Purification and characterization of basic glucosyltransferase from Streptococcus mutans serotype c

Streptococcus mutans Ingbritt (serotype c) was found to secrete basic glucosyltransferase (sucrose: 1,6-alpha-D-glucan 3-alpha and 6-alpha-glucosyltransferase). The enzyme preparation obtained by ethanol fractionation, DEAE Bio-Gel A chromatography, chromatofocusing and preparative isoelectric focusing was composed of three isozymes with slightly different isoelectric points (pI 8.1-8.4). The molecular weight was estimated to be 151000 by SDS-polyacrylamide gel electrophoresis. The specific activity of the enzyme was 9.8 IU per mg of protein and the optimum pH was 6.5. The enzyme was activated 2.4-fold by commercial dextran T10, and had Km values of 7.1 micro M for the dextran and 4.3 mM for sucrose. Glucan was de novo synthesized from sucrose by the enzyme and found to be 1,6-alpha-D-glucan with 17.7% of 1,3,6-branching structure by a gas-liquid chromatography-mass spectroscopy.     

Dextran synthesis by immobilized dextran sucrase

Dextran sucrase has been produced by fermentation of Leuconostoc mesenteroides NRRL B-512, with and without continuous sucrose addition to improve enzyme production. The enzyme preparation has been concentrated from the fermentation broth by ultrafiltration and purified by gel permeation chromatography on Ultrogel. The specific activity of the dextran sucrase was greatly enhanced by calcium chloride addition to the purified enzyme. This enzyme preparation has been immobilized by covalent coupling onto an amino porous silica support (Spherosil) activated with glutaraldehyde. Immobilized dextran sucrase derivatives with an activity up to 830 dextran sucrase units per g. support could thus be obtained. The effect of the support specific area on coupling efficiency and reaction kinetics has been investigated, and the effect of intraparticular diffusion underlined. The molecular weight distribution of the dextran has been determined when varying several parameters.     

Stabilization of basic fibroblast growth factor with dextran sulfate.

Dextran sulfate protected bFGF from heat and acid inactivation and from proteolytic degradation. The protective effect was stronger than that of heparin which is known as a stabilizer of bFGF. Dextran sulfate and bFGF formed a high molecular weight complex via ionic interaction when mixed together in aqueous solution. The complex was dissociated when the ionic strength was increased and the protective effect was completely abolished. Successive digestion of bFGF with Staphylococcus aureus V8 protease and pepsin followed by affinity chromatography on an immobilized dextran sulfate column and reversed-phase high performance liquid chromatography yielded three positively charged fragment peptides, Tyr24-Phe30, Tyr106-Trp114 and Tyr124-Leu138. These results suggest that dextran sulfate stabilizes bFGF by binding close to the putative heparin binding sites of the bFGF molecule.     

Clinical dextran purified by fractional ultrafiltration coupled with water washing

Dextran with extremely narrow molecular weight distribution (MWD) is demanded for clinical use. To elucidate the effect of fractional ultrafiltration on the purification of clinical dextran, a range of ultrafiltration membranes with 100, 30, 5 and 1 kDa molecular weight cut-off (MWCO) were applied. High Performance Gel Permeation Chromatography (HPGPC) analysis indicated that the MWD of ultrafiltrated dextran fractions are wide, with polydispersity index (D) between 2.2 and 5.4, suggesting that the MWD of dextran is hard to be strictly controlled by fractional ultrafitration. However, when coupled with water washing during ultrafiltration process, the homogeneity of dextran was greatly improved. An ultrafiltration fraction of 5-30 kDa (Mw ≈ 35 kDa) with narrow MWD (D = 1.2) was obtained after 5 times of water washing. The results show that fractional ultrafiltration coupled with water washing can be used as a simple and effective method to improve the quality of clinical dextran.     

Purification of ram seminal plasma acid alpha-glucosidase

1. Ram seminal plasma alpha-glucosidase has been purified in order to increase our knowledge of this enzyme and of its role in epididymal physiology. 2. Since the enzyme behaved differently from other known acid alpha-glucosidases and was not affinity-adsorbed on dextran gels, another approach had to be used. 3. The final procedure included an ethanol precipitation step, sequential chromatography on hydroxylapatite and DEAE Sepharose CL-6B, isoelectric focusing in polyacrylamide gels and ultrafiltration. 4. The resulting purification factor of alpha-glucosidase was 9822 with an overall yield of 5%. 5. The purified material consisted of several isoforms with a mol. wt of 105,000 and isoelectric points varying between 4 and 5.     

杂色云芝组成型漆酶Ⅰ的纯化和底物专一性

采用合成培养基培养杂色云芝As5 4 8,从发酵液中纯化出一种组成型漆酶同功酶Ⅰ .经超滤浓缩 ,DEAE SephadexA 5 0离子交换层析 ,Bio gelP 10 0凝胶过滤纯化了该酶 .SDS PAGE分析发现 ,该酶分子量为 6 8kD ,薄层等电聚焦测得等电点为 3 5 .漆酶Ⅰ的底物范围较宽 ,以O2 为电子受体 ,可以氧化多种木素单体模型物 ,包括 2 ,6 二甲氧基酚 ,2 ,2′ 联氮 二 (3 乙基 苯并噻唑 6 磺酸 )(ABTS) ,愈创木酚 ,咖啡酸 ,阿魏酸和邻联茴香胺 .结果表明 ,该酶在木质素的生物降解中可能有重要的作用和应用价值 .     

Calculated molecular weight of proteins in high ionic strengths: contribution of the apparent isopotential specific volume.

Recent sedimentation equilibrium measurements of the molecular weight of tail muscle lactate dehydrogenase from the North American East Coast lobster Homarus americanus show that this enzyme does not dissociate in buffer with high ionic strength (1.2 m ammonium sulfate). However, the apparent isopotential volume φ₂′ increases significantly with increasing ionic strength of the solution. Consequently, molecular weight estimates for proteins using an assumed apparent specific volume equal to that in low salt concentration solutions may lead to erroneously low values under experimental conditions of high ionic strength.     

Separation of beta-casein peptides through UF inorganic membranes.

Ultrafiltration of peptide mixtures is studied under various operating conditions (transmembrane pressure, tangential flow-rate) using two ultrafiltration inorganic membranes M5 and M1 with molecular weight cut-offs, MWCO 10 and 70 kD, respectively. It is shown that the separation of peptides is controlled by a dual mechanism: size exclusion and electrostatic repulsion. When the ionic strength is high enough to screen out the electrostatic interactions, experimental data are in good agreement with a sieving model developed to estimate the intrinsic transmission from the molecular weight of a component and from the MWCO of the membranes. Although the transmission so found is altered by concentration polarisation and pore blocking mechanisms, the results explain the apparent low transmission of peptides by ultrafiltration membranes. If the ionic strength of the fluid is low, electrostatic interactions can influence the transport phenomena, provided that the molecules are highly charged (at pHs away from the pI). For attractive interactions, an apparent partition coefficient larger than 1 is observed. Otherwise, the transmission is lower than predicted by the sieving theoretical equation, as if the partition coefficient were smaller than 1.     

Reversible immobilization of glutaryl acylase on sepabeads coated with polyethyleneimine

The immobilizaton of the enzyme glutaryl-7-aminocephalosporanic acid acylase (GA) was performed via ionic adsorption onto several supports: a new anionic exchange resin, based on the coating of Sepabeads internal surfaces with polyethyleneimine (PEI) of different molecular weights, and conventional EC-Q1A-Sepabeads and DEAE-agarose. Immobilization occurred very rapidly in all cases, but the adsorption strength was much higher in the case of PEI-Sepabeads than in the other supports at pH 7 (e.g., at 150 mM NaCl, 90% of the enzyme was eluted from the DEAE agarose and 15% was eluted from the EC-Q1A-Sepabeads, whereas no desorption was detected with the best PEI-Sepabeads). Interestingly, the adsorption strength of the GA was increased when it was immobilized on PEI-Sepabeads with higher molecular weights. For instance, enzyme desorption was detected from 75 mM NaCl for the derivative prepared onto Sepabeads coated with PEI 700 Da, whereas in the derivative prepared with the highest molecular weight PEI (600 000 Da) no enzyme desorption was detected below 150 mM NaCl. Optimal PEI-Sepabeads (prepared with PEI of 600 000 Da) was even much more thermostable than the covalent derivative prepared onto cyanogen bromide agarose. Moreover, this derivative presented a half-life 26-fold higher than that of the soluble enzyme at 45 degrees C, and the support could be reused 10 times after the full desorption of the enzyme without decreasing loading capacity.     

The mechanism of the dextran-induced red blood cell aggregation

In order to clarify the mechanism of dextran-induced aggregation, the effect of the ionic strength (I) on the minimal shear stress (tau(c)) required to rupture RBC doublets was studied for suspensions with the external media containing 76 and 298 kDa dextrans. At low and high ionic strengths, tau(c) increases with increasing I, whereas at intermediate I values, tau(c) versus I dependencies reveal a plateau step. The non-monotonous shape of these curves disagrees with the depletion model of RBC aggregation and is consistent with the predictions of the bridging mechanism. Literature reports point out that elastic behavior of dextran molecules in low and high I regions is fairly typical of Hookean springs and hence predict an increase in tau(c) with increasing I. A plateau step is accounted for by the enthalpic component of the dextran elasticity due to the shear-induced chair-boat transition of the dextran's glucopyranose rings. A longer plateau step for suspensions with a higher molecular weight dextran is explained by a larger contribution of the enthalpic component to the dextran elasticity. Thus, the results reported in this study provide evidence that RBC aggregation is caused by the formation of dextran bridges between the cells.     

Specific and non-specific affinities of the extracellular glucosyltransferase complex of Streptococcus mutans 6715

Glucosyltransferases (GTF) from different strains of streptococci exhibited different elution profiles when fractionated on insoluble-dextran affinity columns. The proportions of unadsorbed and adsorbed GTF were not related to their extent of stimulation by exogenous dextran, and GTF preparations exposed to, and freed from, clinical dextran prior to fractionation lost their ability to bind to the dextran columns. Different proportions of bound GTF were released by irrigation of columns with different concentrations of salt and clinical dextran, and the “specific” binding and release of GTF exhibited by a column possessing covalently linked, clinical dextran ligands was duplicated on a control column that did not possess the dextran ligands. These results, and the high affinity of GTF for hydrophobic alkyl (Shaltiel) ligands, demonstrate that ionic and hydrophobic properties of impure GTF aggregates may lead to erroneous characterization of the dextran affinity of some protein fractions. Fractionations on DEAE-Sepharose and on hydroxylapatite showed that the two dextran-dependant GTF activities (GTF-S and GTF-I) were present in the major enzyme fraction (Streptococcus mutans 6715) recovered from a Sephacryl S-200 affinity column. A minor, dextran-independent GTF was not adsorbed onto the Sephacryl column. The presence of SDS (0.005%) and Triton X100 (0.01%) stabilized GTF activity during gel filtration and improved the separation of GTF-S and GTF-I in hydroxylapatite fractionation of the highly aggregated enzyme. A comparable separation of the two enzyme forms on DEAE-Sepharose was achieved only if T10 dextran (10 mg/mL) was included with the detergent mixture in the column irrigant.     

The influence of humic substances on the molecular weight distributions of phosphate and iron in epilimnetic lake waters

1. The influence of humic substances on the association of free inorganic iron and phosphate with material of larger molecular weight was investigated in epilimnetic samples from two humus‐rich lakes of contrasting ionic strength. After modification of the molecular weight distribution of the humic substances in the samples using dialysis and ultrafiltration, the molecular weight distribution of added radioisotopes ( Fe3+ and PO43?) was assessed using gel filtration chromatography.
2. The association of Fe3+ and PO43? with larger molecular weight fractions (>50 000 and 10 000–50 000 Da) was not in general related to the quantity of humic substances of the same molecular weight in the samples. However, the proportions of Fe3+ and PO43? observed in higher molecular weight peaks were strongly correlated to the quantity of humic substances of the same molecular weight in (a) the 10 000–50 000 Da peak in the sample of low ionic strength at pH 5.5 and pH 7.0, and (b) the> 50 000 Da peak in the sample of higher ionic strength at pH 4.0.
3. It was concluded that humic substances promote the association of Fe3+ and PO43? with higher molecular weight fractions primarily by acting as peptizing agents for inorganic colloids containing Fe and P. Association of Fe3+ and PO43? with material of higher molecular weight via the formation of humic substance‐Fe3+–PO43? complexes was identified but only at specific pH and within specific molecular weight ranges for each of the epilimnetic lake water samples studied.     

Hypolipidemic effects of orally administered dextran and cellulose anion exchangers in cockerels and dogs

Various cellulose and dextran anion exchangers bind bile salts in vitro under conditions of pH and ionic strength resembling those in the lumen of the small intestine. Of these substances, diethylaminoethyl (DEAE) cellulose, guanidoethyl cellulose, and DEAE Sephadex reduced hypercholesterolemia when added to the diet of cholesterol-fed cockerels. In addition, DEAE Sephadex reduced serum sterols in normocholesterolemic cockerels and dogs, lowered serum phospholipids and triglycerides in cholesterol-fed hypercholesterolemic cockerels and in normocholesterolemic dogs, and increased fecal excretion of bile acids in hypercholesterolemic cockerels. The data indicate that these insoluble cationic polymers exert their hypocholesterolemic effects by interrupting the enterohepatic circulation of bile acids.     

The molecular structure of low and high molecular weight levans synthesized by levansucrase

The levan synthesized by Bacillus subtilis levansucrase in the presence of alcohols was of only high molecular weight, while in solutions of high ionic strength only low molecular weight (MW) levan was produced. The addition of low MW levan to the enzyme reaction mixture at low ionic strength stimulated synthesis of a high MW levan, but the levan added was not incorporated into this high MW levan. Methylation analysis revealed that low MW levans contained glucose, which was isolated as 2,3,46-tetra-O-methyl alditol acetate showing that the glucose units existed as terminal residues. The molecular weight of levan estimated on the basis of glucose content coincided with that determined by the gel filtration method. Methylation analysis also revealed that the number of fructose residues of the linear fraction linked by leads to 6(F)2 leads to type bonds was 22 for levan with a molecular weight of (8.4(-22)) x 10(3), while it was 11 for that of 2,000 x 10(3). The number of (formula: see text) type branched residues increased with increase in the molecular weight of the levan synthesized.     

The Fractionation of Hydrolyzed Dextran by Ultrafiltration Through A Series of Anisotropic Cellulose Acetate Membranes

Dextran, a homopolyner consisting almost solely of 1,6-α- linked glucose units, was separated into five well defined, narrow range, low molecular weight fractions by sequential ultrafiltration, after controlled lydrolysis. A commercially available purified dextran preparation, D 10, with a weight average molecular weight (Mw) of 10,980, was hydrolyzed with HCl to an average molecular weight of 4200. By ultrafiltration through a series of graded anisotropic cellulose acetate membranes of decreasing sore sizes, molecular weight fractions having Mw's of 7100, 6175, 4320, 2810 and 1565 were obtained. From Mw and sedimentation (S) values, the frictional coefficients were calculated for each fraction and the asymmetry ratios obtained therefrom.     

Purification and characterization of basic glucosyltransferase from Streptococcus mutans serotype c

Streptococcus mutans Ingbritt (serotype c) was found to secrete basic glucosyltranserase (sucrose: 1,6-α-^D-glucan 3-α- and 6-α- glucosyltransferase). The enzyme preparation obtained by ethanol fractionation, DEAE Bio-Gel A chromatography, chromatofocusing and preparative isoelectric focusing was composed of three isozymes with slightly different isoelectric points (pI 8.1–8.4). The molecular weight was estimated to be 151 000 by SDS-polyacrylamide gel electrophoresis. The specific activity of the enzyme was 9.8 IU per mg of protein and the optimum pH was 6.5. The enzyme was activated 2.4-fold by commercial dextran T10, and had K_m values of 7.1 μM for the dextran and 4.3 mM for sucrose. Glucan was de novo synthesized from sucrose by the enzyme and found to be 1,6- α-D-glucan with 17.7% of 1,3,6-branching structure by a gas-liquid chromatography-mass spectroscopy.     

Purification and properties of extracellular glucosyltransferase synthesizing 1,3-alpha-D-glucan from Streptococcus mutans serotype a

Extracellular 1,3-alpha-D-glucan synthase (sucrose: 1,3-alpha-D-glucan 3-alpha-D-glucosyltransferase, EC 2.4.1.-) of Streptococcus mutans HS6 (serotype a) was purified from culture supernatant by ultrafiltration, DEAE-Sepharose chromatography and preparative isoelectric focusing. The enzyme had a molecular weight of 158 000 by SDS-PAGE and an isoelectric point of pH 5.2. The specific activity of the enzyme was 48.3 i.u. (mg protein)-1. The Km for sucrose was 1.2 mM and the activity was optimal at pH 6.0. The enzyme activity was stimulated about 20-fold in the presence of dextran T10. Glucan was synthesized de novo from sucrose by the enzyme and characterized as a linear 1,3-alpha-D-glucan by GC-MS.     

Integration of bioconversion and downstream processing: starch hydrolysis in an aqueous two-phase system

Integration of bioconversion and the first step(s) of down stream processing can be used as a means to increase the productivity of bioprocesses. This integration also gives the possibility to run the bioconversion in a continuous mode. We demonstrate the use of an aqueous two-phase system in combination with ultrafiltration to accomplish this. Conversion of native starch to glucose by alpha-amylase and glucoamylase was carried out in an aqueous two-phase system in connection with a membrane filtration unit. In this way, a continuous stream of glucose in buffer solution was obtained; the phase-forming polymers as well as the starch-degrading enzymes were recycled, and clogging of the ultrafiltration membrane was avoided. The process was carried out continuously in a mixer-settler reactor for a period of 8 days. The enzyme activities in the top and bottom phases and in the mixing chamber were monitored intermittently throughout the experiment. The optimum pH, temperature, and ionic strength for the activity of the enzyme mixture were determined. The settling time of phase systems containing varying amounts of PEG, crude dextran, and solid starch was studied. The activity and stability of enzyme mixtures was studied both in buffer medium and in the medium containing the polymers. The enzymes were found to be more active and stable in medium containing polymers than in the buffer solutions.     

Effect of protein adsorption on the transport characteristics of asymmetric ultrafiltration membranes.

The effects of bovine serum albumin adsorption on the transport characteristics of asymmetric poly(ether sulfone) ultrafiltration membranes were determined using polydisperse dextrans with gel permeation chromatography. Actual dextran sieving coefficients were evaluated from observed sieving data for both the clean and preadsorbed membranes using a stagnant film model. The flux dependence of the actual dextran sieving coefficients was used to evaluate the intrinsic membrane hindrance factors for convective (i.e., sieving) and diffusive transport for the different molecular weight dextrans using classical membrane transport theory. Protein adsorption caused a reduction in both dextran sieving and diffusion, with the magnitude of the reduction a function of the dextran molecular weight and pore size. The effects of adsorption on the specific pore area and the membrane porosity were then determined using a recent model for solute transport through asymmetric ultrafiltration membranes. The data indicate that protein adsorption occurs preferentially in the larger membrane pores, causing a greater reduction in solute sieving compared to the membrane hydraulic permeability and porosity than would be predicted on the basis of either a simple pore blockage or pore constriction model.     

Extracellular glucose-producing exodextranase of the yeast Lipomyces lipofer

A dextran-hydrolysing enzyme from Lipomyces lipofer IGC 4042 was purified from the supernatant of cultures grown on a mineral medium with dextran, by ultrafiltration and gel filtration on Bio Gel A-0.5 m. This preparation gave only one band by disc gel electrophoresis. Glucose was the only product of dextran hydrolysis. Optimum pH and temperature for the activity of the enzyme were pH 4.5–5.0 and 45°C, respectively. The enzyme was most stable over a pH range of 4.5–6.0, and after 2 hours at 50°C maintained over 60% of its original activity. The molecular weight was 29,000 daltons and the isoelectric point was at pH 7. K_m (45°C, pH 5) for dextran T-40 was 1.2×10^–5 M. Glucose inhibited the enzyme competitively with a K_i (45°C, pH 5) of 0.5 mM.