agar and neoagarotetraose

The oligosaccharides from agar hydrolysis have special biological activities, and exhibit application prospects in cosmetic, food and pharmaceutical industry. In this study, two novel β-agarases (AgaA and AgaB) were screened and characterized. It was found that the AgaA was an endo-type agarase which could efficiently hydrolyzed agar or agarose to form neoagarobiose (NA2), neoagarotetraose (NA4) and neoagarohexaose (NA6), while the AgaB was an exo-type and bifunctional enzyme that showed activities towards both agarose and porphyran. Based on the properties of the two enzymes, we developed modular strategy for enzymatic production of neoagarobiose through a two-stage hydrolysis reaction. The cheap substrate agar was first liquefied by AgaA at high temperature to form neoagaroligosaccharides, which together with the sulfated polysaccharides were homogenized by AgaB to form neoagarobiose as the final product. High concentration of agar (10 g/L) was almost completely converted into neoagarobiose with high purity.

Topics: Agar; Disaccharides; Galactosides; Glycoside Hydrolases; Hydrolysis; Oligosaccharides; Sepharose

Topics: Acclimatization; Agar; Alteromonadaceae; Bacterial Proteins; Cold Temperature; Disaccharides; Flavobacteriaceae; Galactosidases; Galactosides; Glycoside Hydrolases; Oligosaccharides; Protein Sorting Signals; Recombinant Proteins; Salt Tolerance; Sequence Alignment; Temperature

A novel gene aga3027 from the genome of Flammeovirga sp. OC4, isolated from the deep sea, was screened and expressed in Escherichia coli BL21. This gene encoded the genetic information of a potential agarase that consists of 851 amino acids and belongs to 16 β-agarase family of glycoside hydrolase. Purified recombinant Aga3027 demonstrated the maximum activity of agarase at 40 °C and pH 9.0, displaying excellent thermostability and pH-stability. The agarase retained more than 80% of its maximum activity after incubation at 30-40 °C for 48 h, or after incubation at pH 6.0-9.0 for 60 min, which indicated that this agarase was suitable for industrial applications. Silica gel chromatography was used to purify the hydrolysates of agar treated by agarase from the recombinant Aga3027. The hydrolysates were identified as neoagarotetraose and neoagarohexaose by thin layer chromatography and further confirmed by ion chromatography.

Topics: Agar; Bacteroidetes; Chromatography, Thin Layer; Enzyme Stability; Escherichia coli; Galactosides; Glycoside Hydrolases; Hydrogen-Ion Concentration; Hydrolysis; Oligosaccharides; Recombinant Proteins; Temperature

Agar, a heterogeneous polymer of galactose, is the main component of the cell wall of marine red algae. It is well established as a safe, non-digestible carbohydrate in Oriental countries. Although neoagarooligosaccharides (NAOs) prepared by the hydrolysis of agar by β-agarase have been reported to exert various biological activities, the safety of these compounds has not been reported to date. For safety evaluation, NAOs containing mainly neoagarotetraose and neoagarohexaose were prepared from agar by enzymatic hydrolysis using β-agarase DagA from Streptomyces coelicolor. Genotoxicity tests such as the bacterial reverse mutation assay, eukaryotic chromosome aberration assay, and in vivo micronucleus assay all indicated that NAOs did not exert any mutational effects. The toxicity of NAOs in rat and beagle dog models was investigated by acute, 14-day, and 91-day repeated oral dose toxicity tests. The results showed that NAO intake of up to 5,000 mg/kg body weight resulted in no significant changes in body weight, food intake, water consumption, hematologic and blood biochemistry parameters, organ weight, or clinical symptoms. Collectively, a no-observed-adverse-effect level of 5,000 mg/kg body weight/day for both male and female rats was established for NAO. These findings support the safety of NAO for possible use in food supplements and pharmaceutical and cosmetic products.

Topics: Agar; Animals; Body Weight; Cell Line; Cosmetics; Cricetulus; Dietary Supplements; Dogs; Female; Galactosides; Glycoside Hydrolases; Hydrolysis; Male; Mice; Mice, Inbred ICR; Models, Animal; Mutagenicity Tests; No-Observed-Adverse-Effect Level; Oligosaccharides; Rats; Rats, Sprague-Dawley

Streptomyces coelicolor can degrade agar, the main cell wall component of red macroalgae, for growth. To constitute a crucial carbon source for bacterial growth, the alternating α-(1,3) and β-(1,4) linkages between the 3,6-anhydro-L-galactoses and D-galactoses of agar must be hydrolyzed by α/β-agarases. In S. coelicolor, DagA was confirmed to be an endo-type β-agarase that degrades agar into neoagarotetraose and neoagarohexaose. Genomic sequencing data of S. coelicolor revealed that Sco3487, annotated as a putative hydrolase, has high similarity to the glycoside hydrolase (GH) GH50 β-agarases. Sco3487 encodes a primary translation product (88.5 kDa) of 798 amino acids, including a 45-amino-acid signal peptide. The sco3487 gene was cloned and expressed under the control of the ermE promoter in Streptomyces lividans TK24. β-Agarase activity was detected in transformant culture broth using the artificial chromogenic substrate p-nitrophenyl-β-D-galactopyranoside. Mature Sco3487 (83.9 kDa) was purified 52-fold with a yield of 66% from the culture broth. The optimum pH and temperature for Sco3487 activity were 7.0 and 40°C, respectively. The K(m) and V(max) for agarose were 4.87 mg/ml (4 × 10(-5) M) and 10.75 U/mg, respectively. Sco3487 did not require metal ions for its activity, but severe inhibition by Mn(2+) and Cu(2+) was observed. Thin-layer chromatography analysis, matrix-assisted laser desorption ionization-time of flight mass spectrometry, and Fourier transform-nuclear magnetic resonance spectrometry of the Sco3487 hydrolysis products revealed that Sco3487 is both an exo- and endo-type β-agarase that degrades agarose, neoagarotetraose, and neoagarohexaose into neoagarobiose.

Topics: Agar; Bacterial Proteins; Cloning, Molecular; Disaccharides; Galactosides; Gene Expression Regulation, Bacterial; Glycoside Hydrolases; Oligosaccharides; Sepharose; Streptomyces coelicolor; Streptomyces lividans; Substrate Specificity

A gene (agrP) encoding a beta-agarase from Pseudoalteromonas sp. AG4 was cloned and expressed in Escherichia coli. The agrP primary structure consists of an 870-bp open reading frame (ORF) encoding 290 amino acids (aa). The predicted molecular mass and isoelectric point were determined at 33 kDa and 5.9, respectively. The signal peptide was predicted to be 21 aa. The deduced aa sequence showed 98.6% identity to beta-agarase from Pseudoalteromonas atlantica. The recombinant protein was purified as a fusion protein and biochemically characterized. The purified beta-agarase (AgaP) had specific activity of 204.4 and 207.5 units/mg towards agar and agarose, respectively. The enzyme showed maximum activity at 55 degrees C and pH 5.5. It was stable at pH 4.5 to 8.0 and below 55 degrees C for 1 h. The enzyme produced neoagarohexaose and neoagarotetraose from agar and in addition to that neoagarobiose from the agarose. The neoagarooligosaccharides were biologically active. Hence, AgaP is a useful enzyme source for use by cosmetic and pharmaceutical industries.

Topics: Agar; Amino Acid Sequence; Base Sequence; Disaccharides; Escherichia coli; Galactosides; Glycoside Hydrolases; Molecular Sequence Data; Oligosaccharides; Phylogeny; Pseudoalteromonas; Recombinant Proteins; Republic of Korea; Rhodophyta; Seawater; Sepharose; Sequence Alignment

Whitening effect, tyrosinase inhibitions, and cytotoxicity of neoagarotetraose were measured after purification from hydrolyzed agar by gel filtration chromatography. In melanoma B16F10 cells, melanin content of neoagarotetraose-treated cells was as same as that treated by kojic acid or arbutin. In addition, tyrosinase of melanoma cells was strongly inhibited by neoagarotetraose at a concentration of 1 micron/ml and similarly inhibited at 10 and 100 microg/ml compared to those by arbutin or kojic acid. The activity of mushroom tyrosinase showed a 38% inhibition by neoagarotetraose at 1 microg/ml, and this inhibitory effect was more efficient than that by kojic acid. Neoagarotetraose revealed similar IC(50) (50% inhibition concentration) value for mushroom tyrosinase as that by kojic acid. These data suggest that neoagarotetraose generated from agar by recombinant beta-agarase might be a good candidate as a cosmetic additive for whitening effect.

Topics: Agar; Agaricales; Animals; Cell Line, Tumor; Cell Survival; Enzyme Inhibitors; Galactosides; Hydrolysis; Melanins; Mice; Monophenol Monooxygenase; Oligosaccharides; Protein Binding; Pyrones

The gene for beta-agarase of an Agarivorans sp. JA-1 was expressed in Bacillus subtilis strain DB104 for efficient and economical mass-production of the enzyme. We isolated 360 mg protein with a specific activity of 201 U/mg from the culture broth. The efficiency of production was approximately 130-fold higher than that in E. coli. The enzyme produced neoagarohexaose, neoagarotetraose and neoagarobiose from agar. Neoagarooligosaccharides produced by the enzyme had a whitening effect and inhibited tyrosinase activity in the murine melanoma cell line, B16F10. Neoagarooligosaccharides were not cytotoxic to B16F10 or normal cells. beta-Agarase could therefore be a good whitening, cosmetic additive.

Topics: Agar; Alteromonadaceae; Animals; Bacillus subtilis; Cell Line, Tumor; Cell Survival; Disaccharides; Galactosides; Glycoside Hydrolases; Melanins; Melanocytes; Mice; Monophenol Monooxygenase; Oligosaccharides; Recombinant Proteins