Agarase Activity Research Articles

Isolation and characterization of an agarase-producing bacterial strain, Alteromonas sp. GNUM-1, from the West Sea, Korea.

The agar-degrading bacterium GNUM-1 was isolated from the brown algal species Sargassum serratifolium, which was obtained from the West Sea of Korea, by using the selective artificial seawater agar plate. The cells were Gram-negative, 0.5-0.6 micrometer wide and 2.0-2.5 micrometer long curved rods with a single polar flagellum, forming nonpigmented, circular, smooth colonies. Cells grew at 20 degrees C- 37 degrees C, between pH 5.0 and 9.0, and at 1-10% (w/v) NaCl. The DNA G+C content of the GNUM-1 strain was 45.5 mol%. The 16S rRNA sequence of the GNUM-1 was very similar to those of Alteromonas stellipolaris LMG 21861 (99.86% sequence homology) and Alteromonas addita R10SW13 T (99.64% sequence homology), which led us to assign it to the genus Alteromonas. It showed positive activities for agarase, amylase, gelatinase, alkaline phosphatase, esterase (C8), lipase (C14), leucine arylamidase, valine arylamidase, alpha-chymotrypsin, acid phosphatase, naphthol- AS-BI-phosphohydrolase, alpha-galactosidase, beta-galactosidase, beta-glucosidase, catalase, and urease. It can utilize citrate, malic acid, and trisodium citrate. The major fatty acids were summed feature 3 (21.5%, comprising C16:1omega7c/iso- C15:0 2-OH) and C16:0 (15.04%). On the basis of the variations in many biochemical characteristics, GNUM-1 was considered as unique and thus was named Alteromonas sp. GNUM-1. It produced the highest agarase activity in modified ASW medium containing 0.4% sucrose, but lower activity in rich media despite superior growth, implying that agarase production is tightly regulated and repressed in a rich nutrient condition. The 30 kDa protein with agarase activity was identified by zymography, and this report serves as the very first account of such a protein in the genus Alteromonas.

Optimization of Pseudoalteromonas sp. JYBCL 1 culture conditions, medium composition and extracellular β-agarase activity

Extracellular agarase produced by the Pseudoalteromonas strain JYBCL 1 is used in a variety of applications in the biotechnology, pharmaceutical, cosmetic, and food industries. The optimization of culture conditions for agarase-producing microbes and agarase activity is thus an important consideration in many industrial applications. In this study, the optimum medium composition and culture conditions for the JYBCL 1 strain were determined using the “one factor at a time” (OFAT) method and a Plackett-Burman design. Optimal cell growth was obtained at a temperature of 25°C and when 10 g/L tryptone was present in the culture medium. Optimal agarase activity occurred at a temperature of 40°C and at pH 6. The presence of carbonyl groups in the extracellular agarase hydrolysis products was verified using FT-IR. LC-MS identified the hydrolyzates as neoagarohexaose, neoagarotetraose, and neoagarobiose. The extracellular agarase produced by the JYBCL 1 strain used in this study was identified as β-agarase by 13C-NMR spectroscopy.

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.

Enhanced Activity of Intracellular Agarase from a Novel Marine Strain <i>Agarivorans gilvus</i> WH0801

A marine bacterium strain Agarivorans gilvus WH0801 with the efficient agar degradation ability isolated from fresh seaweed samples of Weihai coast was found to be potential in producing agarase. We studied on the optimal medium composition and culture conditions of Agarivorans gilvus WH0801 by statistical methods in shake flasks. First, several more important factors influencing agarase activity were selected by Plackett-Burman design. They are agar concentration, yeast extract concentration and seed age. Then the optimum levels of these three variables were further determined using Box-Behnken design. The highest agarase activity is obtained in the medium consisting of 2.49 g L-1 agar and 0.88 g L-1 yeast extract when the seed age is 25.64 h. The levels of other factors are 1 g L-1 peptone, 0.01 g L-1 ironic citrate at initial pH 7.0 and 28 °C. The whole optimization strategy results in the activity of agarase reaches 1.158 U mL-1, which is about 6.2-fold increase compares with the control.

Rhodococcus sp. Q5, a novel agarolytic bacterium isolated from printing and dyeing wastewater

An agar-degrading bacterium, Rhodococcus sp. Q5, was isolated from printing and dyeing wastewater using a mineral salts agar plate containing agar as the sole carbon source. The bacterium grew from pH 4.0 to 9.0, from 15 to 35°C, and in NaCl concentrations of 0-5 %; optimal values were pH 6.0, 30°C, and 1 % NaCl. Maximal agarase production was observed at pH 6.0 and 30°C. The bacterium did not require NaCl for growth or agarase production. The agarase secreted by Q5 was inducible by agar and was repressed by all simple sugars tested except lactose. Strain Q5 could hydrolyze starch but not cellulose or carboxymethyl cellulose. Agarase activity could also be detected in the medium when lactose or starch was the sole source of carbon and energy. Strain Q5 could grow in nitrogen-free mineral media; an organic nitrogen source was more effective than inorganic carbon sources for growth and agarase production. Addition of more organic nitrogen (peptone) to the medium corresponded with reduced agarase activity.

Isolation and Identification of Agarose-degrading Bacterium, Pseudoalteromonas sp. GNUM08122.

This study's aim was to isolate microorganisms producing agarase with a high activity, with possible applications in improving the performance of the pretreatment processes for bioethanol production. Marine algaes were collected from the south coast of Korea, from which three kinds of microorganisms were isolated. After a 4-day culture of these strains at , crude enzymes were obtained from culture supernatant or cell-free extract by ammonium sulfate precipitation and membrane dialysis. Agarase activity was observed in these crude enzymes. Notably higher specific activity was observed in the crude enzyme obtained from the culture supernatant rather than that from the cell-free extract. This indicates that a secreted enzyme has a much greater activity than a cellular enzyme. Crude enzymes from the GNUM08122 strain were inferred to have -agarase activity because release of p-nitrophenol was observed, possibly due to the cleavage of p-nitrophenyl--D-galactopyranoside. The 16S rRNA sequence of GNUM08122 showed a close relationship to Pseudoalteromonas issachenkonii KMM 3549 (99.8%) and Pseudoalteromonas tetraodonis IMA 14160 (99.7%), which led us to assign it to the genus Pseudoalteromonas. Biochemical and physiological study revealed that this strain can grow well at under a wide range of pH (pH 4~8) in high-salt conditions (10% NaCl).

Gene cloning, expression and characterization of a neoagarotetraose-producing β-agarase from the marine bacterium Agarivorans sp. HZ105

A β-agarase gene hz2 with 2,868 bp was cloned from the marine agarolytic bacterium Agarivorans sp. HZ105. It encoded a mature agarase HZ2 of 102,393 Da (920 amino acids). Based on the amino acid sequence similarity, agarase HZ2 was assigned to the glycoside hydrolase family 50. The β-agarase shared a gene sequence identity of 98.6% with the reported but much less characterized β-agarase agaB from Vibrio sp. JT0107. Its recombinant agarase rHZ2 was produced in E. coli cells and purified to homogeneity. The agarase rHZ2 degraded agarose and neoagarooligosaccharides with degrees of polymerization above four, to yield neoagarotetraose as the dominant product, which was different from β-agarase agaB of Vibrio sp. JT0107. The agarose hydrolysis pattern suggested that rHZ2 was an endo-type β-agarase. Beta-mercaptoethanol (90 mM) and dithiothreitol (9 mM) increased the agarase activity of rHZ2 by 72.9% and 17.3% respectively, while SDS (9 mM) inhibited the activity completely. The agarase activity was independent of Na(+), K(+), Mg(2+) and Ca(2+). The maximal enzyme activity was observed at 40°C and pH 7. The kinetic parameters K (m), V (max), K (cat), and K (cat)/K (m) values toward agarose of agarase rHZ2 were 5.9 mg ml(-1), 235 U mg(-1), 401 s(-1) and 6.8 × 10(5) M(-1) s(-1), respectively. Agarase rHZ2 could have a potential application in the production of bioactive neoagarotetraose.

Pseudoalteromonas sp. 배양액으로부터 아가레이즈 분리를 위한 음이온교환 크로마토그래피 최적화

Degradation products of agarose are biologically active and thus used as an ingredient in pharmaceuticals or functional cosmetics. Furthermore, it has been strongly considered as a substrate for bio-ethanol fermentation. Recently, we isolated new agarase-producing microorganism, Pseudoalteromonas sp. from south sea of Korea. In this study, we aimed to separate and purify the agarase from culture broth of this strain. Separation of agarase was performed by ion- exchange chromatography on DEAE-Sepharose resin. Equilibrium pH and volume ratio of resin to the amount of protein were optimized for the efficient adsorption of protein. 410 of protein was completely adsorbed to 3 mL of resin at pH 7.5. The total amount of eluted protein increased as NaCl concentration increased to 400 mM at isocratic elution. Agarase was separated by linear gradient elution of NaCl (0~1,000 mM). Three major protein peaks were observed and the presence or absence of agarase in these eluted proteins was measured by Lugol`s staining technique. Only six eluted protein fractions showed strong agarase activity.

English

A bacterium producing agarase was isolated from the vellar estuary and potential strain was identified as Bacillus subtilis. The ideal parameters was found to be of pH 8, temperature of 40°C and 3% of NaCl concentration for optimal growth and agarase activity. Agar as carbon source and yeast extract as nitrogen source was found to be suitable for optimum growth and agarase activity. The activity of enzyme obtained from the cell free filtrate was 15.2 U/L and the activity of the partially purified enzyme was 5.3 U/L and the purified enzyme activity was found to be 2.3 U/L. The stability of the partially purified enzyme on pH profile was found to be pH 8 and thermostability was found to be up to 40°C. The purified enzyme was determined to be homogeneous on the basis of SDS PAGE and had a molecular weight of 20 KDa.   Key words: Agarase, Bacillus subtilis, optimization, stability, SDS PAGE.

Isolation and identification of an agar-liquefying marine bacterium and some properties of its extracellular agarases

Faturrahman, Meryandini A, Junior MZ, Rusmana I (2011) Isolation and identification of an agar-liquefying marine bacterium and some properties of its extracellular agarases. Biodiversitas 12: 192-197. A new agar-liquefying bacterium, designated Alg3.1, was isolated from Gracilaria samples collected from the Kuta Coast at Central Lombok in West Nusa Tenggara and was identified as Aeromonas sp. on the basis of morphology, biochemical-physiological character and 16S rDNA gene sequencing. The bacterium appeared capable of liquefying agar in nutrient agar-plate within 48 hours of incubation and the agar was completely liquefied after l5 days at 29oC. When the isolate was grown in basal salts solution medium B supplemented with peptone and yeast extract, producedextracellular agarases within a short period of time (4-16 h) and the maximum agarase activity was 0.489 nkat/mL at 36h after incubation.

Simiduia areninigrae sp. nov., an agarolytic bacterium isolated from sea sand

During a study intended to screen for agar-degrading bacteria, strain M2-5T was isolated from black sand off the shore of Jeju Island, Republic of Korea. Strain M2-5T exhibited agarase activity; the β-agarase gene of the isolate had 62 % amino acid sequence identity to the β-agarase gene of Microbulbifer thermotolerans JAMB A94T. The isolate was closely related to members of the genus Simiduia but was clearly discernible from reported Simiduia species, based on a polyphasic analysis. Cells of strain M2-5T were Gram-negative, catalase- and oxidase-positive, motile rods. The DNA G+C content was 53.3 mol%. The predominant isoprenoid quinone was Q-8. The major cellular fatty acids were C17:1ω8c (25.9 %), summed feature 3 (iso-C(15 : 0) 2-OH and/or C16:1ω7c; 17.2 %) and C17:0 (15.0 %). Phylogenetic analysis using 16S rRNA gene sequences showed that strain M2-5T had 96.6 % gene sequence similarity to Simiduia agarivorans SA1T, the most closely related type strain of the genus Simiduia. These results suggest that strain M2-5T represents a novel species in the genus Simiduia, for which the name Simiduia areninigrae sp. nov. is proposed; the type strain is M2-5T (=KCTC 23293T=NCAIM B 02424T).

Overexpression and biochemical characterization of DagA from Streptomyces coelicolor A3(2): an endo-type β-agarase producing neoagarotetraose and neoagarohexaose

The DagA product of Streptomyces coelicolor is an agarase with a primary translation product (35 kDa) of 309 amino acids, including a 30-amino acid signal peptide. Although dagA expression in Streptomyces lividans under the control of its own set of promoters was previously reported, its enzymatic properties have never been elucidated. To develop an improved expression system for dagA, three types of strong promoters for the Streptomyces host were linked to dagA, and their efficiencies in DagA production were compared in S. lividans TK24. All of the transformants with dagA grew at improved rates and produced larger amounts of DagA in the modified R2YE medium containing 0.5% agar as the sole carbon source. Of the three transformants, the S. lividans TK24/pUWL201-DagA (ermE promoter) produced the highest agarase activity (A (540)=4.24), and even the S. lividans TK24/pHSEV1-DagA (tipA promoter) and S. lividans TK24/pWHM3-DagA (sprT promoter) produced higher agarase activity (A (540)=0.24 and 0.12, respectively) than the control (A (540)=0.01) in the modified R2YE medium. The mature form of DagA protein (32 kDa) was successfully purified by one-step affinity column chromatography by using agarose beads with excellent yield. The purified DagA was found to exhibit maximal agarase activity at 40 °C and pH 7.0. The K(m), V(max), and K(cat) values for agarose were 2.18 mg/ml (approximately 1.82 × 10(-5) M), 39.06 U/mg of protein, and 9.5 × 10(3)/s, respectively. Thin layer chromatography (TLC) analysis, matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry, and Fourier transform nuclear magnetic resonance (FT-NMR) spectrometry of the hydrolyzed products of agarose by DagA revealed that DagA is an endo-type β-agarase that degrades agarose into neoagarotetraose and neoagarohexaose.

Purification and characterization of β-agarase from seaweed decomposing bacterium Microbulbifer sp. Strain CMC-5

Microbulbifer strain CMC-5 was isolated from decomposing seaweeds, and was found to degrade agar, alginate, carboxymethyl cellulose, carrageenan, xylan, and chitin. The extracellular agarase enzyme from strain CMC-5 was purified 103-fold by ultrafiltration, ion-exchange chromatography, using diethylaminoethyl sepharose FF, and gel filtration, using sephacryl S-300HR, with a yield of 6.7%. Zymogram and protein staining of the purified agarase on a SDS-polyacrylamide gel revealed a single band, with an apparent molecular weight of 59 kDa. The purified enzyme was endo-type β-agarase, as it was able to hydrolyze the β-1, 4 glycosidic linkages of agarose, releasing neoagarotetraose and neoagarohexaose as the end products. The optimum pH and temperature of agarase were 7 and 50°C, respectively. Thermal stability studies indicated that the agarase retained 62% of its activity after incubating at 50°C for 30 min. Treatment with EDTA reduced the agarase activity by 54%. The agarase activity was stimulated by the presence of Ca2+ and Mg2+ ions; whereas, Zn2+, Hg2+, Cu2+, Fe2+, and Co2+ abolished the activity. Further, the presence of NaCl at concentrations lower than 100 mM caused a decrease in the agarase activity; whereas, the activity was enhanced up to a concentration of 500 mM.

Evaluation of HPSEC‐ELSD method for precise measurement of β‐agarase activity

β-agarase activity was monitored by traditional reducing sugar content methods: Somogyi-Nelson's arsenomolybdate, Miller's dinitrosalicylic acid and Kidby and Davidson's ferricyanide methods, as well as by high-performance size exclusion chromatography coupled with a refractive index detector and an evaporative light scattering detector (ELSD). Calibration curves were established separately for each method to measure the amounts of the neoagaro-oligosaccharides (NAOS) in the reaction mixtures, which are the products from 1-10 units (U) of β-agarase cleavage activity on agarose. Product quantities from each monitoring method were compared with the isolated NAOS products. The graphs plotted by agarase activity unit and product concentration clearly displayed that the ELSD method closely followed the results of the isolated products. The percentage deviation of results measured by the five methods away from those of the isolated NAOS product mixture amounted to -13.1-35.1, -21.1-25.5, -27.1-23.81, 6.1-24.3 and 16.2-22.8%, respectively. When the loss during product isolation, about 15-17%, was taken into account, the high precision of the ELSD method was confirmed. HPSEC-ELSD methods also accurately measured the enzyme kinetics as well as enabling partial identification of oligosaccharides assembled in the NAOS product mixture. This study established the HPSEC-ELSD system as an alternative method for monitoring agarase activity.

Agar-based pellets as feed for sea urchins (Paracentrotus lividus): rheological behaviour, digestive enzymes and gonad growth

The algal polysaccharide agar has long been used as a food binder due to its structure, rheological behaviour, stability and interactions – properties that help to generate firm, round, disk-shaped pellets that may be used in recirculating sea urchin-rearing systems. Three algae-based diets (Ulva lactuca, Gracilaria gracilis, Cystoseira sp.) containing 3% and 6% agar were tested on the sea urchin Paracentrotus lividus in order to examine the effect of varying percentages of agar on pellet stability in water and sea urchin gonad growth. The kinetics of water absorption and solute leaching of pellets were measured by immersing quadruplicate samples of the pellets in water for 1, 2, 3, 4, 5 and 6 days. Our results show that the pellets had good water stability, were readily consumed by sea urchins and the presence of agar did not hamper sea urchin gonad growth. Animals fed Ulva-containing pellets reached a more advanced gametogenic stage with respect to animals fed Cystoseira- and Gracilaria-containing pellets. Moreover, the presence of agarase activity in the digestive system indicated that agar may be an energy source. Pellets are relatively low cost and easy to prepare and store. They may represent a useful resource for rearing sea urchins under intensive conditions.

Production of agarase from a novel Micrococcus sp. GNUM-08124 strain isolated from the East Sea of Korea

The agar degrading bacterial strain GNUM-08124 was isolated from Enteromorpha compressa collected in the East Sea of Korea by using a selective artificial sea water (ASW) agar plate containing agar as the sole carbon source. GNUM-08124 grows to produce a circular, smooth, yellow-colored, and raised colony. Its ability to hydrolyze agar was confirmed by staining the ASW agar plate with Lugol’s solution. In liquid culture, the cell density (A600) increased exponentially and reached a maximum level on the third day of cultivation. The specific agarase activity also increased in proportion to the cell density and reached maximum agarolytic activity on the third day. The 16S rRNA sequence of GNUM-08124 showed a close relationship to Micrococcus luteus (99.65%) and Micrococcus endophyticus (99.15%), which led us to assign it to the genus Micrococcus. Physiological studies indicated that optimal growth conditions were between 30 and 40°C, pH 4 and 7, using media containing between 5 and 10% NaCl (w/v), respectively. The GNUM-08124 strain was a grampositive, urease-positive, and catalase-positive bacterium. It could not hydrolyze gelatin, cellulose, xylan, or starch, but fermented a broader range of substrates, including Dglucose, D-galactose, D-fructose, D-lactose, D-trehalose, D-mannitol, D-melibiose, D-raffinose, D-xylose, methyl-α-D-glucopyranoside, N-acetyl-glucosamine, and xylitol, than those fermented by M. luteus or M. endophyticus, suggesting GNUM-08124 is a novel agar hydrolyzing microorganism belonging to Genus Micrococcus. Micrococcus sp. GNUM-08124 showed the highest agarase activity when it was cultured in ASW-YP medium supplemented with 0.4% glucose, but demonstrated lower activity in rich media (LB or TSB), in spite of superior cell growth, implying that agarase production is tightly regulated in an agar-dependent manner and repressed in rich conditions.

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 °C, 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, encoding β-agarases, had been cloned from genomic DNA of Agarivorans albus strain QM38. The open reading frame of agaD01, 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% 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.

Production of 3,6-anhydro- l-galactose from agarose by agarolytic enzymes of Saccharophagus degradans 2-40

Saccharophagus degradans 2-40 is capable of hydrolyzing agarose, a red macroalgae-derived polymer, into d-galactose and 3,6-anhydro- l-galactose (L-AHG). Its agarase system is receiving much attention because it can be used to produce fermentable sugar from agarose. L-AHG is commercially unavailable and is considered a rare sugar with a high value. In this study, cells grown on agarose, agar or red macroalgae biomass were found to have a significantly higher agarase activity and AHG-generating activity than those grown on glucose or galactose. From agar-grown cells, both the volumetric activities of agarases and AHG generation in the cell-free lysate were much higher than in the extracellular fraction. Based on the analyses of the enzyme reaction products, from the reaction with the crude enzymes from cell-free lysate, neoagarobiose with a degree of polymerization (DP) 2 appeared to be the only major product in the initial reaction period, but sugars with DPs 2, 4 and 6 were found to be all predominantly produced by the extracellular enzymes in the initial reaction period. Quantitative analysis of AHG using gas chromatography–mass spectrometry with a derivatization step was also found to be highly reproducible and reliable. These results will be useful for producing L-AHG as a rare sugar to investigate its metabolic fate and commercial utilization.

한천분해세균 Cellvibrio mixtus SC-22의 분리 및 효소적 특성

한천분해세균 SC-22균을 대전 대청댐부근의 담수에서 분리하였다. 생화학적 분석 및 16S rRNA 염기서열 분석을 통한 계통학적 분류를 통해 SC-22는 Cellvibrio mixtus로 동정되었다. 분리균의 생육 및 agarase 효소 생성능을 검토한 결과, SC-22는 탄소원으로 0.2% agar를 첨가한 배지에서 배양 36시간에 최대 생육을, 배양 48시간에 58.5 units/mL의 최대 효소활성을 나타내었다. 분리균은 세포외 및 세포내 agarase를 생성하였으며, zymogram 실험에 의해 P1, P2 및 P3의 isoenzyme을 분비하는 것으로 확인 되었다. 배양여액으로부터 겔여과법과 이온교환수지법을 단계적으로 이용하여 SC-22 균주로부터 SDS-PAGE에 의해 25 kDa의 효소를 정제하였으며, 정제효소는 zymogram에서 확인된 주 단백질인 P2(29 kDa)과 동일한 단백질임이 확인되었다. 또한 정제한 agarase의 효소학적 성질을 검토한 결과, 효소의 최적 pH와 최적온도는 pH 7.0과 <TEX>$50^{\circ}C$</TEX>이었으며, 금속이온 효과의 경우 1 mM 농도의 수준에서도 <TEX>$Fe^{2+}$</TEX>, <TEX>$Na^+$</TEX>, <TEX>$Ca^{2+}$</TEX> 이온 등은 정제효소의 활성을 10-20% 증가시킨 반면 <TEX>$Hg^{2+}$</TEX>, <TEX>$Mn^{2+}$</TEX> 및 <TEX>$Cu^{2+}$</TEX> 이온들은 효소 활성을 크게 저해하였다. 또한 TLC 분석을 통해 정제효소는 한천 분해 올리고당으로 주로 단당류와 이당류를 생성하므로 <TEX>$\beta$</TEX>-agarase의 작용특성을 보였다. 기질특이성 실험에서는 정제효소는 agar와 agarose만을 이용 하였고 유사 해조 다당류인 alginate는 물론 다른 다당류를 분해하지 못하였다. An agar-liquefying bacteria (SC-22), which produces a diffusible agarase that caused agar softening around the colony was isolated from Daecheong lake in Korea. Chemotaxanomic and phylogenetic analyses based on 16S rRNA gene sequences revealed the strain was classified as Cellvibrio mixtus SC-22. The isolate SC-22 showed maximal extracellular agarase activity with 58.5 U/mL after 48 h cultivation in the presence of 0.2% agar. It was observed that the isolate produced two kinds of extracellular and three kinds of intracellular isoenzymes. The major agarase was purified from the culture filtrate of agarolytic bacteria by ammonium sulfate precipitation, anion exchange and gel filtration column chromatographic methods. The molecular mass of the purified enzyme was estimated to be 25 kDa by SDS-PAGE. The optimum pH and temperature of the purified enzyme were pH 7.0 and <TEX>$50^{\circ}C$</TEX>, respectively. The agarase activity was activated by <TEX>$Fe^{2+}$</TEX>, <TEX>$Na^+$</TEX> and <TEX>$Ca^{2+}$</TEX> ions while it was inhibited by <TEX>$Hg^{2+}$</TEX>, <TEX>$Mn^{2+}$</TEX> and <TEX>$Cu^{2+}$</TEX> at 1 mM concentration. The predominant hydrolysis product of agarose by the enzyme was galactose and disaccharide on TLC, indicating the cleavage of <TEX>$\beta$</TEX>-1,4 linkage in a random manner. The enzyme showed high substrate specificity for only agar and agarose among various polysaccharides.

Pseudozobellia thermophila gen. nov., sp. nov., a bacterium of the family Flavobacteriaceae, isolated from the green alga Ulva fenestrata

Two novel aerobic, dark-orange-pigmented, Gram-negative bacterial strains, designated KMM 3531(T) and KMM 3953, were isolated from the green alga Ulva fenestrata. Analysis of the 16S rRNA gene sequences showed that the strains represented a novel lineage within the family Flavobacteriaceae. The most closely related genera with validly published names were Costertonia, Flagellimonas, Kriegella, Maribacter, Muricauda and Zobellia, with 16S rRNA gene sequence similarities of 93.3-91.8 %. Cells of strains KMM 3531(T) and KMM 3953 were rod-shaped, motile by gliding and grew at temperatures up to 49 degrees C. They produced acid from carbohydrates and possessed oxidase, catalase, beta-galactosidase and agarase activities. The predominant cellular fatty acids were iso-C(15 : 0,) iso-C(17 : 0) 3-OH, iso-C(15 : 1) G, summed feature 3 (comprising C(16 : 1)omega7c and/or iso-capital ES, Cyrillic(15 : 0) 2-OH), iso-C(17 : 1)omega9c and iso-C(15 : 0) 3-OH. The DNA G+C content was 47-49 mol%. On the basis of phenotypic and genotypic characteristics, strains KMM 3531(T) and KMM 3953 represent a novel genus and species, for which the name Pseudozobellia thermophila gen. nov., sp. nov. is proposed. The type strain is KMM 3531(T) (=DSM 19858(T)=JCM 11733(T)=KCTC 22016(T)).