Characterization of the agarase system of a multiple carbohydrate degrading marine bacterium

A marine bacterium strain 2-40 (2-40) degraded numerous complex carbohydrates, such as agar, chitin and alginate. It may play an important role in altering carbon fluxes in marine environments. End-product analyses revealed that 2-40 synthesized an agarase system that consisted of at least three enzymes, beta-agarase I, beta-agarase II and alpha-agarase, which acted in concert to degrade polymeric agar to D-galactose and 3,6-anhydro-L-galactose. The agarase system was shown to be both cell envelope-associated and extracellular, with the relative concentrations depending on the growth phase. The principal depolymerase, a beta-agarase I, hydrolysed agar to both neoagarotetrose and neoagarobiose, as identified by thin layer chromatography. This agarase had a mass of 98 kD and a Pi of 4.3. The agarase system was repressed by D-glucose and D-galactose and induced by agar, agarose, neoagarobiose, neoagarotetrose and neoagarohexose.     

Purification and characterization of a novel beta-agarase from Vibrio sp. AP-2

beta-Agarase was purified from the culture fluid of a porphyran-decomposing marine bacterium (strain AP-2) by ammonium sulfate precipitation, successive column chromatography and DNase and RNase treatment. The final enzyme preparation appeared to be homogeneous on polyacrylamide gel electrophoresis. The enzyme had a molecular mass of 20 kDa, a pH optimum of 5.5, and was stable in the pH region 4.0-9.0 and at temperatures below 45 degrees C. The beta-agarase was a novel endo-type enzyme which hydrolyzed neoagarotetraose, larger neoagarooligosaccharides and agar to give neoagarobiose [3,6-anhydro-alpha-L-galactopyranosyl-(1----3)-D-galactose] as the predominant product. The enzyme did not act on kappa-carrageenan. According to the criteria of Bergey's Manual of Systematic Bacteriology, the strain was assigned to the genus Vibrio.     

Enzymatic hydrolysis of agar: purification and characterization of beta-neoagarotetraose hydrolase from Pseudomonas atlantica.

Agarose is degraded by a beta-agarase from Pseudomonas atlantica to neoagarooligosaccharides of degree of polymerization (DP), 4, 6, 8, and 10. A beta-neoagarotetraose hydrolase cleaves the central beta-linkage in neoagarotetraose and the beta-linkage near the nonreducing end in neoagarohexaose and -octaose to yield neoagarobiose. The beta-neoagarotetraose hydrolase was localized on or outside the cytoplasmic membrane, in the cell wall region. The enzyme was activated by NaCl, KCl, CaCl2, MnCl2, and MgSO4, has a Km of 3.4 X 10(-3) M for neoagarotetraose, was free from beta-agarase and alpha-neoagarobiose hydrolase activity, and showed no transglycosidic activity.     

Separation and characterization of two activities from HeLa cell nuclei that degrade double-stranded RNA

Two enzymatic activities that degrade double-stranded RNA have been partially purified from HeLa cell nuclei using reoviral [3H]RNA as the substrate. The two active fractions, separated by chromatography on phosphocellulose, are designated PC I and PC II. Both fractions degrade a variety of double-stranded RNAs with an absolute requirement for a divalent cation. However, they are distinct by at least five criteria. 1)PC I degrades a variety of single- and double-stranded RNAs, single- and double-stranded DNAs, and DNA.RNA hybrids, in addition to double-stranded reoviral RNA. In contrast, PC II has maximal activity with reoviral RNA, some activity with rRNA, and much less activity with the other substrates. 2) Analyses of reaction products by sucrose gradient centrifugation and chromatography on Sephadex G-100 and DEAE-cellulose indicate that, PC I cleaves reoviral RNA endonucleolytically to a final mixture of mono- and oligonucleotides, whereas the only acid- or alcohol-soluble products of PC II are 5'-XMPs produced exonucleolytically. 3) PC I activity is stimulated 2-fold more by MnCl2 than by MgCl2, whereas PC II activity is stimulated 3-fold more by MgCl2 than by MnCl2. 4) PC I activity is inhibited by NaCl concentrations as low as 10 mM, whereas PC II requires 50 to 80 mM NaCl for optimal activity. 5)Estimated by their sedimentation rates in glycerol gradients, PC I and PC II have apparent molecular weights of 55,000 and 20,000, respectively.     

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.     

Beta-agarases I and II from Pseudomonas atlantica. Substrate specificities

Beta-Agarase I and II were characterised by their action on agar-type polysaccharides and oligosaccharides. Beta-Agarase I, an endo-enzyme, was specific for regions containing a minimum of one unsubstituted neoagarobiose unit [3,6-anhydro-alpha-L-galactopyranosyl-(1 leads to 3)-D-galactose], hydrolysing at the reducing side of this moiety. Yaphe demonstrated that agar was degraded by this enzyme to neoagaro-oligosaccharides limited by the disaccharide but with a predominance of the tetramer [Yaphe, W. (1957) Can. J. Microbiol. 3, 987-993]. Beta-Agarase I slowly degraded neoagarohexaose but not the homologous tetrasaccharide. [1-3H]Neoagarohexaitol was cleaved to neoagarotetraose and [1-3H]neoagarobiitol. The highly substituted agar, porphyran was degraded to methylated, sulphated and unsubstituted neoagaro-oligosaccharides which were invariably terminated at the reducing end by unsubstituted neoagarobiose. The novel enzyme, beta-agarase II, was shown to be an endo-enzyme. Preliminary evidence indicated this enzyme was specific for sequences containing neoagarobiose and/or 6(1)-O-methyl-neoagarobiose. It degraded agar to neoagaro-oligosaccharides of which the disaccharide was limiting and predominant. Beta-Agarase II rapidly degraded isolated neogarotetraose and neoagarohexaose to the disaccharide. With [1-3H]neoagarohexaitol, exo-action was observed, the alditol being cleaved to neoagarobiose and [1-3H]neoagarotetraitol. Neoagarotetraitol was hydrolysed at 4% of the rate observed for the hexaitol. Porphyran was degraded to oligosaccharides, the neutral fraction comprising 24% of the starting carbohydrate. This fraction was almost exclusively disaccharides (22.4%) containing neoagarobiose (7.4%) and 6(1)-O-methyl-neoagarobiose (15%). Beta-Agarase II is probably the 'beta-neoagarotetraose hydrolase' reported by Groleau and Yaphe as an exoenzyme against neoagaro-oligosaccharides [Groleau, D. and Yaphe, W. (1977) Can. J. Microbiol. 23, 672-679].     

Partial purification of pepsins from adult and juvenile salmon fish Oncorhynchus keta. Effect of NaCl on proteolytic activities

1. Two pepsins, designated Pepsin I and Pepsin II, were isolated and partially characterized from the stomach of the adult stage salmon Oncorhynchus keta. This stage is developed in a marine environment. 2. One pepsin, designated Pepsin II, was isolated from the stomach of the juvenile stage salmon Oncorhynchus keta. This stage is developed in an estuarine environment. 3. The enzymes were partially purified by ammonium sulfate precipitation, ion exchange chromatography and gel filtration. 4. Pepsins I and II from adults and Pepsin II from juvenile showed proteolytic activity on acid-denatured hemoglobin with a pH optimum of 3. 5. The mol. wt determined by gel filtration on Sephadex G-100 of Pepsin I from juvenile species was found to be 32,000 whereas a value of 27,000 was determined for Pepsin II from juvenile and adult fish. 6. In contrast with Pepsin II, Pepsin I was activated by NaCl. It is suggested that the appearance of NaCl-activated pepsin would represent and adaptive response of the organism to the change from a low to a high salinity environment.     

Genomic and proteomic analyses of the agarolytic system expressed by Saccharophagus degradans 2-40

Saccharophagus degradans 2-40 (formerly Microbulbifer degradans 2-40) is a marine gamma-subgroup proteobacterium capable of degrading many complex polysaccharides, such as agar. While several agarolytic systems have been characterized biochemically, the genetics of agarolytic systems have been only partially determined. By use of genomic, proteomic, and genetic approaches, the components of the S. degradans 2-40 agarolytic system were identified. Five agarases were identified in the S. degradans 2-40 genome. Aga50A and Aga50D include GH50 domains. Aga86C and Aga86E contain GH86 domains, whereas Aga16B carries a GH16 domain. Novel family 6 carbohydrate binding modules (CBM6) were identified in Aga16B and Aga86E. Aga86C has an amino-terminal acylation site, suggesting that it is surface associated. Aga16B, Aga86C, and Aga86E were detected by mass spectrometry in agarolytic fractions obtained from culture filtrates of agar-grown cells. Deletion analysis revealed that aga50A and aga86E were essential for the metabolism of agarose. Aga16B was shown to endolytically degrade agarose to release neoagarotetraose, similarly to a beta-agarase I, whereas Aga86E was demonstrated to exolytically degrade agarose to form neoagarobiose. The agarolytic system of S. degradans 2-40 is thus predicted to be composed of a secreted endo-acting GH16-dependent depolymerase, a surface-associated GH50-dependent depolymerase, an exo-acting GH86-dependent agarase, and an alpha-neoagarobiose hydrolase to release galactose from agarose.     

Purification and Characterization of Soymilk-clotting Enzymes from Bacillus sp. K-295G-7

Enzymes I and II, which have a high soymilk-clotting activity, produced from K-295G-7 were purified by chromatographies on Sephadex G-100, CM-cellulose, hydroxylapatite, and 2nd Sephadex G-100.

The two purified enzymes were found to be homogeneous by polyacrylamide gel elec-trophoresis (PAGE) at pH 4.3. The molecular weights of enzymes I and II were 28,000 and 29,500 by SDS-PAGE, and their isoelectric points were 9.22 and 9.45, respectively. Enzymes I and II coagulated soymilk optimally at 65°C and were stable up to 45°C. Both enzymes were most active at pH 5.8, for soymilk coagulation between pH 5.8 to 6.7, and were stable with about 50 ~ 100% of the original activity from pH 5 to 10.

Each of the purified enzymes was a serine protease with an optimum pH of 9.0 for soy protein isolate (SPI) and casein digestions, because these enzymes were inhibited completely by diisopropylfluoro-phosphate (DFP).

The soymilk-clotting activity to proteolytic activity ratio of the enzyme II was 3 times higher than that of enzyme I. Enzymes I and II were more sensitive to the calcium ion concentration in soymilk than bromelain is.     

对硫磷真菌降解酶的分离纯化和性质

黑曲霉AspergillusnigerY－8在液体培养基中30℃培养5d,其菌体用超声波破碎后,经硫酸铵沉淀、DEAE-纤维素层析、Sephadex－100凝胶过滤,得到凝胶电泳均一的对硫磷降解酶,其比活为6.94,提纯倍数为13.6倍,收率为17．4％。酶作用的最适温度是50℃,最适pH为7.5,在40℃以下和pH6.0～9.0之间稳定,此酶为单亚基蛋白,凝胶过滤法测得分子量为42000,含糖14.6％,SDS、Hg2+、Ag+、Fe3+对酶有强烈的抑制作用,金属螫合剂EDTA对酶活无影响。此酶对甲基对硫磷、敌敌畏、亚胺硫磷也有较好的降解作用,当以对硫磷为底物时,Km为0.43mmol／L,Vmax为1.24μmol·mg-1·min-1。     

Cytosolic protein phosphotyrosine phosphatases from rabbit kidney. Purification of two distinct enzymes that bind to Zn2+-iminodiacetate agarose

Cytosolic protein phosphotyrosine (PPT) phosphatase was measured using a new substrate, Tyr(32P)-labeled bovine serum albumin. Kidney was found as a particularly rich tissue source of PPT-phosphatase activity, containing twice as much as liver and over 10-fold more than brain, heart, lung, or skeletal muscle. An affinity column of Zn2+-iminodiacetate agarose adsorbed up to 60% of the PPT-phosphatase present in kidney extracts. Subsequent chromatography on DEAE-Sepharose separated the phosphatase into two peaks, labeled I and II, that had Mr = 34,000 and 37,000, respectively, upon gel filtration with Sephadex G-75 Superfine. Overall purification of 850- and 1100-fold was achieved with a net 4% yield. Both phosphatases hydrolyzed p-nitrophenylphosphate as well as the protein substrate in the presence of EDTA. Peak I phosphatase activity displayed a neutral pH optimum, had an absolute requirement for sulfhydryl compounds, and was sensitive to trypsin, whereas Peak II activity had an acidic pH optimum and was active without mercaptans. The two proteins also gave different fragmentation patterns by gel electrophoresis after digestion with S. aureus V8 protease. The results show that multiple forms of PPT phosphatase specifically interact with Zn2+ and provide a basis for further structural and functional comparisons among different members of the phosphoprotein phosphatase family.     

Purification and characterization of, and preparation of an antibody to, transketolase from human red blood cells

Transketolase (sedoheptulose-7-phosphate: D-glyceraldehyde-3-phosphate glycolaldehydetransferase, EC 2.2.1.1) was purified 16 000-fold from human red blood cells, using DEAE-Sephadex A-50, Sephadex G-150, FPLC on Mono P, and Sephadex G-100. The purified enzyme migrated as a single protein band on SDS-polyacrylamide gel electrophoresis. The FPLC step resolved transketolase into three peaks, designated I, II and III. From results of re-FPLC on Mono P, SDS-polyacrylamide gel electrophoresis, gel filtration, catalytic studies, amino acid analysis and immunological studies, it was concluded that I, II and III were originally the same protein, modified during storage and purification. Transketolase had a subunit (Mr 70 000) and appeared to be composed of two identical subunits. 1 mol of subunit contained 0.9 mol of thiamine pyrophosphate. The pH optimum of the reaction lay within the range 7.6-8.0, and the Km values were determined to be 1.5 X 10(-4) M for xylulose 5-phosphate and 4.0 X 10(-4) M for ribose 5-phosphate. Hg2+ and p-chloromercuribenzoate inhibited the enzyme reaction, and the inhibition of the latter disappeared upon the addition of cysteine. Thiamine and its phosphate esters did not, but cysteine (1 X 10(-2) M) and ethanol (10% and 1% v/v) did activate the enzyme reaction. Antibody prepared to II bound all forms of transketolase in the hemolysate, but inhibited the reaction only about 20%.     

Cloning of the novel gene encoding beta-agarase C from a marine bacterium, Vibrio sp. strain PO-303, and characterization of the gene product

The beta-agarase C gene (agaC) of a marine bacterium, Vibrio sp. strain PO-303, consisted of 1,437 bp encoding 478 amino acid residues. beta-Agarase C was identified as the first beta-agarase that cannot hydrolyze neoagarooctaose and smaller neoagarooligosaccharides and was assigned to a novel glycoside hydrolase family.     

Purification and properties of GM1 ganglioside beta-galactosidases from bovine brain

Two GM1-beta-galactosidases, beta-galactosidases I, and II, have been highly purified from bovine brain by procedures including acetone and butanol treatments, and chromatographies on Con A-Sepharose, PATG-Sepharose, and Sephadex G-200. beta-Galactosidase I was purified 30,000-fold and beta-galactosidase II 19,000-fold. Both enzymes appeared to be homogeneous, as judged from the results of polyacrylamide disc gel electrophoresis. Enzyme I had a molecular weight of 600,000-700,000 and enzyme II one of 68,000, as determined on gel filtration. On sodium dodecyl sulfate polyacrylamide slab gel electrophoresis under denaturing conditions, enzyme II gave a single band with a molecular weight of 62,000, while enzyme I gave two minor bands with molecular weights of 32,000 and 20,000 in addition to the major band at 62,000. Both enzymes liberated the terminal galactose from GM1 ganglioside and lactosylceramide but not from galactosylceramide. Enzyme I showed a pH optimum of 4.0 and was heat stable, while enzyme II showed a pH optimum of 5.0 and lost 50% of its activity in 15 min at 45 degrees C. Enzyme I showed a pI of 4.2 and enzyme II one of 5.9.     

Purification and properties of amylase produced by a moderately halophilic Acinetobacter sp.

A moderately halophilic Acinetobacter sp., capable of producing dextrinogenic amylase, was isolated from sea-sands. Maximum enzyme production was obtained when the bacterium was cultivated aerobically in media containing 1 to 2M NaCl or 1M KCl. Two kinds of amylase, amylases I and II were purified from the culture filtrate to an electrophoretically homogenous state by glycogen-complex formation, DEAE-Sephadex A-50 chromatography, and Sephadex G-200 gel filtration. Both enzymes had maximal activity at pH 7.0 in 0.2 to 0.6 M NaCl or KCl at 50 to 55 degrees C. The activities were lost by dialysis against distilled water. Molecular weights for amylases I and II were estimated to be 55 000 and 65 000 respectively by SDS-gel electrophoresis. The action pattern on amylose, soluble starch, and glycogen showed that the products were maltose and maltotriose.     

Purification and characterization of two types of alkaline serine proteases produced by an alkalophilic actinomycete

Two types of alkaline serine proteases were isolated from the culture filtrate of an alkalophilic actinomycete, Nocardiopsis dassonvillei OPC-210. The enzymes (protease I and protease II) were purified by acetone precipitation, DEAE-Sephadex A-50, CM-Sepharose CL-6B, Sephadex G-75 and phenyl-Toyopearl 650 M column chromatography. The purified enzymes showed a single band on sodium dodecyl sulphate polyacrylamide gel electrophoresis. The molecular weights of proteases I and II were 21,000 and 36,000, respectively. The pIs were 6.4 (protease I) and 3.8 (protease II). The optimum pH levels for the activity of two proteases were pH 10-12 (protease I) and pH 10.5 (protease II). The optimum temperture for the activity of protease I was 70 degrees C and that for protease II was 60 degrees C. Protease I was stable in the range of pH 4.0-8.0 up to 60 degrees C and protease II was stable in the range of pH 6.0-12.0 up to 50 degrees C.     

Purification and properties of beta-mannanases I and II from the germinated seeds of Trifolium repens. Mode of galactomannan degradation in vitro.

Two beta-mannanases (beta-mannosidases, EC 3.2.1.25) purified from the germinated seeds of Trifolium repens by a procedure that included chromatography on hydroxyapatite, gel filtration on acrylamide/agarose (Ultragel 5/4) and preparative polyacrylamide-gel-electrophoresis. The final purification step completely resolved two beta-mannanases with distinct specificities, which were termed beta-mannanase I and beta-mannanase II. beta-Mannanase I was purified 1400-fold and beta-mannanase II 1000-fold. The purified enzymes showed a single protein band when examined by polyacrylamide-gel disc electrophoresis. beta-Mannanase I, apparent mol.wt. 43 000, accounted for 49% of the total activity recovered from the final step of purification. beta-Mannanase II, apparent mol.wt. 38 000, accounted for the remaining 51% of activity. Molecular-weight determinations were by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and by the electrophoretic method of Hendrick & Smith [(1968) Arch. Biochem. Biophys. 126, 155-164]. The substrate specificities of both enzymes were examined with the galactomannans of T. repens and of Medicago sativa, as well as with manno-oligosaccharides. The pH optimum was between pH 5.1 and 5.6 for both enzymes.     

Production and characteristics of some new β-agarases from a marine bacterium,Vibrio sp. strain JT0107

Vibrio sp. strain JT0107 is one of the marine bacteria that secrete β-agarases which catalyze the hydrolysis of agarose. The optimum culture conditions for the production of some β-agarases have been determined. To increase agarase activity, aeration and a sufficient concentration of agarose are needed. One of the enzymes that the bacteria secreted into the culture medium was isolated and purified 39-fold using a combination of ultrafiltration and subsequent anion exchange column chromatography. The purified protein migrated as a single band (72 kDa) on sodium dodecyl sulfate polyacrylamide gel electrophoresis and its isoelectric point was 4.7. Amino acid sequence analysis revealed a single N-terminal sequence that had no sequence identity to other marine bacterial agarases. This novel enzyme was found to be an endo-type β-agarase (EC 3.2.1.81) that catalyzes the hydrolysis of the β-1,4 linkage of agarose to yield neoagarotetraose [O-3,6-anhydro-α-l-galactopyranosyl(1→3)-O-β-d-galactopyranosyl(1→4)-O-3,6-anhydro-α-l-galactopyranosyl(1→3)-d -galactose] and neoagarobiose [O-3,6-anhydro-α-l-galactopyranosyl(1→3)-d-galactose]. The optimum pH and temperature for obtaining high activity of the enzyme were at around 8 and 30°C, respectively. The enzyme did not degrade sodium alginate, λ-carrageenan, ι-carrageenan or κ-carrageenan.     

Molecular cloning and characterization of a novel beta-agarase, AgaB, from marine Pseudoalteromonas sp. CY24

Agarases are generally classified into glycoside hydrolase families 16, 50, and 86 and are found to degrade agarose to frequently generate neoagarobiose, neoagarotetraose, or neoagarohexaose as the main products. In this study we have cloned a novel endo-type beta-agarase gene, agaB, from marine Pseudoalteromonas sp. CY24. The novel agarase encoded by agaB gene has no significant sequence similarity with any known proteins including all glycoside hydrolases. It degrades agarose to generate neoagarooctaose and neoagarodecaose as the main end products. Based on the analyses of enzymatic kinetics and degradation patterns of different oligosaccharides, the agarase AgaB appears to have a large substrate binding cleft that accommodates 12 sugar units, with 8 sugar units toward the reducing end spanning subsites +1 to +8 and 4 sugar units toward the non-reducing end spanning subsites -4 to -1, and enzymatic cleavage taking place between subsites -1 and +1. In addition, 1H NMR analysis shows that this enzyme hydrolyzes the glycosidic bond with inversion of anomeric configuration, in contrast to other known agarases that are retaining. Altogether, AgaB is structurally and functionally different from other known agarases and appears to represent a new family of glycoside hydrolase.     

Purification and characterization of an extracellular polygalacturonase from Lactobacillus plantarum strain BA 11

Lactobacillus plantarum produced extracellular polygalacturonase in a medium containing 1.5% low methyl-pectin (w/v) and 0.5% glucose (w/v) as inducers. The enzyme was purified (approximately 70-fold) by ammonium sulphate fractionation, Sephadex G-100 gel filtration and DEAE-cellulose ion exchange chromatography. Two peaks (PG I and PG II) of enzymic activity were obtained from the DEAE-cellulose column. The molecular mass of PG I was similar to that of PG II (32 000 Da). The K _m values of PG I and PG II for sodium polypectate were calculated to be 1.63 mg/ml and 1.78 mg/ml respectively. Their isoelectric points were about pH 5.5. The pH optimum was 4.5, while the optimum temperature was 35°C for both PG I and PG II. The two purified enzymes had similar endo modes of action on polygalacturonic acid, as determined by comparison of viscosity reduction and reducing group release.