添加核苷对肝素黄杆菌发酵产肝素酶的影响

研究了添加核苷对肝素黄杆菌发酵产肝素酶的影响,结果发现,单种核苷的添加会抑制产酶,而复合核苷的添加则促进产酶,当4种核苷的添加比例与肝素酶mRNA中4种相应核苷酸的比例一致时,促进作用最强。通过HPLC检测,证实添加后核苷很快进入了菌体内。HPLC的结果还表明,菌体内嘧啶核苷酸和嘌呤核苷酸的合成代谢可能不平衡,这对产酶是不利的。为此,还研究了通过添加天冬氨酸以增强嘧啶核酸合成代谢的调节方式,使产酶得到了提高。     

Heparinase II from Flavobacterium heparinum. Action on chemically modified heparins

Five chemically modified heparins were derived from native pig mucosal heparin (pig heparin Is). These were de-N-sulphated heparin (heparin IH), N-acetylheparin (heparin IA), de-N/O-sulphated heparin (heparin IVH), de-O-sulphated heparin (heparin IVs) and de-O-sulphated N-acetyl-heparin (heparin IVA). Their structures were studied by 13C-NMR spectroscopy at 90.56 MHz. Native heparin and the derivatives were incubated with Flavobacterium heparinase II at 25 degrees C. The progress of degradation was followed by the delta A235 and the final composition examined by gel filtration with Bio-Gel P-4. Native heparin (Is) was readily degraded by heparinase II and, with the exception of heparin IVH for which degradation was negligible, the chemically modified derivatives were also degraded. Approximately 90% of the saccharides from heparins Is, IA, IVs and IVA were disaccharides and tetrasaccharides. For heparin IH, which was degraded more slowly, the proportion was 65%. Heparins Is, IVs and IVA underwent initial rapid degradation. The digestion of heparin Ia proceeded rapidly after an initial lag phase. The undegraded polymers produced similar elution profiles from Bio-Gel P-4. Following the action of heparinase II on heparins Is, IA, IVs and IVA, the elution profiles revealed a major peak of disaccharides and minor peaks of higher oligomers. The profile of heparin IH revealed a greater proportion of intermediate-molecular-mass saccharides. Our results demonstrate a broad specificity for heparinase II. It is capable of lysing both N-acetylated and N-sulphated heparins independent of O-sulphation. Heparinase II will also degrade heparin derivatives that are non-N-substituted provided that they are O-sulphated.     

Heparinase II from Flavobacterium heparinum. HPLC analysis of the saccharides generated from chemically modified heparins.

Saccharides produced by the action of heparinase II on native pig mucosal heparin (heparin IS), de-N-sulphated heparin (heparin IH), N-acetylheparin (heparin IA), de-N/O-sulphated heparin (heparin IVH), de-O-sulphated heparin (heparin IVS) and de-O-sulphated N-acetylheparin (heparin IVA) were analysed by reversed-phase HPLC using Spherisorb ODS2. Fractions obtained by gel filtration with Bio-Gel P-4 were similarly examined. Heparin IS gave delta UA-2S----GlcNS-6S (IS) as the major unsaturated disaccharide and lesser amounts of delta UA----GlcNS-6S (IIS), delta UA-2S----GlcNS (IIIS), delta UA----GlcNS (IVS), delta UA-2S----GlcNAc-6S (IA), delta UA----GlcNAc-6S (IIA), delta UA-2S----GlcNAc (IIIA) and delta UA----GlcNAc (IVA). Heparins IA, IVA and IVS gave as the predominant unsaturated disaccharide that corresponding to the major repeat structure of the polymer. These were respectively delta UA-2S----GlcNAc-6S (IA), delta UA-GlcNAc (IVA) and delta UA----GlcNS (IVS). Minor disaccharides from the heterogeneous structure in native pig heparin and from residual O-sulphates after the de-O-sulphating process were detected. Heparin IH was degraded more slowly than any of the N-substituted heparins. The predominant unsaturated disaccharide was IH, which was derived from the major repeating unit. In addition, disaccharides IIH, IIIH, IA, IIA and IVA were detected. Heparin IVH showed little degradation, the unsaturated disaccharide IVH not being detected after 24 h. Disaccharide IVA was obtained from the heterogeneous sequence in heparin IVH. Several higher oligosaccharides were identified in the gel-filtration fractions including saccharides from the linkage region (for heparin IS and IVA) and the anti-thrombin binding site (for heparin IS only). A tetrasaccharide and hexasaccharide, with the structures delta UA----GlcNAc----UA----GlcNAc and delta UA----GlcNAc----UA----GlcNAc----UA----GlcNAc, were present in the HPLC profiles of heparins IA and IVA.     

Chondroitinase C from Flavobacterium heparinum.

A chondroitinase that acts upon chondroitin sulfate C and hyaluronic acid was isolated from Flavobacterium heparinum. This enzyme was seperated from constitutional chondroitinase AC and an induced chondroitinase B also present in extracts of F. heparinum previously grown in the presence of chondroitin sulfates A, B or C. The enzyme acts upon chondroitin sulfate C producing tetrasaccharide plus an unsaturated 6-sulfated disaccharide (delta Di-6S), and upon hyaluronic acid producing unsaturated nonsulfated disaccharide (delta Di-OS). Chondroitin sulfate A is also degraded producing oligosaccharides and delta Di-6S but not delta Di-4S. The chondroitinase C is also distinguished from the chondroitinases B and AC by several properties, such as effect of ions, temperature for optimal activity, and susceptibility to increasing salt concentrations. The substrate specificity of the chondroitinase C is different from that of any other chondroitinase or hyaluronidase described so far.     

Regulation of heparinase synthesis in Flavobacterium heparinum

The effect of various carbon, nitrogen and sulfur sources on the production of heparinase by Flavobacterium heparinum in defined medium in the presence and absence of heparin as the inducer has been studied. Carbon catabolite repression has been observed in defined medium containing one of several carbon sources including simple sugars, alcohols and organic acids. Fed batch fermentations result in 10 g/l of cells and heparinase titers as high as 100,000 U/l by avoiding carbon catabolite repression. Growth on heparin as a sole carbon source resulted in both a high growth rate of 0.12 h^–1 and a high specific activity of 18 U/mg. Specific heparinase activity was markedly reduced when the end products of heparin catabolism were used as carbon, nitrogen or sulfur sources in defined medium. In defined medium with a low sulfate concentration, of less than 10^–3 M, specific activities as high as 8 U/mg have been observed even in the absence of the normally required inducer, heparin.     

Flavobacterium heparinum sulphamidase for D-glucosamine sulphamate. Purification and characterisation

A novel assay has been developed for 2-deoxy-2-sulphamido-D-glucose (GlcNS) sulphamidase from Flavobacterium heparinum. This has enabled the 1930-fold purification of the enzyme from a soluble fraction of bacterial homogenate. From SDS/polyacrylamide gel electrophoresis the enzyme was shown to have a relative molecular mass of 81,500. Ca2+ was essential for enzyme activity. Inorganic phosphate and sulphate inhibited activity by 28% and 29% respectively at 5 mmol dm-3. The purified sulphamidase had a pH optimum of 7.0 and a Km of 8.32 mumol dm-3 for GlcNS. The degradation of 2-deoxy-2-sulphamido-6-O-sulpho-D-glucose (GlcNS-6S) was also re-investigated. The two sulphate groups were hydrolysed sequentially in a single non-bifurcate manner, in contrast to previous reports [Dietrich, C.P., Silva, M.E. and Michelacci, Y.M. (1973) J. Biol. Chem. 248, 6408-6415].     

Sulfur regulation of heparinase and sulfatases in Flavobacterium heparinum.

Sulfur regulation of heparinase synthesis and sulfatase synthesis was studied in Flavobacterium heparinum. Heparinase synthesis was strongly repressed by sulfate and L-cysteine, while the activity of this enzyme showed little or no inhibition by these compounds. Heparinase was synthesized in the absence of heparin when L-methionine was used as the sole sulfur source. The sulfatases produced by F. heparinum, which include the sulfatases involved in heparin catabolism, were also studied. At least some of the sulfatase activity was regulated by sulfur compounds in a manner similar to heparinase regulation. L-Cysteic acid and taurine were not suitable sulfur sources to support the growth of F. heparinum.     

Purification and characterization of heparin lyases from Flavobacterium heparinum.

Heparin lyase I has been purified from Flavobacterium heparinum and has been partially characterized (Yang, V. C., Linhardt, R. J., Berstein, H., Cooney, C. L., and Langer, R. (1985) J. Biol. Chem. 260, 1849-1857). There has been no report of the purification of the other polysaccharide lyases from this organism. Although all three of these heparin/heparan sulfate lyases are widely used, with the exception of heparin lyase I, there is no information on their purity or their physical and kinetic characteristics. The absence of pure heparin lyases and a lack of understanding of the optimal catalytic conditions and substrate specificity has stood in the way of the use of these enzymes as reagents for the specific depolymerization of heparin and heparan sulfate into oligosaccharides for structure and activity studies. This paper describes a single, reproducible scheme to simultaneously purify all three of the heparin lyases from F. heparinum to apparent homogeneity. Heparin lyase I (heparinase, EC 4.2.2.7), heparin lyase II (no EC number), and heparin lyase III (heparitinase, EC 4.2.2.8) have molecular weights (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and isoelectric points (by isoelectric focusing) of M(r) 42,800, pI 9.1-9.2, M(r) 84,100, pI 8.9-9.1, M(r) 70,800, pI 9.9-10.1, respectively. Their amino acid analyses and peptide maps demonstrate that while these proteins are different gene products they are closely related. The kinetic properties of the heparin lyases have been determined as well as the conditions to optimize their activity and stability. These data should improve the application of these important enzymes in the study of heparin and heparan sulfate.     

Purification and characterization of heparinase from Flavobacterium heparinum

Heparinase (EC 4.2.2.7) isolated from Flavobacterium heparinum was purified to homogeneity by a combination of hydroxylapatite chromatography, repeated gel filtration chromatography, and chromatofocusing. Homogeneity was established by the presence of a single band on both sodium dodecyl sulfate and acid-urea gel electrophoretic systems. Amino acid analysis shows that the enzyme contains relatively high amounts of lysine residues (9%) consistent with its cationic nature (pI 8.5) but contains only 4 cysteine residues/polypeptide. The molecular weight of heparinase was estimated to be 42,900 +/- 1,000 daltons by gel filtration and 42,700 +/- 1,200 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is very specific, acting only on heparin and heparan monosulfate out of 12 similar polysaccharide substrates tested. It has an activity maximum at pH 6.5 and 0.1 M NaCl and a stability maximum at pH 7.0 and 0.15 M NaCl. The Arrhenius activation energy was found to be 6.3 kcal/mol. However, the enzyme is very sensitive to thermal denaturation and loses activity very rapidly at temperatures over 40 degrees C. Kinetic studies of the heparinase reaction at 37 degrees C gave a Km of 8.04 X 10(-6) M and a Vm of 9.85 X 10(-5) M/min at a protein concentration of 0.5 microgram/ml. By adapting batch procedures of hydroxylapatite and QAE (quaternary aminoethyl)-Sephadex chromatography, gram quantities of heparinase that is nearly free of catalytic enzyme contaminants can be purified in 4-5 h.     

Sulfur regulation of heparinase and sulfatases in Flavobacterium heparinum

Sulfur regulation of heparinase synthesis and sulfatase synthesis was studied in Flavobacterium heparinum. Heparinase synthesis was strongly repressed by sulfate and L-cysteine, while the activity of this enzyme showed little or no inhibition by these compounds. Heparinase was synthesized in the absence of heparin when L-methionine was used as the sole sulfur source. The sulfatases produced by F. heparinum, which include the sulfatases involved in heparin catabolism, were also studied. At least some of the sulfatase activity was regulated by sulfur compounds in a manner similar to heparinase regulation. L-Cysteic acid and taurine were not suitable sulfur sources to support the growth of F. heparinum.     

Flavobacterium heparinum 3-O-sulphatase for N-substituted glucosamine 3-O-sulphate

A novel bacterial sulphatase has been discovered in an extract of Flavobacterium heparinum. The enzyme hydrolyses the 3-O-sulphate from 2-deoxy-2-sulphamido-3-O-sulpho-D-glucose and 2-acetamido-2-deoxy-3-O-sulpho-D-glucose. The activity was purified 10 800-fold by chromatography successively on CM-Sepharose CL-6B, hydroxyapatite, taurine-Sepharose CL-4B and CM-Sepharose CL-6B. Sodium dodecylsulphate/polyacrylamide gel electrophoresis showed the enzyme to be homogeneous and of relative molecular mass 56 000. Two novel assays were developed using 2-[14C]acetamido-2-deoxy-3-O-sulpho-D-glucose and 2-deoxy-2-sulphamido-3-O-sulpho-D-glucose as respective substrates. The purified 3-O-sulphatase was shown to be free of all other known heparin-degrading enzymes. Optimal activity was at pH 7.5 for the disulphated substrate and pH 8.0 for the N-acetylated substrate. Enzyme activity was virtually unaffected by Na+, K+ or Mg2+ ions. A 1.2-fold enhancement of activity was effected by 0.002 mol dm-3 Ca2+. Inorganic phosphate and sulphate inhibited 3-O-sulphatase activity. The Km value of the N-acetylated substrate was determined to be 42 mumol dm-3. No activity was detected with 2-amino-2-deoxy-3-O-sulpho-D-glucose.     

Isolation and characterization of an induced chondroitinase ABC from Flavobacterium heparinum

During the investigation of alternative methods for the large scale preparation of chondroitinases AC, B and C from Flavobacterium heparinum, a new chondroitinase activity was observed. This new enzyme, like the other chondroitinases, acts as an eliminase, forming unsaturated sulfated disaccharides from dermatan and chondroitin sulfates. In contrast to the chondroitinases previously described, which are endoglycosidases, this chondroitinase ABC cleaves the glycosidic linkages in an exolytic fashion, beginning at the reducing end of the substrate molecules. The oligosaccharides formed as transient products by the action of either chondroitinases or testicular hyaluronidase upon dermatan and chondroitin sulfates are also rapidly degraded by the chondroitinase ABC, regardless of their size or the presence of delta-4,5 unsaturation in the terminal uronic acid residue. The maximum activity of the chondroitinase ABC occurs at 30 degrees C and at pH 6.0-7.5. Only 15% of the activity was observed at 37 degrees C, indicating that the enzyme is very sensitive to thermal denaturation. It is strongly inhibited by phosphate ions and is also inhibited by the unsaturated disaccharides formed.     

Enzymic degradation of heparin A sulphamidase and a sulphoesterase from Flavobacterium heparinum

A sulphamidase and a sulphoesterase were isolated from adapted cells of Flavobacterium heparinum. These enzymes were partially purified from the ;heparinases' present in the bacterial extracts and characterized. The sulphamidase has a high specificity for glucosamine N-sulphate and glucosamine 2,6-disulphate. The activity decreases sharply with increasing molecular weight of the substrates tested. The sulphamidase and the sulphoesterase activities were distinguished from each other by their different sensitivities to concentration of phosphate ion and to temperature. The importance of these enzymes in the study of the structure of heparin is discussed.     

Flavobacterium heparinum 2-O-sulphatase for 2-O-sulphato-delta 4,5-glycuronate-terminated oligosaccharides from heparin

The glycosulphatase which hydrolyses the 2-O-sulphate of the disaccharide, 4-deoxy-2-O-sulphato-alpha-L-threohex-4-enopyranosyl uronic acid-(1----4)-2-deoxy-2-sulphamido-6-O-sulphato-D-glucose (delta UA-2S----GlcNS-6S), has been isolated from the soluble fraction of disrupted Flavobacterium heparinum. The activity was purified 3300-fold by chromatography on CM-Sepharose CL-6B, hydroxyapatite, taurine-Sepharose CL-4B and blue-Sepharose CL-6B. From sodium dodecylsulphate/polyacrylamide gel electrophoresis, the enzyme was homogeneous and of 62000 Mr. A novel assay was devised using the de-N-sulphonated [1-3H]alditol, 4-deoxy-2-O-sulphato-alpha-L-threo-hex-4-enopyranosyl uronic acid-(1----4)-2-amino-2-deoxy-6-O-sulphato-D-[1-3H]glucitol (delta UA-2S----[1-3H]GlcNH2-ol-6S). This alditol was shown by 13C-NMR to be desulphated in the analogous manner to the original reducing trisulphated disaccharide. The purified 2-O-sulphatase was completely free of heparinase I, heparinase II (heparitinase), chondroitinases AC, chondroitinase B, the delta 4,5-glycuronidase for heparin delta 4,5-disaccharides, the 6-O-sulphatase and the 2-sulphamidase. It was optimally active over the range pH 5.5-6.5 and was practically unaffected by Na, K, Ca or Mg ions. Inorganic phosphate inhibited the activity. The Km value for the alditol substrate was 1.22 mmol dm-3. Using 13C-NMR, the 2-O-sulphatase was found to hydrolyse the analogous esters of higher delta 4,5-oligosaccharides from heparin. This contrasts with the findings of other authors [Dietrich, C. P., Silva, M. E., and Michelacci, Y. M. (1973) J. Biol. Chem. 248, 6408-6415].