Purification and characterization of β-agarase from seaweed decomposing bacterium <Emphasis Type="Italic">Microbulbifer</Emphasis> 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.     

Gene cloning,expression and characterization of a neoagarotetraose-producing β-agarase from the marine bacterium <Emphasis Type="Italic">Agarivorans</Emphasis> 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⁺, Mg²⁺ and Ca²⁺. 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 × 10⁵ M⁻¹ s⁻¹, respectively. Agarase rHZ2 could have a potential application in the production of bioactive neoagarotetraose.     

New Sulfated Oligosaccharides Produced by Pseudomonas β-Agarase from Gracilaria verrucosa Polysaccharide

Polysaccharide (partially sulfated agarose) with macrophage-stimulation activity, derived from Gracilaria verrucosa, was decomposed by two types of β-agarase (agarases II and IV) from Pseudomonas sp. O-148. The hydrolysates were fractionated with ethanol precipitation and anion-exchange chromatography. The resulting anionic oligosaccharides with sulfate groups were investigated by ¹³C-NMR spectroscopy. While the spectra of oligosaccharides produced by agarase IV showed identical patterns with those by β-agarase I from Pseudomonas atlantica and indicated the location of a sulfated saccharide unit on the non-reducing end, another new type of saccharide was found in the products by agarase II. The novel oligosaccharides by agarase II had a neoagarobiose unit on their non-reducing end and had sulfated units internally. This indicated the novelty of agarase II in cleavage fashion.     

Interaction mechanism between Cd2+ ions and DNA from the kidney of the silver crucian carp

Cadmium is one of the most toxic heavy metals and is known to accumulate in freshwater food chains. The underlying mechanism for its genotoxicity has not been investigated for any freshwater fish. It has, however, been suggested that cadmium-induced carcinogenesis might involve either direct or indirect interaction of Cd²⁺ with DNA. The interaction between Cd²⁺ and DNA from the kidney of the silver crucian carp (Carassius auratus gibelio) in vitro and in vivo is investigated by spectrophotometric methods and agarose gel electrophoresis methods. Cd²⁺ could insert into DNA basepairs, bind to nucleic acid, and result in notable hypochromicities. The analysis of agarose gel electrophoresis, proves that Cd²⁺ at different concentrations does not cause DNA cleavage in vitro; however, kidneys display the classical laddering degradation of DNA in vivo, which is the result of the promotion of deoxyribonuclease activity or inhibition of superoxide dismutase and catalyse activity and the accumulation of reactive oxygen species caused by Cd²⁺ ions in vivo.     

Cloning and sequencing of agaA, a unique agarase 0107 gene from a marine bacterium, Vibrio sp. strain JT0107.

An agarase gene (agaA) was cloned from genomic DNA of Vibrio sp. strain JT0107. An open reading frame of 2,985 nucleotides gave a primary translation product composed of the mature protein, agarase 0107 (975 amino acid residues, with a molecular weight of 105,271) and a signal peptide of 20 amino acid residues at the N terminus. Comparison of the deduced amino acid sequence of agarase 0107 with those of Streptomyces coelicolor and Pseudomonas atlantica suggests that these enzymes share two regions in common. The AgaA protein which was expressed in Escherichia coli had the agarase activity. Agarase 0107 hydrolyzes not only agarose but also neoagarotetraose [O-3,6-anhydro-alpha-L-galactopyranosyl (1-->3)-O-beta-D-galactopyranosyl(1-->4)-O-3,6-anhydro-alpha-L-galact opy ranosyl (1-->3)-D-galactose] to yield neoagarobiose [O-3,6-anhydro-alpha-L-galactopyranosyl(1-->3)-D-galactose]. This is a quite unique characteristic for a beta-agarase.     

The removal of cell surface material by enzymes used to dissociate mammary-gland cells

Summary Treatment of mouse mammary epithelial cells (MMEC) with various enzymes used for dispersing and transferring cells results in extensive digestion of materials on the cell surfaces. MMEC biosynthetially labeled with [³H]fucose, [¹⁴C]fucose and [³H]amino acids or with¹²⁵I by the lactoperoxidase method were exposed to either collagenase plus hyaluronidase, followed by pronase, or to trypsin in concentrations and conditions currently used for cell dispersion. Whereas the latter enzyme preparation solubilized 76% of the trichloroacetic acid precipitable radioactive fucose and 96% of the protein-bound¹²⁵I, collagenase plus hyaluronidase treatment released lesser amounts of each label. Subsequent treatment of the cells with pronase removed additional surface-labeled materials, but the total amounts released were still less than when the trypsin preparation alone was employed. Released cell surface materials were analyzed by gel chromatography. Some of the peaks obtained also were examined by polyacrylamide gel electrophoresis. The labeled materials that remained attached to the MMEC after enzymatic treatment were investigated by these two methods as well. We could show that collagenase plus hyaluronidase solubilized three main glycoprotein components from the cell surface. In addition, we could show that the extensive cell surface damage caused by these two enzyme preparations was due to the high proteolytic activity present in these preparations as judged by their ability to hydrolyze rabbit gamma globulin labeled with¹²⁵I. Even though their membranes were extensively damaged by the enzyme treatments, the dispersed cells could be cultured successfully in vitro and could incorporated fucose into their surfaces in a manner similar to that by intact tissue. Through the use of gel-filtration (cochromatography of [¹⁴C]fucose and [³H]fucose cell surface materials), we could demonstrate the identity of cell surface glycoproteins synthesized by cultured cells and by intact tissue. This work was supported by Grant Nos. CA 11736 and CA 19455 from the National Cancer Institute, and Biomedical Research Support Grant No. RR05467 from the National Institutes of Health, DHEW.     

Physical and nutritional factors in gel culture of mammalian cells

Summary The growth of human glioma cells, cultured as spherical colonies in agarose gel, stopped after about 10 days for both large and small colonies apparently due to an increased osmolality in the gel. When osmolality was kept under control by addition of distilled water, growth continued. However, a continuous increase in the population-doubling period, similar for both large and small colonies, then was observed. The increase persisted although excess amounts of nutrition were added. When the cells were cultured in liquid suspension above a thin layer of agarose gel and most of the medium was repeatedly changed, the colonies continued to grow beyond the limits in gel culture. HeLa and hamster embryonic lung cell colonies showed a growth pattern in agarose gel similar to the glioma cells. The results imply that the osmolality must be kept under precise control to prevent growth inhibition. However, it seems difficult to ascertain optimal growth in gel culture for more than about 2 weeks probably because of the accumulation of toxic products. The work was supported financially by the Swedish Cancer Society and the Swedish Natural Science Research Council.     

Hybridoma cells in a protein-free medium within a composite gel perfusion bioreactor

A composite gel system has been developed combining the chemical and physical properties of calcium alginate and agarose gels. The results of growing composite gel immobilized hybridoma SPO1 cells in a protein-free medium within a fluidized-bed perfusion bioreactor are presented in this paper. During the continuous operation of this system, the total cell density reached 3.9×10⁷ cells per ml of beads (viability 79.6%). The specific productivity of monoclonal antibody of the immobilized hybridoma cells reached more than 1.5 g per 10⁶ viable cells per hour, compared with 0.5 for non-immobilized viable cells grown in a one liter agitated bioreactor with the same medium. Significant increases in cell metabolic activities, including substrate utilization and byproduct formation, were also observed. Leaching of materials from the beads was evident and the major fraction of released materials was alginate.     

A new β-agarase from marine bacterium <Emphasis Type="Italic">Janthinobacterium</Emphasis> sp. SY12

An agar-degrading bacterium, strain SY12, was identified as the genus Janthinobacterium, which is a member of the class Betaproteobacteria. A β-agarase gene agaY was cloned from SY12, and it is the first reported agarase from the Betaproteobacteria. AgaY consisted of 1,338 bp encoding 445 amino acid residues, and it was assigned to GH16 family. AgaY has an N-terminal secretary leader peptide preceding a GH16 catalytic domain and a CBM13 carbohydrate binding module. The recombinant agarase AgaY overexpressed in Escherichia coli displayed a molecular mass of 50.2 kDa and the optimum temperature and pH for the activity of the enzyme was 40°C and pH 7.0, respectively. It degraded agarose to give neoagarotetraose and neoagarobiose as the main products. Interestingly, in contrast to other agarases of GH16, the enzymatic activity of AgaY is Na⁺ and Ca²⁺ independent.     

Repeated batch production of agar-oligosaccharides from agarose by an amberlite IRA-900 immobilized agarase system

The production of agar-oligosaccharides from agarose by free and immobilized agarase, obtained from a Pseudomonas aeruginosa AG LSL-11 was investigated and the activity, longevity and the operational stability of immobilized enzyme was compared with that of the free enzyme. The agar hydrolyzed products of free enzyme and immobilized enzyme were neoagarobiose, neoagarotetraose and neoagarohexaose as evidenced by LC-MS analysis. The immobilization of agarase was confirmed by SEM and also by the enzymatic transformation of agarose into agaroligosaccharides. The free agarase showed maximum activity at 40°C, whereas it’s immobilized counterpart showed maximum activity at 45oC, however, the optimum pH for both systems remained unchanged (pH 8.0). The relative activities of free agarase at pH 9.0 and 10.0 were 90 and 74%, respectively, whereas, the corresponding activities of the immobilized system were determined to be 97 and 90%. The stabilities of free agarase at pH 9.0 and 10.0 were 80 and 60% respectively, but for the immobilized system the respective residual activities were estimated to be 97 and 85%. Immobilized agarase appears to be more tolerant to high temperatures in terms of its activity and stability as it is compared to that of the free enzyme which retained 74 and 50.84% of relative activity at 55 and 60°C while, free agarase retained only 40 and 16.79% of its original activity. Furthermore, the immobilized agarase could be reused in batches efficiently for eight cycles, and could be stored for 3 months at 4°C as wet beads and for more than 6 months as dry beads.     

Purification, characterization and application of a novel extracellular agarase from a marine Bacillus megaterium

A marine, gram positive, aerobic, spore forming, and non flagellated bacterium which degrades low melting point (LMP) — agarose was isolated from the west coast of India and identified as Bacillus megaterium based on its morphological, biochemical, and molecular characterization. This bacterium produced clear haloes or zone of clearance on agar containing plates which was a clear indication of its agarolytic property. The extracellular agarase thus obtained was purified 8.8 and 78 fold from the culture supernatant by ammonium sulfate precipitation and gel filtration, respectively. Molecular mass by gel filtration and SDS-PAGE gave values of 15 and 12 kDa, respectively. The optimum temperature and pH for maximum agarase activity were 40°C and 6.6. The activity of agarase was drastically reduced by addition of metal ions in the assay system. This agarase, gave a K _m and V _max value of 4 mg/mL and 2.75 μmol/min/mg. The isolation of protoplast from agarophyte like Gelidiella acerosa using indegenous agarase is reported for the first time.     

The thermophilic bacteria hydrolyzing agar: Characterization of thermostable agarase

Three strains of anaerobic thermophilic bacteria capable of growing on agarose as a source of energy and carbon were isolated from hot springs near Lake Baikal (Barguzin National Park) and the caldera Uzon (Kamchatka). Cells of all the three strains were spore bacilli with peritrichous flagellation. These isolates grew at a temperature of 55–60°C and pH 6.5–7.0 and fermented a wide range of organic substrates. Analysis of the 16S rRNA sequences allowed us to ascribe the strains B5 and K14 to the genus Thermoanaerobacter and the strain K67 to the genus Caldoanaerobacter. According to the results of DNA-DNA hybridization, B5 was determined as belonging to the species Thermoanaerobacter wiegelii. Agarase was isolated by preparative PAGE and subsequent gel chromatography from the culture liquid of strain B5 grown on the medium containing 0.5% agarose and 0.3% galactose. The molecular weight of this enzyme amounted to 67 kDa and pI, to 4.2. The T. wiegelii B5 agarase was active in the pH range of 3.5 to 7.0 (optimum, 5.2) and temperature range of 50 to 80°C (optimum, 70°C). The preincubation of this enzyme at 90° C for 60 min did not reduce the agarase activity. This activity increased in the presence of metal ions; the maximal effect was observed in the presence of 5 mM Mg²⁺ and 25 mM Co²⁺.     

Superporous agarose monoliths as mini-reactors in flow injection systems

A new type of agarose material, superporous agarose, was used as a support material in an analytical system designed for monitoring of bioprocesses with respect to metabolites and intracellular enzymes. The superporous agarose was used in the form of miniaturised gel plug columns (15×5.0 mM I.D. monolithic gel bed). The gel plugs were designed to have one set of very large pores (about 50 m in diameter) through which cells, cell debris and other particulate contaminants from the bioreactor could easily pass. The material also had normal diffusion pores (300 Å) characteristic of all agarose materials, providing ample surface for covalent attachment of antibodies and enzymes used in the analytical sequence. The superporous agarose gel plug columns were characterised with respect to flow properties and handling of heavy cell loads as well as dispersion of injected samples (a Bodenstein number of about 40 was observed with acetone tracer at a flow rate of 1 ml min^–1). To evaluate the practical performance of the superporous gel plug columns, two applications were studied: (1) on-line determination of glucose in cultivation broth (gel plug with immobilized glucose oxidase) and (2) immunochemical quantification of intracellular -galactosidase in E. coli (gel plug with lysozyme to achieve cell lysis and gel plug with antibodies against -galactosidase).     

Short protocol for pulsed field gel electrophoresis of a variety of Clostridia species

While pulsed field gel electrophoresis has become an important tool for genotyping of bacteria, one of its drawbacks is that standard methods are rather time-consuming. In order to overcome this problem, shortened procedures for DNA preparation have been developed for some bacterial species. The aim of this study was to examine if a short procedure used for pulsed field gel electrophoresis of Clostridium botulinum could be applied to other Clostridia species. For this, the protocol was modified and used to prepare the DNA of 34 strains of 25 different Clostridia species. In contrast to a standard procedure, which takes at least 5 days from DNA extraction to completion of the electrophoresis, this protocol yielded results within 2 days. In order to directly compare the results of the short protocol with those of the standard, long procedure, parallel DNA preparations were performed using both methods and the two DNA samples thus obtained per strain were then run on the same gel. Briefly, the procedure was as follows. After embedding the bacterial cells in agarose, the agarose blocks were incubated for 1 h in lysis solution containing lysozyme, mutanolysin, lysostaphin and RNase. This was followed by a 1-h proteinase K treatment. Then, slices were cut from the agarose blocks and washed for 15 min in TE buffer, these washes were repeated four times with fresh TE. After a 2-h restriction with SmaI, electrophoresis was carried out overnight.     

Modifications of Δ5-3-ketosteroid isomerase induced by ultraviolet irradiation in the presence of the solid-phase photoaffinity reagent Δ6-testosterone agarose

The nature of the products formed during the photoinactivation of Δ⁵-3-ketosteroid isomerase in the presence of the solid-phase photoaffinity reagent Δ⁶-testosterone succinyl agarose has been investigated after ultraviolet irradiation. The polypeptide products eluted from the agarose phase by sodium cholate, sodium dodecyl sulfate, and pH 10.5 triethylamine buffer have been characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis, pH 4–6 gel isoelectric focusing, and amino acid analysis. The amino acid compositions of the cholate eluted and SDS eluted products are found to be similar to that of native isomerase, whereas the covalently bound polypeptide eluted by pH 10.5 triethylamine possesses a distinetly different composition. Digestion of the covalently bonded isomerase polypeptide with trypsin yields an agarose-bound peptide fraction that has been characterized by its amino acid composition. This composition is different from that of the undigested covalently bound polypeptide and suggests that the site of covalent attachment lies somewhere between residues 28 and 45 of the isomerase polypeptide.     

Isolation of a novel freshwater agarolytic Cellvibrio sp. KY-YJ-3 and characterization of its extracellular beta-agarase

A novel agarolytic bacterium KY-YJ-3, producing extracellular agarase, was isolated from the freshwater sediment of the Sincheon River in Daegu, Korea. On the basis of gram-staining data, morphology, and phylogenetic analysis of the 16S rDNA sequence, the isolate was identified as Cellvibrio sp. By ammonium sulfate precipitation followed by Toyopearl QAE-550C, Toyopearl HW-55F, and Mono-Q column chromatography, the extracellular agarase in the culture fluid could be purified 120.2-fold with yield of 8.1%. The specific activity of the purified agarase was 84.2 U/mg. The molecular mass of the purified agarase was 70 kDa as determined by dodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal temperature and pH of the purified agarase were 35 degrees C and pH 7.0, respectively. The purified agarase failed to hydrolyze the other polysaccharide substrates, including carboxymethyl (CM)-cellulose, dextran, soluble starch, pectin, and polygalacturonic acid. Kinetic analysis of the agarose-hydrolysis catalyzed by the purified agarase using thin layer chromatography (TLC) exhibited that the main products were neoagarobiose, neoagarotetraose, and neoagarohexaose. These results demonstrated that the newly isolated freshwater agarolytic bacterium KY-YJ-3 was a Cellvibrio sp., and could produce an extracellular beta-agarase, which hydrolyzed agarose to yield neoagarobiose, neoagarotetraose, and neoagarohexaose as the main products.     

A Novel β-Agarase with High pH Stability from Marine Agarivorans sp. LQ48

A novel endo-type β-agarase gene, agaA, was cloned from a newly isolated marine bacterium, Agarivorans sp. LQ48. It encodes a protein of 457 amino acids with a calculated molecular mass of 51.2 kDa. The deduced protein contains a typical N-terminal signal peptide of 25 amino acid residues, followed by a catalytic module, which is homologous to that of glycoside hydrolase family 16. A sequence similar to a carbohydrate-binding module is found in the C-terminal region of the enzyme. The overall amino acid sequence shares a highest identity of 73% with the sequence of beta-agarase AgaB from Pseudoalteromonas sp. strain CY24. The mature agarase was highly expressed extracellularly in Escherichia coli. At pH 7.0 and 40°C, the purified recombinant AgaA had a high specific activity of 349.3 μmol min⁻¹ mg⁻¹, a K _m of 3.9 mg ml⁻¹, and a V _max of 909.1 μmol min⁻¹ mg⁻¹ for agarose. The recombinant enzyme hydrolyzed the β-1,4-glycosidic linkages of agarose, yielding neoagarotetraose and neoagarohexaose as the main products. Enzyme activity analysis revealed that the optimal temperature and pH of the recombinant AgaA were 40°C and 7.0, respectively. Notably, AgaA still retained more than 95% activity after incubation at pH 3.0–11.0 for 1 h, a characteristic much different from other agarases reported. It is the first agarase identified to have so wide a pH range stability. This favorable property could make AgaA to be attractive to the food, cosmetic, and medical industrial applications.     

A support for affinity chromatography that covalently binds amino groups via a cleavable connector arm.

The preparation of a new succinimidyl ester agarose derivative (SEPE-Agarose) is described. This agarose derivative can be used for covalently linking proteins and other ligands containing amino groups to agarose via phenyl ester linkages that can later be broken under mild conditions which should not alter other groups which may be present in proteins such as cystinyl residues and glycosyl residues. SEPE-Agarose is prepared by the reaction of bis[4-[2-(N-succinimidoxycarbonyl)ethyl]phenyl]succinate with an aminoethylcarbamylmethyl derivative of agarose. Studies of the covalent binding and release of trypsin and myoglobin to SEPE-Agarose indicate that gels containing 0.1 to 0.6 μmol protein/ml of gel are obtained by reacting protein (0.5–5 mg/ml) with the N-succinimidyl ester groups in SEPE-Agarose. Protein-linked gel is reasonably stable in dilute phosphate buffers (pH ≤ 7.4, ≤ 25 °C). Protein is released from the gel, however, by treatment at 25 °C with solutions containing nucleophiles such as 1 m imidazole-glycine, pH 7.4, for 4 h, or 1 m hydroxylamine, pH 7, for 10 min. Protein is also released from the gel by treatment with 1 m Tris pH 8.2 for 24 h. SEPE-Agarose should prove useful in affinity chromatography and immunoabsorption when it is difficult or impractical to elute material bound to conventional affinity supports.     

Purification and characterization of β-agarase from agar-liquefying soil bacterium, Acinetobacter sp., AG LSL-1

The extracellular β-agarase LSL-1 produced by an agar-liquefying, soil bacterium Acinetobacter sp., AG LSL-1 was purified to homogeneity by combination of ion-exchange and size exclusion chromatography with final yield of 44%. The enzyme has a specific activity of 397 U mg⁻¹ protein and with a molecular mass of 100 kDa. The agarase was active in the pH range of 5.0–9.0, optimally at pH 6.0 and temperature between 25 °C and 55 °C and optimal at 40 °C. The enzyme retained 63% of native activity at 50 °C suggesting it is a thermostable. The activity of the agarase was completely inhibited by metal ions, Hg²⁺, Ag⁺ and Cu²⁺, whereas 25–40% of native activity was retained in the presence of Zn²⁺, Sn²⁺ and SDS. Neoagarobiose was the final product of hydrolysis of both agarose and neoagarohexaose by the purified agarase LSL-1. Based on the molecular mass and final products of agarose hydrolysis, the β-agarase LSL-1 may be further grouped under group III β-agarases and may be a member of GH-50 family. This is the first report on the purification and biochemical characterization of β-agarase from an agar-liquefying Acinetobacter species.