal conditions for Aga3 activity. The data was analyzed using first-order kinetics, where $\ln (RA)$ (residual activity) was plotted against time to determine the inactivation rate constant (k_d). The enzyme half-life (t_1/2) was subsequently calculated using the equation shown below.

t_{1 / 2} = \frac{\ln 2}{k_{d}}

t_{1 / 2}

represents the time needed for the residual activity to decline to 50%, and k_d denotes the deactivation rate constant.^29,30

To examine the effect of metal cofactors on the Aga3 activity, various metals including CoCl₂, NiCl_2, CuSO₄, CaCl₂, ZnSO₄, MgCl₂, and KCl as well as the chelating reagent (EDTA) were added to each enzyme solution containing 2 g/L low melting agarose at a final concentration of 5 mM. To examine the interactions between Aga3 and its cofactors, we employed the MIB2 server (Metal Ion Binding site prediction and docking) (https://combio.life.nctu.edu.tw/MIB2/), which predicts metal-binding sites through template-based structural comparisons combined with type-specific scoring functions.³¹ The relative activity was calculated by defining the activity of the sample without addition of any metal cofactor or chelating reagent as 100%.

Analysis of enzyme reaction products

The final products hydrolyzed by the Aga3 enzyme were estimated by thin-layer chromatography (TLC).³² For this purpose, the reaction mixture containing 50 μg of Aga3 enzyme and 2 g/L low-melting agarose gel was prepared in 50 mM Na₂HPO₄/citric acid buffer and incubated at 50 °C for 15 min. After the completion of the reaction, 10 μL of sample was loaded onto a silica gel 60 F₂₅₄ plate and eluted with a solvent mixture (n-butanol: ethanol: H₂O = 3:1:1, v/v).²³ The spots were visualized with 10% sulfuric acid (v/v) in ethanol at 120 °C for 5 min. The NAOs including neoagarohexaose (NA6), neoagarotetraose (NA4), and neoagarobiose (NA2) were purchased from Biosynth, South Korea and used as standards. To examine the substrate specificity of Aga3, NA2, NA4, and NA6 were reacted with 50 μg of Aga3, respectively.

NA2 and NA4 production using Aga3 from agarose

The production of NA2 and NA4 was evaluated using 50 μg of recombinant Aga3 and different concentrations of low-melting agarose (2–40 g/L) under optimized reaction conditions for 240 min. After enzyme reaction, the resulting hydrolysates, NA2 and NA4, were quantified by high-performance liquid chromatography (HPLC). The concentration of NAOs was measured using an HPLC system equipped with a refractive index detector (RID, Agilent technologies, USA) and an Aminex HPX-89H column (300 × 7.8 mm, 9 μm, Bio-Rad Laboratories, Inc. Hercules, CA, USA) with 0.005 N H₂SO₄ as the mobile phase. The sample (5 μL) was injected and analyzed at a flow rate of 0.5 mL/min at 60 °C. The conversion rate (%) was calculated using following equation:

Conversion rate (%) = \frac{NAOs (NA 2 or NA 4) concentration (g / L)}{Initial agarose concentration (g / L)} \times 100

Results and discussion

Characterization of Aga3 from C. omnivescoria MSK1 strain

Genome-wide identification of β-agarase genes in C. omnivescoria MSK1

Whole-genome sequencing of C. omnivescoria MSK1¹⁵ (NCBI accession number: NZ_CP115819) was performed to identify genes encoding β-agarases responsible for agarose hydrolysis. Comprehensive analysis revealed that the genome possesses four putative β-agarases initially annotated as two glycosyl hydrolase family 16 (GH16)-type β-agarase (MSK_00419, MSK_00448), a GH86-type β-agarase (MSK_00434), and a T9SS domain containing β-agarase (MSK_02872). Since the endolytic GH16 β-agarases family has been well-known for producing various NAOs which can be used as high-valued compounds,³ the investigation of MSK_00448 gene, which encodes a putative GH16 β-agarase was performed. The hypothetical route of enzymatic hydrolyzation of Aga3, encoded by MSK_00448 gene, is presented in Supplemental Figure S1.

In silico characterization of the GH16 β-agarase Aga3

The MSK_00448 gene encoding the Aga3 enzyme consists of 936 bp and translates into 329 amino acids with a theoretical molecular mass of approximately 37.9 kDa. Signal peptide prediction using SignalP 6.0 (https://services.healthtech.dtu.dk/services/SignalP-6.0/)³³ identified a 17-residue signal peptide (MMKFNVAIVLLLFYVVS) at the N-terminus, which is associated with the Sec-dependent secretion pathway via signal peptidase II. The predicted cleavage site between Ser¹⁷ and Cys¹⁸ indicates that Aga3 is secreted through the Sec-mediated secretion system.

BLASTp analysis showed that Aga3 shares more than 97% sequence identity with β-agarases from Cellulophaga lytica (WP_013620028.1) and Cellulophaga geojensis KL-A (EWH13636.1). In contrast, relatively lower identity (36–47%) were observed with β-agarases from Zobellia galactanivorans (CAZ98338.1), Agarivorans albus (ABW77762.1), and Aquimarina agarivorans (WP_010523251.1).^34,35

The phylogenetic relationship of Aga3 was further analyzed through multiple sequence alignment with other β-agarases from the GH16, GH50, and GH86 families. As shown in Figure 1A, the Aga3 was closely clustered with the β-agarases of the GH 16 family. Moreover, the conserved catalytic residues (ExDxxE), a unique feature of GH16 family β-agarase, were identified in Aga3 protein (Glu¹⁷⁵, Asp¹⁷⁷, and Glu¹⁸⁰) (Figure 1B).³⁶ These findings indicated that Aga3, derived from C. omnivescoria MSK1, is a distinct extracellular β-agarase within the Cellulophaga genus and represents a novel member of the GH16 family.

Figure 1.

The profiles of amino acid sequence of Aga3 protein in Cellulophaga omnivescoria MSK1. (A) Phylogenetic analysis of Aga3 (highlighted in bold) with other β-agarases belonging to the glycoside hydrolase (GH) families 16, 50, and 86. The scale bar indicates a genetic distance of 0.20. The number shown in each node indicates the percentage bootstrap value of 1000 replicates. (B) Multiple alignment of conserved catalytic residues of Aga3 with other GH16 β-agarases. Catalytic residues were highlighted with inverted triangles. Protein and strain names were listed in labels.

SDS-PAGE analysis of recombinant Aga3

To verify the molecular mass of Aga3, the recombinant protein was heterologously expressed and purified, followed by SDS-PAGE analysis. As shown in Supplemental Figure S2, lanes 4 and 5 display a distinct band at approximately 36.5 kDa, which is consistent with the predicted molecular mass of the mature Aga3 protein (35.9 kDa) including a histidine hexamer (0.84 kDa). These results confirm the successful expression and purification of the target enzyme.

Investigation of optimal conditions for β-agarase

To evaluate the effect of pH on agarose hydrolysis, the agarose-degrading activity of Aga3 was measured across a range of pH values. As shown in Figure 2A, Aga3 retained > 6 U/mL activity from pH 4 to 9, with a maximum activity of 10.74 U/mL at pH 5. Even under strongly acidic conditions (pH 3), activity remained at 1.15 U/mL, demonstrating that Aga3 is active over a broad pH range (pH 3–9). The effect of temperature on agarose-degrading activity was also assessed. As shown in Figure 2B, Aga3 activity increased progressively with temperature from 20 °C to 50 °C, reaching a maximum of 5.97 U/mL at 50 °C. Although activity decreased to 3.32 U/mL at 65 °C, the enzyme remained active above 50 °C, indicating high thermostability.

Figure 2.

Biochemical characterization of the purified Aga3. (A) Effect of different pH on Aga3 activity. The reaction buffer used was 50 mM Na₂HPO₄/citric acid buffer (pH 3–9, closed circles). (B) Effect of temperature on Aga3 activity. (C) Linear regression analysis for determining the kinetic parameters of Aga3 acting on agarose using Lineweaver-Burk plot. All data was presented as the means and standard deviations from at least three independent experiments. V, reaction rate; S, substrate concentration.

Industrial bioprocessing of marine biomass containing agar typically involves washing, bleaching, and acid hydrolysis. Consequently, agarases with stable catalytic activity at elevated temperatures and resistance to acidic conditions are crucial for the biological treatment of such biomass.³⁷ However, most β-agarases from marine microorganisms exhibit maximum activity at neutral pH (7) or slightly alkaline pH (8–9), limiting their suitability for acidic processing environments.³⁸ Only a few agarases, such as those from Alteromonas sp. C-1 and Vibrio sp. AP-2, have been reported to retain agarolytic activity under mildly acidic conditions, and their thermostability has not been established.^39,40 In this context, the newly identified Aga3 described in this study exhibits both acid tolerance and thermostability, highlighting its strong potential for the efficient conversion of agar or agarose from pretreated marine waste into NAOs.

Kinetic properties of Aga3

The kinetic properties of Aga3 were also evaluated using a Lineweaver-Burk plot, based on the tests conducted at different concentrations of agarose substrate (0.2 g to 2 g/L (v/w)) (Figure 2C). As listed in Table 1, β-agarase Aga3 exhibited higher substrate affinity and turnover rate compared to other β-agarases. The K_m, V_max, and k_cat values of Aga3 enzyme were determined to be 0.93 mg/L, 2.1 U/mg, and 1300 s⁻¹, respectively. Notably, Aga3 showed the highest turnover number, which was at least 6.5-fold higher than that of previously reported β-agarase (Table 1).

Table 1.

Comparison of biochemical characteristics of Aga3 with other GH16 β-agarases.

Agarase	Size (kDa)	Optimal pH	Optimal temperature (°C)	Metal cofactor	K_m(mg mL⁻¹)	V_max(U mg⁻¹)	K_cat(S⁻¹)	Major product	Reference
Gaa16B	65	6–7	55	Mn²⁺, Ca²⁺, Mg²⁺	6.4	953	201.2	NA4	41
Aga2	37.8	8	45	Zn²⁺	2.59	275.48	173	NA4, NA6	42
rmAga0917	32.5	6	60	Co²⁺	39.6	334	178	NA2, NA4	43
Aga3	37.9	5	50	Co²⁺, Mg²⁺	0.93	2.1	1300	NA2, NA4	This study

Analysis of thermostable properties of Aga3

In general, various industrial processes for the utilization of algal biomass were conducted under the harsh conditions such as fluctuating temperatures and different pH values. Thermostable agarases are advantageous in these environments, as they retain their activity and stability under stress, making them suitable for use in continuous bioreactor systems and high-temperature pretreatment steps. Protein thermostability is known to be associated with amino acid sequences and structures. Two predictive models, the Instability Index (II, < 40 of values) and the Aliphatic Index (AI, ≈ 100 of values), are widely used to determine whether a protein is stable or unstable.^44,45 From these index models, the II and AI values of Aga3 amino acid sequence were predicted to be 35.9_II and 66.6_AI, respectively. These findings suggest that the Aga3 may possess relatively greater thermal stability than other reported β-agarases such as AgaP (38.7_II, 64.1_AI) from Pseudoalteromonas sp. AG4⁴⁵ and AgaB (44.3_II, 66.1_AI) from Zobellia galactanivorans.³⁴ To exam if the Aga3 was indeed thermostable, purified Aga3 was pre-incubated for 0 to 60 min at temperatures ranging from 20 °C to 65 °C, followed by the measurement of residual agarase activity using 2 g/L agarose at its optimal temperature and pH. As shown in Figure 3A, Aga3 maintained more than 10 U/mL of activity across this temperature range, whereas a decline was observed at temperatures exceeding 50 °C. Likewise, Aga3 preserved over 80% of its initial activity after pre-incubation for 0 to 60 min at 20 to 50 °C, relative to the control without pre-incubation (Figure 3B). The calculated half-lives at 20 °C, 30 °C, 37 °C, 50 °C, and 65 °C were 288.81, 128.36, 210.04, 182.41, and 18.84 min, respectively (Figure 3C). Notably, at 50 °C, Aga3 exhibited a half-life of 182.41 min (3.04 h), representing a 6.4-fold improvement in thermal stability compared with Pseudoalteromonas fuliginea Aga0917 ( $t_{1 / 2}$ = 28.4 min)¹⁰ and 28-fold more stable than Vibrio fluvialis A8 (gene3152) ( $t_{1 / 2}$ = 6.3 min).²⁹ These results indicate that Aga3 is thermostable and suitable for industrial agarose hydrolysis, as it remains active across a temperature range that includes the typical gel-sol transition of agarose (32 °C–47 °C).⁴⁵

Figure 3.

Thermostability of Aga3 at different pre-incubation temperatures and time. (A) Thermostability of Aga3 at different pre-incubation temperatures (0 °C, 10 °C, 20 °C, 30 °C, 37 °C, 50 °C, and 65 °C). The thermostability of Aga3 was determined by pre-incubating the Aga3 enzyme at the indicated temperature for 1 hour. The enzyme reaction was performed with 2 g/L agarose (w/v) at pH 5 and 50 °C for 15 min. (B) Thermostability of Aga3 at different pre-incubation temperatures and time (0–60 min). Residual activity was evaluated by comparison with non pre-incubated Aga3. (C) Kinetic parameters of thermal inactivation of Aga3 at different temperatures and times. Inactivation rate constant (k_d) and half-life ( $t_{1 / 2}$ ) were shown.

Co²⁺ as an enhancer for Aga3 activity

There had been a lot of previous reports investigating the influence of metal ions to agarase activity. Previous studies on GH16 β-agarases suggest that divalent metal ions such as Ca²⁺ and Mg²⁺ may enhance activity, while transition metals like Cu²⁺ and Zn²⁺ can be inhibitory.⁴¹ To assess the positive effects of metal ions on agarase activity, Aga3 was evaluated under optimal reaction conditions supplemented with various metal ions. As shown in Figure 4A, the agarase activity was significantly enhanced by 5 mM Co²⁺ and Mg²⁺, increasing by approximately 604% and 159.8%, respectively, compared to the control without metal ions and chelating agents, while most other metal ions exhibited inhibitory effects. Furthermore, Co²⁺ showed a dose-dependent increase in agarase activity up to 5 mM, followed by a gradual decline at higher concentrations (Figure 4B).

Figure 4.

Investigation of metal cofactors to enhance Aga3 activity. (A) Effects of metal ions and chelating agent on Aga3 activity. (B) The profiles of Aga3 activity at different concentrations of cobalt ion. The Aga3 activity in the absence of metal ion was set as 100%. All data were presented as the mean and standard deviation from three independent replicates.

The effect of Co²⁺ is likely due to its interaction with potential cobalt-binding residues in Aga3, as predicted by MIB2. Glu¹⁷⁵ and Asp¹⁷⁷, the conserved residues in the GH16 β-agarase active site motif (ExDxxE), were identified as primary cobalt-docking sites, exhibiting a high MIB2 docking score (3.129), indicating strong binding potential. Additionally Co²⁺ is particularly effective probably because of its optimal ionic radius (0.74 Å)⁴⁶ and intermediate Lewis acid strength (borderline acid in the HSAB concept),⁴⁷ which allow stable coordination with Asp/Glu side chains.⁴⁸ These interactions likely involve both electrostatic attraction and maintenance of the preferred octahedral geometry, stabilizing the active site in a conformation favorable for catalysis.

This result somewhat contrasts with previous reports of marine- and terrestrial-derived β-agarases in which Ca²⁺ was identified as the most effective metal ion for GH16 β-agarases. In conclusion, Aga3 from the MSK1 strain exhibited more than a six-fold increase in activity in the presence of Co²⁺, representing the highest agarase activity reported among β-agarases to date.

Analysis of NAOs produced by Aga3

In terms of cleavage type, β-agarases are classified into endo-type or exo-type based on how they break down agarose into oligosaccharides. To determine whether Aga3 functions as an endolytic or exolytic β-agarase and to identify its hydrolysis products, purified Aga3 was incubated with agarose under optimal conditions, followed by product analysis using TLC. As shown in Figure 5A, the major hydrolysis products were NA2, NA4, and NA6. Within 15 min of reaction initiation, agarose was predominantly cleaved into NA2, NA4, and NA6 (Figure 5A, lane 1). As the reaction progressed, the proportion of higher molecular weight oligosaccharides, including NA6, gradually decreased, while the levels of NA2 and NA4 increased. Notably, Aga3 specifically and efficiently hydrolyzed NA6 into NA4 and NA2 (Figure 5B, lane 3) but was unable to further degrade NA4 or NA2 (Figure 5B, lanes 1 and 2). These results indicate that Aga3 acts as an endolytic β-agarase, cleaving internal β-1,4-glycosidic bonds and primarily releasing NA4 and NA2 as final products. These results are consistent with the endolytic activity characteristic of GH16 family β-agarases. However, most reported GH16 endo-type β-agarases typically produce NA4 and NA6, or primarily NA6, as the smallest major products.¹¹ In contrast, only a few studies have reported NA2 or a combination of NA2 and NA4 as the predominant end products from agar or agarose.^36,48 Notably, NA2 is generally produced by exo-type β-agarases belonging to the GH50 and GH86 families.⁴⁹ In this regard, Aga3 differs from typical GH16 β-agarases by predominantly generating NA2 and NA4, suggesting that it represents a novel variant within the GH16 family.

Figure 5.

Analysis of the hydrolyzed products of the Aga3 enzyme. Lanes: M1, neoagarobiose standard (NA2); M2, neoagarotetraose standard (NA4); M3, neoagarohexaose standard (NA6). (A) TLC analysis of hydrolyzed products of Aga3 when incubated with 2 g/L agarose. Lanes 1 and 2 indicate the hydrolyzed products that were incubated for 15 and 240 min, respectively. (B) TLC analysis of the Aga3 activity on NA2, NA4, and NA6 as the specific substrates. Aga3 (50 μg) was incubated with 1 g/L of NA2 (lane 1), NA4 (lane 2), or NA6 (lane 3) in 50 mM Na₂HPO₄/citric acid buffer (pH 5) at 50 °C for 15 min. (C) The effect of agarose concentrations on the production of NA2 and NA4 by the purified Aga3 enzyme. (D) Conversion rates of NA2 and NA4 by the purified Aga3 enzyme at different initial concentrations of agarose. All data were presented as the mean and standard deviation from three independent replicates. TLC: thin-layer chromatography.

Production of NA2 and NA4 from agarose

Finally, to evaluate the agarose-degrading performance of Aga3, NA2, and NA4 production was quantified at varying agarose concentrations. As shown in Figure 5C, NA2 and NA4 yields increased markedly in an agarose concentration-dependent manner, reaching 6.61 ± 0.55 g/L and 10.4 ± 1.30 g/L, respectively, from 40 g/L agarose after a 240 min reaction. In contrast, the overall conversion rate declined gradually with increasing agarose concentrations but remained at approximately 45% for concentrations above 20 g/L (Figure 5D). The highest conversion rate, 88.8%, was observed at 2 g/L agarose.

Previous studies have reported biological production of NAOs, particularly NA2 and NA4, using β-agarases. For instance, the recombinant β-agarase AgaG1 from Alteromonas sp. GNUM1 produced 3.8 g/L NA2 and 6.4 g/L NA4 from 10 g/L agarose with a 98% conversion rate.⁵⁰ However, its optimal activity at 40 °C and pH 7 limits industrial applicability. Likewise, the exolytic GH50 β-agarase from Cellvibrio sp. KY-GH-1 achieved a 54% conversion rate, producing 216 mg NA2 from 4 g/L agarose, but did not generate NA4.¹² In contrast, Aga3 can simultaneously produce NA2 and NA4 at high yields, while maintaining activity under acidic conditions and elevated temperatures. These features highlight its potential as an eco-friendly and cost-effective biocatalyst for the valorization of marine algal biomass.

Conclusion

In this study, a novel GH16 β-agarase, Aga3, was isolated and characterized from the agarose-degrading marine bacterium C. omnivescoria MSK1. Aga3 exhibited superior catalytic turnover rate (1300 s⁻¹) and strong substrate affinity (0.93 mg/mL) compared with previously reported GH16 β-agarases, enabling efficient production of NA2 (6.61 ± 0.55 g/L) and NA4 (10.4 ± 1.30 g/L) with a maximum conversion rate of 88.8% at 2 g/L agarose. Enzyme activity was enhanced more than six-fold in the presence of cobalt ions. In addition, Aga3 retained optimal activity at 50 °C above the sol-gel transition temperature of agarose, a key factor in marine biomass processing and under mildly acidic conditions (pH 5). These properties demonstrate Aga3 as a promising eco-friendly biocatalyst for the sustainable and high-yield production of NA2 and NA4 to meet growing industrial demands.

Supplemental Material

sj-docx-1-eae-10.1177_0958305X261421369 - Supplemental material for High-efficiency and thermostable β-agarase Aga3 for sustainable production of neoagarooligosaccharides (NAOs) from algal biomass

Supplemental material, sj-docx-1-eae-10.1177_0958305X261421369 for High-efficiency and thermostable β-agarase Aga3 for sustainable production of neoagarooligosaccharides (NAOs) from algal biomass by Yerin Kim, Sun-Wook Jeong, Ji Hyeon Lee and Yong Jun Choi in Energy & Environment

Footnotes

ORCID iDs

Ji Hyeon Lee

Yong Jun Choi

Author contributions

Yerin Kim: Conceptualization, Data curation, Formal analysis, Investigation, Visualization, Methodology, Writing-original draft, Writing-reviewing & editing

Sun-Wook Jeong: Conceptualization, Investigation, Methodology

Ji-Hyeon Lee: Formal analysis, Visualization

Yong Jun Choi: Supervision, Validation, Writing-review & editing

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) (NRF-2023R1A2C1006302) and (RS-2024-00440478).

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data availability

Data will be made available on request.

Supplemental material

Supplemental material for this article is available online.

Yerin Kim is a student in master's course in School of Environmental engineering, University of Seoul. Her research field focuses on bioconversion and metabolic engineering.

Sun-Wook Jeong is presently working as a research professor in School of Environmental engineering, University of Seoul. In 2016, He received his PhD degree in Molecular & cellular biology from College of Bioscience & Biotechnology, Chungnam National University. His research interests include environmental microbiology, microbial production of high-valued metabolites, and bioremediation & management.

Ji Hyeon lee is a student in master's course in School of Environmental engineering, University of Seoul. Her research focuses on bioconversion and microbial production of high-valued metabolites.

Yong Jun Choi is presently working as professor at the School of Environmental engineering, University of Seoul. His research areas of interest are metabolic engineering, bioremediation using extremophilic microorganisms, microbial fermentation and optimization of cultivation process. He has about eleven years of teaching experience for environmental ecology and environmental microbiology.

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High-efficiency and thermostable β-agarase Aga3 for sustainable production of neoagarooligosaccharides (NAOs) from algal biomass

Abstract

Keywords

Introduction

Materials and methods

Whole genome sequencing of C. omnivescoria MSK1

Amino acid sequence analysis

Cloning, expression, and purification of the recombinant Aga3 protein

β-agarase activity assay

Biochemical properties of Aga3

Analysis of enzyme reaction products

NA2 and NA4 production using Aga3 from agarose

Results and discussion

Characterization of Aga3 from C. omnivescoria MSK1 strain

Genome-wide identification of β-agarase genes in C. omnivescoria MSK1

In silico characterization of the GH16 β-agarase Aga3

SDS-PAGE analysis of recombinant Aga3

Investigation of optimal conditions for β-agarase

Kinetic properties of Aga3

Analysis of thermostable properties of Aga3

Co2+ as an enhancer for Aga3 activity

Analysis of NAOs produced by Aga3

Production of NA2 and NA4 from agarose

Conclusion

Supplemental Material

sj-docx-1-eae-10.1177_0958305X261421369 - Supplemental material for High-efficiency and thermostable β-agarase Aga3 for sustainable production of neoagarooligosaccharides (NAOs) from algal biomass

Footnotes

ORCID iDs

Author contributions

Funding

Declaration of conflicting interests

Data availability

Supplemental material

References

Co²⁺ as an enhancer for Aga3 activity