Biological activities of a neutral water-soluble agar polysaccharide prepared by agarase degradation

Depolymerization of agar was performed using agarase, which was extracted from the cell-free medium of a culture of marine bacterial Alterornonas sp. nov. SY 37-12. After ethanol fractionation and lyophilization, the water-soluble agar polysaccharide （WSAP3） was collected. The anti-tumor activity of the product was determined by using Sarcoma 180 tumor in mouse. The tumor inhibition rate of WSAP3 the product was determined by using Sarcoma 180 tumor in mouse. The tumor inhibition rate of WSAP3 reached 48.7% at a dose of 64mg kg^-1 after 15 days treatment. WSAP3 enhanced the aetivities of superoxide dismutase and catalase, which suggests that WSAP3 was effective in promoting the antioxidation ability and eliminating danger from free radicals. The result of flow cytometry showed that the WSAP3 had no activities of cell cycle inhibition or apoptosis-inducing activities. The anti-oxidation of WSAP3 was further confirmed by test in vitro, which might play an important role in anti-tumor activity. The immunological regulation of WSAP3, especially its effect on the phagocytosis ratio and phagocytosis index of rophage was also assayed in test in vivo.

Identification of a marine agarolytic bacterium Agarivorans albus QM38 and cloning and sequencing its beta-agarase genes

A total of 117 agar-decomposing cultures were isolated from coastal seawater around Qingdao, China. The phenotypic and agarolytic features of an agarolytic isolate, QM38, were investigated. The strain was gram negative, strictly aerobic, curved rod and polar flagellum. On the basis of several phenotypic characters, biochemical and morphological characters and phylogenetic analysis of the gene coding for the 16S rRNA, the strain was identified as Agarivorans albus strain QM38. This strain can liquefy the agar on the solid agar plate. An excellular agarase activity was determined in liquid culture. The enzyme exhibited maximal activity at 40 ℃, pH 7.6. Its activity was greatly affected by different concentrations of agarose. The highest activity 32 U/ml was achieved in the culture supernatant. The hydrolytic product was analyzed by fluorophore-assisted carbohydrate electrophoresis （FACE）. After complete hydrolysis of agarose, a series of agaro-oligosaccharides were produced. The main products of the enzymes were oligosaccharides in the degree of polymerization （DP） of 2, 4, 6 and 8. Three genes agaD01, agaD02 and agaD03, encodingβ-agarases, had been cloned from genomic DNA of Agarivorans albus strain QM38. The open reading frame of agaDOl, consisted of 2 988 bp, and shared 95.5%-98.9% identity to the β-agarase genes of some strains of Vibrio and Agarivorans. Gene agaD02 comprised 2 868 bp and encoded a 955- amino-acid protein. It showed 97.4% and 98.7~0 identity to the β-agarase genes of strain Vibrio sp. PO-303 and strain Vibrio sp. JT0107, respectively. Only partial sequence of agaD03 gene has been cloned. It showed 96.5% identity to β-agarase gene （agaB） of Pseudoalteromonas sp. CY24, and shared 96.8% identity to β-agarase-c gene of Vibrio sp. PO-303.

Expression and enzymatic characterization of a cold-adapted β-agarase from Antarctic bacterium Pseudoalteromonas sp.NJ21

An agar-degrading bacterium, designated as Pseudoalteromonas sp. NJ21, was isolated from an Antarctic sediment sample. The agarase gene aga1161 from Pseudoalteromonas sp. NJ21 consisting of a 2 382-bp coding region was cloned. The gene encodes a 793-amino acids protein and was found to possess characteristic features of the Glyco_hydro 42 family. The recombinant agarase （rAgal 161） was overexpressed in Escherichia coli and purified as a fusion protein. Enzyme activity analysis revealed that the optimum temperature and pH for the purified recombinant agarase were 30--40℃ and 8.0, respectively. rAga 1161 was found to maintain as much as 80% of its maximum activity at 10℃, which is typical of a cold- adapted enzyme. The pattern of agar hydrolysis demonstrated that the enzyme is an β-agarase, producing neoagarobiose （NA2） as the final main product. Furthermore, this work is the first proof of an agarolytic activity in Antarctic bacteria and these results indicate the potential for the Antarctic agarase as a catalyst in medicine, food and cosmetic industries.

Deletion of a Non-Catalytic Region Increases the Enzymatic Activity of a β-Agarase from Flammeovirga sp. MY04

A Glycoside hydrolase （GH） typically contains one catalytic module and varied non-catalytic regions （NCRs）. However, effects of the NCRs to the catalytic modules remain mostly unclear except the carbohydrate-binding modules （CBMs）. AgaG4 is a GH16 endo-β-agarase of the agarolytic marine bacterium Flammeovirga sp. MY04. The enzyme consists of an extra sugar-binding peptide within the catalytic module, with no predictable CBMs but function-unknown sequences in the NCR, which is a new characteristic of agarase sequences. In this study, we deleted the NCR sequence, a 140-amino acid peptide at the C-terminus and expressed the truncated gene, agaG4-T140, in Escherichia coli. After purification and refolding, the trtmcated agarase rAgaG4-T140 retained the same catalytic temperature and pH value as rAgaG4. Using combined fluorescent labeling, HPLC and MS/MS techniques, we identified the end-products of agarose degradation by rAgaG4-T140 as neoagarotetraose and neoagarohexaose, with a final molar ratio of 1.53：1 and a conversion ratio of approximately 70%, which were similar to those of rAgaG4. However, the truncated agarase rAgaG4-T140 markedly decreased in protein solubility by 15 times and increased in enzymatic activities by 35 times. The oligosaccharide production of rAgaG4-T140 was approximately 25 times the weight of that produced by equimolar rAgaG4. This study provides some insights into the influences of NCR on the biochemical characteristics of agarase AgaG4 and implies some new strategies to improve the properties of a GH enzyme.

A Polysaccharide-Degrading Marine Bacterium Flammeovirga sp.MY04 and Its Extracellular Agarase System

Bacteria of the genus Flammeovirga can digest complex polysaccharides (CPs), but no details have been reported regarding the CP depolymerases of these bacteria. MY04, an agarolytic marine bacterium isolated from coastal sediments, has been identified as a new member of the genus Flammeovirga. The MY04 strain is able to utilize multiple CPs as a sole carbon source and grows well on agarose, mannan, or xylan. This strain produces high concentrations of extracellular proteins (490 mg L-1 ± 18.2 mg L-1 liquid culture) that exhibit efficient and extensive degradation activities on various polysaccharides, especially agarose. These proteins have an activity of 310 U mg-1 ± 9.6 U mg-1 proteins. The extracellular agarase system (EAS) in the crude extracellular enzymes contains at least four agarose depolymerases, which are with molecular masses of approximately 30-70 kDa. The EAS is stable at a wide range of pH values (6.0-11.0), temperatures (0-50℃), and sodium chloride (NaCl) concentrations (0- 0.9 mol L-1). Two major degradation products generated from agarose by the EAS are identified to be neoagarotetraose and neoagarohexaose, suggesting that β-agarases are the major constituents of the MY04 EAS. These results suggest that the Flammeovirga strain MY04 and its polysac-charide-degradation system hold great promise in industrial applications.

Agarase Production by Marine Pseudoalteromonas sp.MHS:Optimization,and Purification

Agar is an essential polysaccharide that has been utilized in numerous fields.Many kinds of literature have been published regarding agarolytic microorganisms’isolation and agarases biochemical studies.In this search,a local marine agarolytic bacterium associated with marine alga Ulva lactuca surface was isolated and identified as Pseudoalteromonas sp.MHS.The agarase production was parallel to the growth of Pseudoalteromonas sp.MHS as cells displayed a lag phase(2 h),subsequently an exponential growth that prolonged till 10 h where maximum growth(OD550nm=3.9)was achieved.The enzyme activity increased rapidly as cells increased exponentially where the maximum activity of 0.22 U/mL was achieved after 8h and remained constant till 12 h during the stationary phase of growth.Agarase production was optimized using Plackett-Burman statistical design by measuring enzyme activity as a response and the design was validated using a verification experiment;the activity of the enzyme increased from 0.22 U/mL to 0.29 U/mL.Pseudoalteromonas sp.MHS agarase was partially purified and its molecular weight(MW)was determined by SDS-PAGE(15-25 kDa).Agarase showed approximately 94% of its activity at 40℃.The enzyme stability decreased as the temperature increased;the enzyme could retain about 98,90,80,75,and 60% of its activity at 20,30,40,50,and 60℃,respectively.Biomass of the red alga Pterocladia capillacea proved to be a suitable substrate for agarase production using Pseudoalteromonas sp.MHS;the enzyme activity recorded after 24 h of incubation was 0.35 U/mL compared to 0.29 U/mL from the optimized medium.

Applying Both Chemical Liquefaction and Enzymatic Catalysis Can Increase Production of Agaro-Oligosaccharides from Agarose

Red algae represents an important marine bioresource.One high-value utilization of red algae is the production of agarooligosaccharides which have many positive biological effects.However,the lack of an efficient production route seriously limits the application of agaro-oligosaccharides.In this study,we established a green route that combines chemical liquefaction and enzymatic catalysis for the efficient production of agaro-oligosaccharides,and we used the production of neoagarotetraose(NA4)as an example.Agarose(150 g L^−1)liquefaction by citric acid was controlled with respect to two targets:a 100%liquefaction rate and a high average degree of polymerization(>4)of the liquesced agaro-oligosaccharides,which were then catalyzed byβ-agarase into an oligosaccharides mixture with a high concentration of NA4(30.8 g L^−1)in a 1-L reaction volume.After purification,we obtained 25.5 g of NA4 with a purity of 92%.This work establishes an easy route for the efficient production of pure agaro-oligosaccharides from agarose.