Agar Degradation Research Articles

Improving Agar Degradation Activity of Vibrio natriegens WPAGA4 via Atmospheric and Room Temperature Plasma (ARTP)

Agar oligosaccharides from the degradation of agar harbor great potential in the food and pharmaceutical industries. An agar-degrading bacterium, Vibrio natriegens WPAGA4, was isolated from the deep sea in our previous work. However, the agar-degrading activity of WPAGA4 remains to be improved for more production benefits of this strain. The aim of this study was to enhance the agar-degrading activity of WPAGA4 by using atmospheric and room temperature plasma (ARTP) mutagenesis. Three mutant strains, including T1, T2, and T3, with good genetic stability were obtained, and the agar-degrading activities of these strains increased by 136%, 141%, and 135%, respectively. The optimal temperature and pH for agar degradation were slightly changed in the mutant strains. No sequence mutation was detected in all the agarase genes of WPAGA4, including agaW3418, agaW3419, agaW3420, and agaW3472. However, ARPT mutagenesis increased the relative expression levels of agaW3418, agaW3419, and agaW3420 in the mutant strains, which could be the reason for the improvement of degradation activities in the mutant strains. Furthermore, T3 had the lowest consumption rate of agar oligosaccharide, which was 21% less than the wild-type strain. Therefore, T3 possessed a preferable production value due to its higher degrading activity and lower consumption of agar oligosaccharides. The current work enhanced the agar-degrading activity of WPAGA4 and offered strains with greater potential for agar oligosaccharide production, thereby laying the foundation for industrial applications.

Complete genome sequence of a species of the genus Agarivorans from the Sea of Japan.

A strain of Agarivorans sp., named OAG1, predicted to be capable of degrading agar, was isolated from the Sea of Japan. Its 4.7 Mbp genome with 4,224 predicted protein-coding genes offers insight into the varied genes responsible for agar degradation, as observed in the comparative genomic study of Agarivorans species.

Diversity of Culturable Bacteria from the Coral Reef Areas in the South China Sea and Their Agar-Degrading Abilities.

The South China Sea (SCS) is abundant in marine microbial resources with high primary productivity, which is crucial for sustaining the coral reef ecosystem and the carbon cycle. Currently, research on the diversity of culturable bacteria in the SCS is relatively extensive, yet the culturable bacteria in coral reefs has been poorly understood. In this study, we analyzed the bacterial community structure of seawater samples among Daya Bay (Fujian Province), Qionghai (Hainan Province), Xisha Islands, and the southern South China Sea based on culturable methods and detected their abilities for agar degradation. There were 441 bacterial strains, belonging to three phyla, five classes, 43 genera, and 101 species, which were isolated by marine agar 2216E (MA; Becton Dickinson). Strains within Gammaproteobacteria were the dominant group, accounting for 89.6% of the total bacterial isolates. To investigate vibrios, which usually correlated with coral health, 348 isolates were obtained from TCBS agar, and all isolates were identified into three phylum, three classes, 14 orders, 25 families, and 48 genera. Strains belonging to the genus Vibrio had the greatest number (294 strains), indicating the high selectivity of TCBS agar for vibrios. Furthermore, nineteen strains were identified as potentially novel species according to the low 16S rRNA gene similarity (<98.65%), and 28 strains (15 species) had agar-degrading ability. These results indicate a high diversity of culturable bacteria in the SCS and a huge possibility to find novel and agar-degrading species. Our study provides valuable microbial resources to maintain the stability of coral ecosystems and investigate their roles in the marine carbon cycle.

Catalytic hydrolysis of agar using magnetic nanoparticles: optimization and characterization

BackgroundAgar is used as a gelling agent that possesses a variety of biological properties; it consists of the polysaccharides agarose and porphyrin. In addition, the monomeric sugars generated after agar hydrolysis can be functionalized for use in biorefineries and biofuel production. The main objective of this study was to develop a sustainable agar hydrolysis process for bioethanol production using nanotechnology. Peroxidase-mimicking Fe3O4-MNPs were applied for agar degradation to generate agar hydrolysate-soluble fractions amenable to Saccharomyces cerevisiae and Escherichia coli during fermentation.ResultsFe3O4-MNP-treated (Fe3O4-MNPs, 1 g/L) agar exhibited 0.903 g/L of reducing sugar, which was 21-fold higher than that of the control (without Fe3O4-MNP-treated). Approximately 0.0181% and 0.0042% of ethanol from 1% of agar was achieved using Saccharomyces cerevisiae and Escherichia coli, respectively, after process optimization. Furthermore, different analytical techniques (FTIR, SEM, TEM, EDS, XRD, and TGA) were applied to validate the efficiency of Fe3O4-MNPs in agar degradation.ConclusionsTo the best of our knowledge, Fe3O4-MNP-treated agar degradation for bioethanol production through process optimization is a simpler, easier, and novel method for commercialization.

Complete genome of Rossellomorea sp. DA94, an agarolytic and orange-pigmented bacterium isolated from mangrove sediment of the South China Sea.

Rossellomorea sp. DA94, isolated from mangrove sediment in the South China Sea (Beihai, Guangxi province), is an agarolytic and orange-pigmented bacterium. Here, we present the complete genome sequence of strain DA94, which comprises 4.63Mb sequences with 43.5% GC content. In total, 4589 CDSs, 33 rRNA genes and 110 tRNA genes were obtained. Genomic analysis of strain DA94 revealed that 108 CAZymes were organized in 4578 PULs involved in polysaccharides degradation, transport, and regulation. Further, we performed the diversity of CAZymes and PULs comparison among Rossellomorea strains. Less CAZymes were organized more PULs, indicating highly efficiently polysaccharides utilization in Rossellomorea. Meanwhile, PUL0459, PUL0460 and PUL0316 related to agar degradation, and exolytic beta-agarase GH50, endo-type beta-agarase GH86 and arylsulfatase were identified in the genome of strain DA94. We verified that strain DA94 can degrade agar to form a bright clear zone around the bacterial colonies in the laboratory. Moreover, the carotenoid biosynthetic pathways were proposed, which may be responsible for orange-pigment of Rossellomorea sp. DA94. This study represents a thorough genomic characterization of CAZymes repertoire and carotenoid biosynthetic pathways of Rossellomorea, provides insight into diversity of related enzymes and their potential biotechnological applications.

Agarolytic Pathway in the Newly Isolated Aquimarina sp. Bacterial Strain ERC-38 and Characterization of a Putative β-agarase

Marine microbes, particularly Bacteroidetes, are a rich source of enzymes that can degrade diverse marine polysaccharides. Aquimarina sp. ERC-38, which belongs to the Bacteroidetes phylum, was isolated from seawater in South Korea. It showed agar-degrading activity and required an additional carbon source for growth on marine broth 2216. Here, the genome of the strain was sequenced to understand its agar degradation mechanism, and 3615 protein-coding sequences were predicted, which were assigned putative functions according to their annotated functional feature categories. In silico genome analysis revealed that the ERC-38 strain has several carrageenan-degrading enzymes but could not degrade carrageenan because it lacked genes encoding κ-carrageenanase and S1_19A type sulfatase. Moreover, the strain possesses multiple genes predicted to encode enzymes involved in agarose degradation, which are located in a polysaccharide utilization locus. Among the enzymes, Aq1840, which is closest to ZgAgaC within the glycoside hydrolase 16 family, was characterized using a recombinant enzyme expressed in Escherichia coli BL21 (DE3) cells. An enzyme assay revealed that recombinant Aq1840 mainly converts agarose to NA4. Moreover, recombinant Aq1840 could weakly hydrolyze A5 into A3 and NA2. These results showed that Aq1840 is involved in at least the initial agar degradation step prior to the metabolic pathway that uses agarose as a carbon source for growth of the strain. Thus, this enzyme can be applied to development and manufacturing industry for prebiotic and antioxidant food additive. Furthermore, our genome sequence analysis revealed that the strain is a potential resource for research on marine polysaccharide degradation mechanisms and carbon cycling.

Whole-Genome Sequencing of Pseudoalteromonas sp. Strain KAN5, an Agar-Degrading Bacterium Isolated from the Gut of an Alga-Eating Fish.

ABSTRACTA strain of Pseudoalteromonas that degrades agar was isolated from the intestines of an alga-eating fish (Andamia tetradactyla). We named the strain KAN5 and report on the genome sequenced with the Oxford Nanopore Technologies platform. The 3.8-Mbp genome contains 3,428 protein-coding genes, and the genes involved in agar degradation were confirmed.

Molecular Characterization of a Novel 1,3-α-3,6-Anhydro-L-Galactosidase, Ahg943, with Cold- and High-Salt-Tolerance from Gayadomonas joobiniege G7.

1,3-α-3,6-anhydro-L-galactosidase (α-neoagarooligosaccharide hydrolase) catalyzes the last step of agar degradation by hydrolyzing neoagarobiose into monomers, D-galactose, and 3,6-anhydro-Lgalactose, which is important for the bioindustrial application of algal biomass. Ahg943, from the agarolytic marine bacterium Gayadomonas joobiniege G7, is composed of 423 amino acids (47.96 kDa), including a 22-amino acid signal peptide. It was found to have 67% identity with the α-neoagarooligosaccharide hydrolase ZgAhgA, from Zobellia galactanivorans, but low identity (< 40%) with the other α-neoagarooligosaccharide hydrolases reported. The recombinant Ahg943 (rAhg943, 47.89 kDa), purified from Escherichia coli, was estimated to be a monomer upon gel filtration chromatography, making it quite distinct from other α-neoagarooligosaccharide hydrolases. The rAhg943 hydrolyzed neoagarobiose, neoagarotetraose, and neoagarohexaose into D-galactose, neoagarotriose, and neoagaropentaose, respectively, with a common product, 3,6- anhydro-L-galactose, indicating that it is an exo-acting α-neoagarooligosaccharide hydrolase that releases 3,6-anhydro-L-galactose by hydrolyzing α-1,3 glycosidic bonds from the nonreducing ends of neoagarooligosaccharides. The optimum pH and temperature of Ahg943 activity were 6.0 and 20°C, respectively. In particular, rAhg943 could maintain enzyme activity at 10°C (71% of the maximum). Complete inhibition of rAhg943 activity by 0.5 mM EDTA was restored and even, remarkably, enhanced by Ca2+ ions. rAhg943 activity was at maximum at 0.5 M NaCl and maintained above 73% of the maximum at 3M NaCl. Km and Vmax of rAhg943 toward neoagarobiose were 9.7 mg/ml and 250 μM/min (3 U/mg), respectively. Therefore, Ahg943 is a unique α-neoagarooligosaccharide hydrolase that has cold- and high-salt-adapted features, and possibly exists as a monomer.

Comparative Genomic and Secretomic Analysis Provide Insights Into Unique Agar Degradation Function of Marine Bacterium Vibrio fluvialis A8 Through Horizontal Gene Transfer

Agarose-oligosaccharide production from agar degradation by agarase exhibits lots of advantages and good application prospects. In this study, a novel agar-degrading bacterium Vibrio sp. A8 was isolated from a red algae in the South China Sea. The whole genome sequencing with comparative genomic and secretomic analysis were used to better understand its genetic components about agar degradation. This strain exhibited good agarase production in artificial seawater after culture optimization. The complete genome (4.88 Mb) of this strain comprised two circular chromosomes (3.19 and 1.69 Mb) containing 4,572 protein-coding genes, 108 tRNA genes and 31 rRNA genes. This strain was identified as Vibrio fluvialis A8 by comparative genomic analysis based on genome phylogenetic tree and average nucleotide identity (ANI) similarity. Different from other 20 similar strains including three strains of the same species, V. fluvialis A8 possessed unique agar degradation ability with four β-agarases (GH50) and one α-1,3-L-NA2 hydrolase (GH117) due to the horizontal gene transfer. Secretomic analysis showed that only β-agarase (gene 3152) was abundantly expressed in the secretome of V. fluvialis A8. This agarase had a good substrate specificity and wide work conditions in complex environments, suggesting its potential application for agarose-oligosaccharide production.

Asp271 is critical for substrate interaction with the surface binding site in β-agarase a from Zobellia galactanivorans.

In the marine environment agar degradation is assured by bacteria that contain large agarolytic systems with enzymes acting in various endo- and exo-modes. Agarase A (AgaA) is an endo-glycoside hydrolase of family 16 considered to initiate degradation of agarose. Agaro-oligosaccharide binding at a unique surface binding site (SBS) in AgaA from Zobellia galactanivorans was investigated by computational methods in conjunction with a structure/sequence guided approach of site-directed mutagenesis probed by surface plasmon resonance binding analysis of agaro-oligosaccharides of DP 4-10. The crystal structure has shown that agaro-octaose interacts via H-bonds and aromatic stacking along 7 subsites (L through R) of the SBS in the inactive catalytic nucleophile mutant AgaA-E147S. D271 is centrally located in the extended SBS where it forms H-bonds to galactose and 3,6-anhydrogalactose residues of agaro-octaose at subsites O and P. We propose D271 is a key residue in ligand binding to the SBS. Thus AgaA-E147S/D271A gave slightly decreasing KD values from 625 ± 118 to 468 ± 13 μM for agaro-hexaose, -octaose, and -decaose, which represent 3- to 4-fold reduced affinity compared with AgaA-E147S. Molecular dynamics simulations and interaction analyses of AgaA-E147S/D271A indicated disruption of an extended H-bond network supporting that D271 is critical for the functional SBS. Notably, neither AgaA-E147S/W87A nor AgaA-E147S/W277A, designed to eliminate stacking with galactose residues at subsites O and Q, respectively, were produced in soluble form. W87 and W277 may thus control correct folding and structural integrity of AgaA.

Heterologous expression of an agarase gene in Bacillus subtilis, and characterization of the agarase

A β-agarase was identified from Pseudoalteromonas sp. Q30F and heterologously expressed in Bacillus subtilis WB800n. The β-agarase, Aga862 encoded by aga862 gene in an open reading frame of 1338 bp is 445 amino acids in length, and has a calculated molecular mass of 50.1 kDa and an estimated isoelectric point of 4.81. Protein sequence analysis showed that Aga862 belongs to family 16 of glycoside hydrolases (GH16) and carbohydrate-binding module family 13 (CBM13). The agarase was expressed in B. subtilis WB800n and purified by precipitation, anion exchange and gel filtration for a specific activity of 4.6 U/mg, a 27.8-fold improvement over the activity of the crude enzyme. Aga862 exhibited optimal activity at 45 °C and pH 6.5, and showed excellent pH stability with retention of over 80% relative activities after preincubation for the pH range of 3.0–10.0 at 4 °C for 3 h. The agarase exhibited a Km value of 14.15 mg/mL toward agarose and a Vmax of 256.41 U/mg. The mass spectrometry analysis revealed that the end products of agar degradation were neoagarotetraose and neoagarohexaose. Recombinant Aga862 has great potential for the manufacture of agaro-oligosaccharides for the non-pathogenic nature and safety of the B. subtilis WB800n.

A novel agaro-oligosaccharide-lytic β-galactosidase from Agarivorans gilvus WH0801.

β-Galactosidases have a wide application in the food and pharmaceutical industries. Recently, β-galactosidase was also found to participate in agar degradation. In this study, the second reported agarolytic β-galactosidase was found in the marine bacterium Agarivorans gilvus WH0801 and characterized. The β-galactosidase named AgWH2A (83kDa) exhibited good activities under optimal hydrolysis conditions of pH8.0 and 40°C. AgWH2A could cleave the first D-galactose of agarooligosaccharides from its nonreducing end to produce neoagarooligosaccharides, but could not act on the neoagarooligosaccharides. AgWH2A has great potential in the comprehensive utilization of marine red algae.

Molecular cloning, expression, and functional characterization of the \u03b2-agarase AgaB-4 from Paenibacillus agarexedens

In this study, a β-agarase gene, agaB-4, was isolated for the first time from the agar-degrading bacterium Paenibacillus agarexedens BCRC 17346 by using next-generation sequencing. agaB-4 consists of 2652 bp and encodes an 883-amino acid protein with an 18-amino acid signal peptide. agaB-4 without the signal peptide DNA was cloned and expressed in Escherichia coli BL21(DE3). His-tagged recombinant AgaB-4 (rAgaB-4) was purified from the soluble fraction of E. coli cell lysate through immobilized metal ion affinity chromatography. The optimal temperature and pH of rAgaB-4 were 55 °C and 6.0, respectively. The results of a substrate specificity test showed that rAgaB-4 could degrade agar, high-melting point agarose, and low-melting point agarose. The Vmax and Km of rAgaB-4 for low-melting point agarose were 183.45 U/mg and 3.60 mg/mL versus 874.61 U/mg and 9.29 mg/mL for high-melting point agarose, respectively. The main products of agar and agarose hydrolysis by rAgaB-4 were confirmed to be neoagarotetraose. Purified rAgaB-4 can be used in the recovery of DNA from agarose gels and has potential application in agar degradation for the production of neoagarotetraose.

Integration of Bacterial Expansin on Agarolytic Complexes to Enhance the Degrading Activity of Red Algae by Control of Gelling Properties.

Expansin act by loosening hydrogen bonds in densely packed polysaccharides. This work characterizes the biological functions of expansin in the gelling and degradation of algal polysaccharides. In this study, the bacterial expansin BpEX from Bacillus pumilus was fused with the dockerin module of a cellulosome system for assembly with agarolytic complexes. The assembly of chimeric expansin caused an indicative enhancement in agarase activity. The enzymatic activities on agar substrate and natural biomass were 3.7-fold and 3.3-fold higher respectively than that of agarase as a single enzyme. To validate the effect on the agar degradation, the regulation potential of parameters related to gel rheology by bacterial expansin was experimentally investigated to indicate that the bacterial expansin lowered the gelling temperature and viscosity of agar. Thus, these results demonstrated the possibility of advancing more efficient strategies for utilizing agar as oligo sugar source in the biorefinery field that uses marine biomass as feedstocks.

Comparative genomic analysis of Paenibacillus sp. SSG-1 and its closely related strains reveals the effect of glycometabolism on environmental adaptation

The extensive environmental adaptability of the genus Paenibacillus is related to the enormous diversity of its gene repertoires. Paenibacillus sp. SSG-1 has previously been reported, and its agar-degradation trait has attracted our attention. Here, the genome sequence of Paenibacillus sp. SSG-1, together with 76 previously sequenced strains, was comparatively studied. The results show that the pan-genome of Paenibacillus is open and indicate that the current taxonomy of this genus is incorrect. The incessant flux of gene repertoires resulting from the processes of gain and loss largely contributed to the difference in genomic content and genome size in Paenibacillus. Furthermore, a large number of genes gained are associated with carbohydrate transport and metabolism. It indicates that the evolution of glycometabolism is a key factor for the environmental adaptability of Paenibacillus species. Interestingly, through horizontal gene transfer, Paenibacillus sp. SSG-1 acquired an approximately 150 kb DNA fragment and shows an agar-degrading characteristic distinct from most other non-marine bacteria. This region may be transported in bacteria as a complete unit responsible for agar degradation. Taken together, these results provide insights into the evolutionary pattern of Paenibacillus and have implications for studies on the taxonomy and functional genomics of this genus.

English

Xylanases have largely been obtained from filamentous fungi and bacteria; few studies have investigated the production of this enzyme by yeasts. The aim of this study was to isolate yeasts from different sources, such as vegetables, cereal grains, fruits, and agro-industrial waste and to obtain yeasts capable of producing celulase-free xylanase. Samples were enriched using yeast malt broth, and yeasts were isolated on Wallerstein nutrient agar. In all, 119 yeast strains were isolated and evaluated in terms of their ability to degrade xylan, which was found in the medium by using agar degradation halos, the basis of this polysaccharide, and Congo red dye. Selected microorganisms were grown in complex medium and the enzymatic activities of endo-xylanase, &beta;-xylosidase, carboxymetilcellulase, and filter paper cellulose were determined over 96 h of cultivation; the pH and biomass concentration were also evaluated. The yeast strain 18Y, which was isolated from chicory and later identified as Cryptococcus laurentii, showed the highest endo-xylanase activity (2.7 U.mL-1). This strain had the ability to produce xylanase with low levels of cellulase production (both CMCase [0.11 U.mL-1] and FPase [0.10 U.mL-1]). This result gives this strain great biotechnological potential since this enzyme can be used for industrial pulp and paper bleaching. &nbsp; Key words: Cryptococcus laurentii, endo-xylanase, xylan.&nbsp

Expression and characterization of a thermostable and pH-stable β-agarase encoded by a new gene from Flammeovirga pacifica WPAGA1

A novel β-agarase gene aga4383 was cloned from Flammeovirga pacifica WPAGA1. The gene consists of 2898 bp and encodes a protein, designated as AgaP4383, with 965 amino acids. The DNA sequence of aga4383 has no significant sequence similarity with any known proteins, including all glycoside hydrolases. AgaP4383 shares a highest amino acid sequence homology of 41% with a putative β-agarase from Agarivorans albus. Phylogenetic analysis showed that AgaP4383 belongs to family 86 of glycoside hydrolases (GH86). The agarase gene was expressed in Escherichia coli and purified by affinity chromatography. The purified AgaP4383 showed endolytic activity on agar degradation, yielding neoagarotetraose and neoagarohexaose as the end products. The Km values for agar and Gracilaria lemaneiformis were 8.53 and 32.41mgmL−1. The optimal temperature and pH for the recombinant AgaP4383 were 50°C and pH 9.0, respectively. Notably, the enzyme exhibited good thermostability. No activity loss was observed after incubation at 50°C for 10h. The recombinant AgaP4383 showed a wide range of pH stability, retaining 90% of activity at pH 5.0–10.0 for 24h at 30°C. These beneficial characteristics of the enzyme provide some advantages for potential application in industry.

Purification and characterization of a novel β-agarase of Paenibacillus sp. SSG-1 isolated from soil

Agar is a polysaccharide polymer material, generally extracted from seaweed. Most agar degradation strains were isolated from seawater. In order to find new species resources and novel agarase from soil, an agar-degrading bacterium Paenibacillus sp. SSG-1 was isolated from soil. Agarase SSG-1a was purified to homogeneity by 30.2 fold with a yield of 4.8% through ammonium sulfate precipitation, DEAE FF chromatography and native-PAGE separation. The tandem mass spectrometry (MS/MS) results indicated that purified SSG-1a should be a novel β-agarase. The molecular mass of SSG-1a was estimated to be 77 kDa. The optimal temperature and pH for SSG-1a were 50°C and pH 6.0, respectively. Moreover, SSG-1a was stable in pH range of 4.0-10.0 and at temperature up to 40°C. It could hydrolyze the β-1,4 linkage of agarose to produce neoagarohexaose (95 mol%) and neoagarooctaose (5 mol%). Metal ion Mn(2+) and reducing reagents (β-Me and DTT) could increase its activity by 150% and 60%, respectively.

An Extra Peptide within the Catalytic Module of a β-Agarase Affects the Agarose Degradation Pattern

Agarase hydrolyzes agarose into a series of oligosaccharides with repeating disaccharide units. The glycoside hydrolase (GH) module of agarase is known to be responsible for its catalytic activity. However, variations in the composition of the GH module and its effects on enzymatic functions have been minimally elucidated. The agaG4 gene, cloned from the genome of the agarolytic Flammeovirga strain MY04, encodes a 503-amino acid protein, AgaG4. Compared with elucidated agarases, AgaG4 contains an extra peptide (Asn(246)-Gly(302)) within its GH module. Heterologously expressed AgaG4 (recombinant AgaG4; rAgaG4) was determined to be an endo-type β-agarase. The protein degraded agarose into neoagarotetraose and neoagarohexaose at a final molar ratio of 1.5:1. Neoagarooctaose was the smallest substrate for rAgaG4, whereas neoagarotetraose was the minimal degradation product. Removing the extra fragment from the GH module led to the inability of the mutant (rAgaG4-T57) to degrade neoagarooctaose, and the final degradation products of agarose by the truncated protein were neoagarotetraose, neoagarohexaose, and neoagarooctaose at a final molar ratio of 2.7:2.8:1. The optimal temperature for agarose degradation also decreased to 40 °C for this mutant. Bioinformatic analysis suggested that tyrosine 276 within the extra fragment was a candidate active site residue for the enzymatic activity. Site-swapping experiments of Tyr(276) to 19 various other amino acids demonstrated that the characteristics of this residue were crucial for the AgaG4 degradation of agarose and the cleavage pattern of substrate.

Characterization of a novel β-agarase from an agar-degrading bacterium Catenovulum sp. X3

An agar-degrading bacterium, Catenovulum sp. X3, was isolated from the seawater of Shantou, China. A novel β-agarase gene agaXa was cloned from the strain Catenovulum sp. X3. The gene agaXa consists of 1,590 bp and encodes a protein of 529 amino acids, with only 40 % amino acid sequence identity with known agarases. AgaXa should belong to the glycoside hydrolase family GH118 based on the amino acid sequence similarity. The molecular mass of the recombinant AgaXa (rAgaXa) was estimated to be 52 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It had a maximal agarase activity at 52 °C and pH 7.4 and was stable over pH 5.0 ~ 9.0 and at temperatures below 42 °C. The K m and V max for agarose were 10.5 mg/ml and 588.2 U/mg, respectively. The purified rAgaXa showed endolytic activity on agarose degradation, yielding neoagarohexaose, neoagarooctaose, neoagarodecaose, and neoagarododecaose as the end products. The results showed that AgaXa has potential applications in agar degradation for the production of oligosaccharides with various bioactivities.