Agarase Activity Research Articles

Isolation, Characterization and Molecular Identification of Bacteria Associated with Green and Brown Seaweeds

Aims: The co-existence of bacterial communities along with macroalgae in marine environment develops a mutualistic relationship resulting in functional advantages for both the groups. The present research studied the composition of epiphytic bacteria associated with green (Ulva spp.) and brown seaweeds (Sargassum spp. and Padina spp.) sampled off Maharashtra coast, India, and their physiological and bioactive properties. Study Design: Green and brown seaweeds were collected from three locations along Maharashtra coast and analyzed for the associated bacteria. Place and Duration of Study: Seaweed samples analyzed in this study were from Kelwa, Manori and Ratnagiri along the Maharashtra coast. The analysis was performed during June to September 2021. Methodology: Bacteria from freshly collected seaweed samples were homogenized in saline, 10-fold serially diluted and plated on Zobell marine agar. Distinct colonies were selected and identified by a series of biochemical tests, followed by partial 16SrRNA gene sequencing. The physiological characteristics of the isolated bacteria were studied by screening for temperature and salinity tolerance, production of protease, lipase, agarose, amylase and biosurfactants. Results: The total seaweed bacterial count ranged from 4.5 ×103 to 2.9 × 104 CFU/g. Seventy-seven bacteria were isolated, 44 (57.14%) and 33 (42.85%) isolates from green and brown seaweeds respectively. The 16SrRNA sequencing of 20 representative isolates revealed the dominance of Bacillus spp., followed by Vibrio spp. Growth at 0°C was exhibited by all bacteria except Bacillus tequilensis, Bacillus altitudinis, Oceanobacillus iheyensis and one isolate of Vibrio spp. A majority of the isolates grew at 45°C. Vibrio spp. exhibited protease, amylase, gelatinase, and agarase activities, whereas the biosurfactant activity was commonly associated with Bacillus spp. Conclusion: The results of this study illustrate the occurrence of seaweed-associated beneficial bacteria exhibiting bioactive properties with potential biomedical applications.

Biochemical characterization of Fsa16295Glu from "Fervidibacter sacchari," the first hyperthermophilic GH50 with β-1,3-endoglucanase activity and founding member of the subfamily GH50_3.

The aerobic hyperthermophile "Fervidibacter sacchari" catabolizes diverse polysaccharides and is the only cultivated member of the class "Fervidibacteria" within the phylum Armatimonadota. It encodes 117 putative glycoside hydrolases (GHs), including two from GH family 50 (GH50). In this study, we expressed, purified, and functionally characterized one of these GH50 enzymes, Fsa16295Glu. We show that Fsa16295Glu is a β-1,3-endoglucanase with optimal activity on carboxymethyl curdlan (CM-curdlan) and only weak agarase activity, despite most GH50 enzymes being described as β-agarases. The purified enzyme has a wide temperature range of 4-95°C (optimal 80°C), making it the first characterized hyperthermophilic representative of GH50. The enzyme is also active at a broad pH range of at least 5.5-11 (optimal 6.5-10). Fsa16295Glu possesses a relatively high kcat/KM of 1.82 × 107 s-1 M-1 with CM-curdlan and degrades CM-curdlan nearly completely to sugar monomers, indicating preferential hydrolysis of glucans containing β-1,3 linkages. Finally, a phylogenetic analysis of Fsa16295Glu and all other GH50 enzymes revealed that Fsa16295Glu is distant from other characterized enzymes but phylogenetically related to enzymes from thermophilic archaea that were likely acquired horizontally from "Fervidibacteria." Given its functional and phylogenetic novelty, we propose that Fsa16295Glu represents a new enzyme subfamily, GH50_3.

A quantitative assay for agarase activity determination using agarose–iodine complex

Rapid and simple spectrophotometric methods are required to detect various oligosaccharides produced by agar-hydrolysing enzymes. Herein, we present a quantitative agarose–iodine assay for agarase activity determination via the detection of the extent of agarose degradation. The agarose–iodine complex becomes reddish orange upon the addition of Lugol solution, and the enzymatic activity can be detected with ultraviolet–visible spectroscopy at 600 nm. The main advantages of this modified Lugol assay are high sensitivity, simple detection, and cost effectiveness. A novel definition of the unit to measure and compare the activities of agarases is also suggested.

Multifunctional alkalophilic \u03b1-amylase with diverse raw seaweed degrading activities

Uncultured microbes are an important resource for the discovery of novel enzymes. In this study, an amylase gene (amy2587) that codes a protein with 587 amino acids (Amy2587) was obtained from the metagenomic library of macroalgae-associated bacteria. Recombinant Amy2587 was expressed in Escherichia coli BL21 (DE3) and was found to simultaneously possess α-amylase, agarase, carrageenase, cellulase, and alginate lyase activities. Moreover, recombinant Amy2587 showed high thermostability and alkali resistance which are important characteristics for industrial application. To investigate the multifunctional mechanism of Amy2587, three motifs (functional domains) in the Amy2587 sequence were deleted to generate three truncated Amy2587 variants. The results showed that, even though these functional domains affected the multiple substrates degrading activity of Amy2587, they did not wholly explain its multifunctional characteristics. To apply the multifunctional activity of Amy2587, three seaweed substrates (Grateloupia filicina, Chondrus ocellatus, and Scagassum) were digested using Amy2587. After 2 h, 6 h, and 24 h of digestion, 121.2 ± 4 µg/ml, 134.8 ± 6 µg/ml, and 70.3 ± 3.5 µg/ml of reducing sugars were released, respectively. These results show that Amy2587 directly and effectively degraded three kinds of raw seaweeds. This finding provides a theoretical basis for one-step enzymatic digestion of raw seaweeds to obtain seaweed oligosaccharides.

Characterization and activity enhancement of a novel exo-type agarase Aga575 from Aquimarina agarilytica ZC1.

The novel β-agarase gene aga575 from the agarolytic bacterium Aquimarina agarilytica ZC1 is composed of 2142bp, and the encoded protein Aga575 has the highest amino acid sequence homology of only 65.2% with known agarases. Though carrying a domain of glycoside hydrolase family 42 in the C-terminal, Aga575 should belong to glycoside hydrolase family 50 according to the phylogenetic analysis. Gene aga575 was successfully cloned and overexpressed in Escherichia coli Rosetta (DE3) cells. The recombinant protein had the maximal agarase activity at pH 8.0 and 37°C. The values Km and Vmax toward agarose were 8.4mg/mL and 52.2 U/mg, respectively. Aga575 hydrolyzed agarose and neoagarooligosaccharides to yield neoagarobiose as the sole product. The agarose hydrolysis pattern of Aga575 indicated that it was an exo-type β-agarase. Random mutagenesis was carried out to obtain two beneficial mutants M1 (R534G) and M2 (S4R-R424G) with higher activities. The results showed that the agarase activity of mutant M1 and M2 reached 162% and 192% of the wild-type agarase Aga575, respectively. Moreover, the activity of the mixed mutant M1/M2 (S4R-R424G-R534G) increased to 227%. KEY POINTS: • Aga575 is a novel exo-type β-agarase degrading agarose to yield neoagarobiose as the sole product. • Though owning a domain of glycoside hydrolase family GH42, Aga575 should belong to family GH50. • The agarase activity of one mutant increased to 227% of the wild-type Aga575.

Shewanella algae and Microbulbifer elongatus from marine macro-algae - isolation and characterization of agar-hydrolysing bacteria.

Macro-algae are a good source of agar oligosaccharides, which can be obtained through bacterial enzymatic hydrolysis. The agarase enzyme secreted by the micro-organisms cleaves the cell wall of the algae and releases agar oligosaccharides as degradation products with various applications. Agarolytic bacteria were isolated from the marine algae Kappaphycus sp., and Sargassum sp., and studied for their agar-degrading properties. Among the 70 isolates, 2 isolates (A13 and Sg8) showed agarase activity in in vitro assays. The maximum agarolytic index was recorded in the isolate Sg8 (3.75 mm and 4.29 µg ml−1 agarase activity), followed by the isolate A13 (2.53 mm and 2.6 µg ml−1 agarase activity). Optimum agarase production of isolate Sg8 was observed at pH7 and at a temperature of 25 °C in 24–48 h, whereas for isolate A13 the optimum production was at pH7 and at a temperature of 37 °C in 48 h. The identities of the agarolytic isolates (Sg8 and A13) were confirmed based on microscopy, morphological, biochemical and molecular analysis as Shewanella algae [National Center for Biotechnology Information (NCBI) GenBank accession number MK121204.1] and Microbulbifer elongatus [NCBI GenBank accession number MK825484.1], respectively.

Isolation of agarase-producing bacteria from seawater and examination of the enzyme activity

This study aimed to isolate agarase-producing bacteria from seawater, and then determine activity of the agarase. Eight coastal surface seawater samples were collected from Ba Ria - Vung Tau province. Twenty-one bacterial strains that are capable of liquefying agar were isolated. These isolates produced disintegration zones around their colonies on agar plates with diameters ranging from 4.0 to 7.0 cm after an incubation period of 2 days at room temperature. Five bacterial strains (M1, M5, M7, M62B, and M71) that produced large halos on plates were identified belonging to Vibrio genus with identity > 96%. The crude enzyme activities of these strains ranged from 0.15 to 0.22 U/mL in reaction with agarose as substrate. Among isolated strains, the strain M71 showed the highest agarase activity, and was used to examine the degradation of seaweed. The hydrolysis of dried Gracilaria seaweed by the crude enzyme of M71 at concentration of 5% (v/v) released 915 μM/mL reducing sugar after a 24-h incubation period at 40oC.

Characterization of Two Thermostable β-agarases from a Newly Isolated Marine Agarolytic Bacterium, Vibrio sp. S1

An agar-degrading bacterium, strain S1, was isolated from the coastal seawater of Jeju Island, Korea, and identified as a novel species of the genus Vibrio. The isolate, Vibrio sp. S1, produced at least five kinds of extracellular agarases in artificial sea water broth containing yeast extract and bacto peptone, and two of them were purified to homogeneity. Both agarases, AgaA33 and AgaA29, with apparent molecular weights of 33 kDa and 29 kDa, respectively, exhibited an optimum temperature and pH of 45°C and 7.0, respectively. AgaA33 and AgaA29 showed acidophilic properties and maintained 93% and 87% of the maximum agarase activity at 50°C, respectively, displaying their thermostability. Moreover, more than 80% activity was retained after heat treatment at 45°C for 1 h. Their agarase activities were inhibited by the presence of EDTA and remarkably stimulated by the presence of Mn2+ in a concentration-dependent manner, indicating that both agarases required the Mn2+ ion as a cofactor. The AgaA33 enzyme exhibited Km and Vmax values of 4.02 mg/mL and 27 U/mg, respectively. AgaA29 exhibited Km and Vmax values of 3.26 mg/mL and 200 U/mg, respectively. The instrumental analysis demonstrated that both are new β-agarases that can hydrolyze agarose and agaro-oligomers into neoagarotetraose and neoagarohexaose. In addition, AgaA33 coproduced neoagarooctaose as the major final product. Both thermostable enzymes are expected to be useful for the industrial application of agar.

Isolation and pathogenicity identification of bacterial pathogens in bleached disease and their physiological effects on the red macroalga Gracilaria lemaneiformis

Marine macroalga, Gracilaria lemaneiformis, is affected by bleached disease in recent years, but the bacterial pathogens for bleached disease in G. lemaneiformis are either known or unknown. In this study, we identified eight bacterial pathogens, of which Agarivorans albus, Aquimarina latercula (T) and Brachybacterium sp. had the strongest pathogenicity on healthy G. lemaneiformis. These pathogens had strong agarase activity, suggesting a possible infection mechanism through the degradation of algal-agar. Furthermore, analysis of the macroalgal defense against pathogens in terms of hydrogen peroxide and malondialdehyde content indicated that oxidative burst started at an early stage during the bacterial infection. On the other hand, the increasing activity of peroxidase and the occurrence of different peroxidases were to protect algae from the oxidative damage, by compensating for the decrease in catalase and total antioxidant capacity in bleached tissue. The photosynthetic pigment – phycobiliprotein was dramatically decreased after the bacterial infection, with the beta subunit but not the alpha subunit of phycoerythrin being degraded first as the disease progressed. The findings here provide very important fundamental information for future research on understanding the algae-bacteria interaction mechanism, the mechanism of algal defense against bacterial infection, which is important for the efficient and healthy cultivation of G. lemaneiformis.

A novel GH16 beta-agarase isolated from a marine bacterium, Microbulbifer sp. BN3 and its characterization and high-level expression in Pichia pastoris

An agar-degrading bacterium, strain BN3, was isolated from a coastal soil sample collected in Taiwan Strait, China and identified to be a novel species of the genus Microbulbifer. The gene (N3-1) encoding for a novel β-agarase from the isolate was cloned and sequenced. It encoded a mature protein with 274 amino acids and a calculated molecular mass of 34.3 kDa. The deduced amino acid sequence manifested sequence similarity (61–84% identity) to characterized β-agarases in the glycoside hydrolase family 16. The recombinant agarase was hyper-produced extracellularly using Pichia pastoris as the host. After induction in a shake flask for 96 h, the yield of recombinant N3–1 protein reached 0.406 mg/mL, and the enzyme activity attained 502.1 U/mL. The enzyme purified by ion exchange chromatography displayed a specific activity of 6447 U/mg at pH 6.0 and 50 °C. The optimal pH and temperature for agarase activity were approximately 6 and 50 °C, respectively. The pattern of agarose hydrolysis showed that the enzyme was an endo-type β-agarase, capable of hydrolyzing agarose and Gracilaria lemaneiformis, with neoagarobiose and neoagarotetraose as the final main products.

Fungal Agarase Production in a Cost-Effective Macroalgal Based Medium and Enzymatic Hydrolysis of the Alkali Extracted Macroalgal Biomass: An Optimization Study

Agarase is a promising biocatalyst that catalyze the hydrolysis of agar or agarose and produce oligosaccharides with several biotechnological applications. Agarase production by Dendryphiella arenaria was optimized using a natural low-cost medium composed of the red alga Palisada perforata and natural seawater. The results showed that seaweed biomass and seawater concentrations were the most important factors influencing agarase production. After optimization, the agarase activity was enhanced to 2.5 ± 0.3 U/mL. The crude agarase exhibited a wide pH (4‒10) stability with residual activity more than 75%. The enzyme showed high thermostability at 40 °C, and a moderate thermostability at 50, 60, 70 °C. Several parameters of thermal inactivation kinetics and thermodynamics were calculated, and suggested that the enzyme would be thermostable. Enzymatic saccharification of alkali extracted polysaccharides from the red macroalgal biomass was also optimized with respect to substrate concentration, enzyme dosage, and temperature and proceeded optimally at 0.15% substrate, 40 °C with enzyme dosage 0.9 mL/mL substrate. Under these conditions, the enzymatic saccharification yielded 647.96 mg reducing sugars/g substrate. This study therefore describes an improved, effective, and low-cost process for agarase and fermentable sugar production from seaweed biomass.

Purification and Characterization of Agarase from Marine Bacteria Acinetobacter sp. PS12B and Its Use for Preparing Bioactive Hydrolysate from Agarophyte Red Seaweed Gracilaria verrucosa.

Acinetobacter strain PS12B was isolated from marine sediment and was found to be a good candidate to degrade agar and produce agarase enzyme. The extracellular agarase enzyme from strain PS12B was purified by ammonium sulfate precipitation followed by DEAE-cellulose ion-exchange chromatography. The specific activity of the crude enzyme which was 1.52U increased to 45.76U, after two-stage purification, with an enzyme yield of 9.76%. Purified enzyme had a molecular mass of 24kDa. The optimum pH and temperature for activity of purified agarase were found to be 8.0 and 40°C, respectively. The Km and Vmax values for agarase were 4.69mg/ml and 0.5μmol/min, respectively. Treatment with EDTA reduced the agarase activity by 58% at 5mM concentration. The enzyme activity was stimulated by the presence of Fe2+, Mn2+, and Ca2+ ions while reducing reagents (β-mercaptoethanol and dithiothreitol, DTT) enhanced its activity by 30-40%. The purified agarase exhibited tolerance to both detergents and organic solvents. Major hydrolysis products of agar were DP4 and also a mixture of longer oligosaccharides DP6 and DP7. The enzyme hydrolysed seaweed (Gracilaria verrucosa) exhibited strong antioxidant activity in vitro. Successful hydrolysis of seaweed indicates the potential use of the enzyme to produce seaweed hydrolysate having health benefits as well as the industrial application like the production of biofuels.

Biochemical Characterization of a Novel GH86 β-Agarase Producing Neoagarohexaose from Gayadomonas joobiniege G7.

A novel β-agarase, AgaJ5, was identified from an agar-degrading marine bacterium, Gayadomonas joobiniege G7. It belongs to the glycoside hydrolase family 86 and is composed of 805 amino acids with a 30-amino-acid signal peptide. Zymogram analysis showed that purified AgaJ5 has agarase activity. The optimum temperature and pH for AgaJ5 activity were determined to be 30°C and 4.5, respectively. AgaJ5 was an acidic β-agarase that had strong activity at a narrow pH range of 4.5-5.5, and was a cold-adapted enzyme, retaining 40% of enzymatic activity at 10°C. AgaJ5 required monovalent ions such as Na+ and K+ for its maximum activity, but its activity was severely inhibited by several metal ions. The Km and Vmax of AgaJ5 for agarose were 8.9 mg/ml and 188.6 U/mg, respectively. Notably, thin-layer chromatography, mass spectrometry, and agarose-liquefication analyses revealed that AgaJ5 was an endo-type β-agarase producing neoagarohexaose as the final main product of agarose hydrolysis. Therefore, these results suggest that AgaJ5 from G. joobiniege G7 is a novel endo-type neoagarohexaose-producing β-agarase having specific biochemical features that may be useful for industrial applications.

Characterization and overexpression of a glycosyl hydrolase family 16 β-agarase Aga0917 from Pseudoalteromonas fuliginea YTW1-15-1.

A gene (aga0917) encoding a putative β-agarase was identified from the genome of Pseudoalteromonas fuliginea YTW1-15-1. The nucleotide sequence analysis revealed that aga0917 had significant homology to the agarase genes of the GH16 family. aga0917 encodes a putative protein of 290 amino acids with an estimated molecular mass of 32.5 kDa, including a 21-amino acid signal peptide. A gene fragment encoding only the putative mature form of Aga0917 (269 amino acids) was overexpressed in Escherichia coli BL21 (DE3) pLysS as a 6 × histine-tagged fusion protein (rmAga0917). The Km, Vmax, and kcat for agarose of rmAga0917 were 39.6 mg/mL, 334 (U/mg) of protein, and 178 (1/s), respectively. According to the results of thin-layer chromatography and mass spectrometry analysis, the main end product from agarose with rmAga0917 was neoagarotetraose, in addition to a small amount of neoagarobiose. Notably, the recombinant protein rmAga0917 showed optimum activity at 60°C and retained approximately 100% agarolytic activity after being kept at 40°C for 1 h and 57% residual activity after incubation at 50°C for 1 h. The rmAga0917 exhibited maximum agarase activity at pH 6.0, and retained more than 80% of activity after incubation over a range of pH 4.0-9.0 for 1 h at 4°C.

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.

Statistical Optimization of Culture Variables for Enhancing Agarase Production by Dendryphiella arenaria Utilizing Palisada perforata (Rhodophyta) and Enzymatic Saccharification of the Macroalgal Biomass.

Agarase is a promising biocatalyst for several industrial applications. Agarase production was evaluated by the marine fungus Dendryphiella arenaria utilizing Palisada perforata as a basal substrate in semi-solid state fermentation. Seaweed biomass, glucose, and sucrose were the most significant parameters affecting agarase production, and their levels were further optimized using Box-Behnken design. The maximum agarase activity was 7.69U/mL. Agarase showed a degree of thermostability with half-life of 99min at 40°C, and declining to 44.72min at 80°C. Thermodynamics suggested an important process of protein aggregation during thermal inactivation. Additionally, the enzymatic saccharification of the seaweed biomass using crude agarase was optimized with respect to biomass particle size, solid/liquid ratio, and enzyme loadings. The amount of biosugars obtained after optimization was 26.15±1.43mg/g. To the best of our knowledge, this is the first report on optimization of agarase in D. arenaria.

Identification and characterization of a thermostable endolytic β-agarase Aga2 from a newly isolated marine agarolytic bacteria Cellulophaga omnivescoria W5C

Research on the enzymatic breakdown of seaweed-derived agar has recently gained attention due to the progress in green technologies for marine biomass utilization. The enzymes known as agarases catalyze the cleavage of glycosidic bonds within the polysaccharide. In this study, a new β-agarase, Aga2, was identified from Cellulophaga omnivescoria W5C. Aga2 is one of four putative agarases from the W5C genome, and it belongs to the glycoside hydrolase 16 family. It was shown to be exclusive to the Cellulophaga genus. Agarase activity assays showed that Aga2 is an endolytic-type β-agarase that produces tetrameric and hexameric neoagaro-oligosaccharides, with optimum activity at 45°C and pH 8.0. Zinc ions slightly enhanced its activity while manganese ions had inhibitory effects even at very low concentrations. Aga2 has a Km of 2.59mgmL−1 and Vmax of 275.48Umg−1. The Kcat is 1.73×102s−1, while the Kcat/Km is 8.04×106s−1M−1. Aga2 also showed good thermostability at 45°C and above, and retained >90% of its activity after repeated freeze-thaw cycles. Bioinformatic analysis of its amino acid sequence revealed that intrinsic properties of the protein (e.g. presence of certain dipeptides and the relative volume occupied by aliphatic amino acids) and tertiary structural elements (e.g. presence of salt bridges, hydrophobic interactions and H-bonding) contributed to its thermostability.

Identification and characterization of a new agar-degrading strain with the novel properties of saccharides inhibition and nitrogen fixation.

In this study, a new agar-degrading strain was isolated from soil with agar as a sole carbon source and energy. Based on its morphological, physiological, biochemical characterization and 16S rDNA sequence, the strain was identified as Streptomyces lavendulae UN-8. The extracellular agarase activity reached 0.03 U/ml after fermentation in shake flask (250 ml), which was close to other reported non-marine microorganisms. Furthermore, it is interesting that the growth of UN-8 would be inhibited by glucose (40 g/L) and maltose (40 g/L) with the inhibitory rate of 100% and 70%, respectively. Besides, UN-8 could be grown on the solid medium without any nitrogen sources, then the possible nitrogen fixation gene nifU was cloned from its genomic DNA. The deduced amino acid sequence of nifU has high similarity (98%) with nitrogen fixation protein NifU from Streptomyces sp. NRRL S-104 (KJY22454.1) and Streptomyces sp. NRRL F-4428 (KJK52526.1) based on NCBI blast. It is suggested that the nifU gene of UN-8 also encoded nitrogen fixation protein NifU. These results provided some new information for the further understanding of agar-degrading strain.

Agar-degrading bacteria isolated from Antarctic macroalgae.

This study describes the taxonomic diversity of pigmented, agar-degrading bacteria isolated from the surface of macroalgae collected in King George Island, Antarctica. A total of 30 pigmented, agarolytic bacteria were isolated from the surface of the Antarctic macroalgae Adenocystis utricularis, Monostroma hariotii, Iridaea cordata, and Pantoneura plocamioides. Based on the 16S rRNA data, the agarolytic isolates were affiliated to the genera Algibacter, Arthrobacter, Brachybacterium, Cellulophaga, Citricoccus, Labedella, Microbacterium, Micrococcus, Salinibacterium, Sanguibacter, and Zobellia. Isolates phylogenetically related to Cellulophaga algicola showed the highest agarase activity in culture supernatants when tested at 4 and 37°C. This is the first investigation of pigmented agar-degrading bacteria, members of microbial communities associated with Antarctic macroalgae, and the results suggest that they represent a potential source of cold-adapted agarases of possible biotechnological interest.

An agarase of glycoside hydrolase family 16 from marine bacterium Aquimarina agarilytica ZC1.

A novel β-agarase gene aga672 was cloned from strain ZC1, the typical strain of agar-degrading Aquimarina agarilytica. Gene aga672 is composed of 2130 bp, and the encoded protein Aga672 showed an amino acid sequence identity of only 42% with reported agarases. Aga672 should belong to glycoside hydrolase family 16 according to the protein sequence similarity. The molecular mass of the recombinant Aga672 was estimated to be 98 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Aga672 decomposed agarose to produce neoagarotetraose, neoagarohexaose and neoagarooctaose as the main products. That is the main difference between Aga672 and other reported agarases of family GH16. The Km and Vmax for agarose degradation were 59.8 mg mL-1 and 154.3 U mg-1, respectively. The activity of Aga672 was stable at temperatures below 40°C and at pH 7.0-11.0 with the maximal agarase activity at 25°C and pH 7.0. The results showed that agarase Aga672 could be suitable to hydrolyze the gelated agarose. Thus, it has potential applications in the production of neoagarooligosaccharides directly from red alga.