Treatment of artificially induced storage disease with lysosomotropic microparticles

An artificial storage disease has been induced in mice by intravenously injecting 14C-labelled crosslinked dextran. The dextran was essentially taken up in the spleen and liver by cells belonging to the reticuloendothelial system and stored there in the lysosomes. Soluble dextranase, when given intravenously, had no effect on the stored dextran as judged by the radioactivity content of the tissues. However, when the dextranase was immobilized in microparticles of polyacryldextran and injected intravenously, the clearance of the radiolabelled dextran was significantly catalyzed. The lysosomotropic microparticles carried the enzyme to the lysosomes, where the microparticles were destroyed by the dextranase, which thereafter digested the stored 14C-dextran. The results illustrate the use of "self-destroying" microparticles as a lysosome-directed carrier of enzymes.     

淡紫拟青霉右旋糖酐酶的形成条件

比较了各种碳水化合物对淡紫拟青霉(Paecilomyces lilacinus)右旋糖酐酶形成的影响,右旋糖酐是最好的碳源,也是最佳诱导物。不同分子量(17．2—1000kD)的右旋糖酐对酶形成的诱导作用不同,酶的产生随右旋糖酐分子量的增大而增加。用分子量为1000kD的右旋糖酐作碳源时比用17.2kD的右旋糖酐作碳源时的产酶量高40％以上。用右旋糖酐和其它糖的混合物作碳源时,酶的形成受到不同程度的抑制。右旋糖酐酶形成的其它适宜条件：氮源为牛肉蛋白胨,培养基初始pH6．0—7.0.种龄为48小时,在250ml三角瓶中装50ml培养基,于28℃在200r／min摇床上培养6天。     

Isolation of a dextranase constitutive mutant of Lipomyces starkeyi and its use for the production of clinical size dextran

A derepressed and partially constitutive mutant for dextranase of Lipomyces starkeyi was selected after ethyl methane sulphonate mutagenesis by zone clearance on blue dextran agar plates. The mutant produced dextranase when grown on glucose, fructose and sucrose as well as on dextran, and more enzyme was produced by the mutant than by the parental strain when grown on 1% dextran. The pH and temperature optima for the mutant dextranase were 5.5 and 55°C, respectively. Dextranase produced on sucrose produced more isomaltose and less glucose after dextran hydrolysis than the equivalent enzyme produced on dextran. The clinical size dextran (average mol. wt of 75000 ± 25000) yield of mixed culture fermentation with the mutant and Leuconostoc mesenteroides was 94% of the total dextran produced.     

Characterization of an Extracellular Dextranase from Fusarium moniliforme

An extracellular dextranase (EC 3.2.1.11) was purified approximately 75-fold from cell-free culture filtrates of Fusarium moniliforme. The purified dextranase was of the endo type, and isomaltose was identified as the primary end product of dextran hydrolysis. The molecular weight of the dextranase was determined to be 39,000 by gel permeation chromatography. The enzyme was most active at pH 5.5, and the temperature optimum was near 55 C. Activity was not inhibited by either ethylenediaminetetraacetic acid or iodoacetate. The Km for dextran with an average molecular weight of 10,000 was estimated to be 1.1 X 10(-4) M. The electrophoretic mobility of the dextranase was distinctly different from that of a Penicillium-derived commercial dextranase. The F. moniliforme dextranase was also found to differ from the commercial preparation by its greater relative activity against glucans isolated from Streptococcus mutans.     

Characterization of a yeast D-mannan with an alpha-D-glucosyl phosphate residue as an important immunochemical determinant

Conditions for the reaction of concanavalin A and dextranase with glutaraldehyde have been established to give a soluble, intermolecularly cross-linked conjugate possessing both dextranase and concanavalin activities. Evidence is presented that the dextranase and concanavalin molecules are linked to each other in the conjugate. The conjugate gives a different pattern of hydrolysis products on incubation with dextran than does dextranase.     

Biochemical characterization of thermophilic dextranase from a thermophilic bacterium, Thermoanaerobacter pseudethanolicus

TPDex, a putative dextranase from Thermoanaerobacter pseudethanolicus, was purified as a single 70 kDa band of 7.37 U/mg. Its optimum pH was 5.2 and the enzyme was stable between pH 3.1 and 8.5 at 70 degrees C. A half-life comparison showed that TPDex was stable for 7.4 h at 70 degrees C, whereas Chaetominum dextranase (CEDex), currently used as a dextranase for sugar milling, was stable at 55 degrees C. TPDex showed broad dextranase activity regardless of dextran types, including dextran T2000, 742CB dextran, and alternan. TPDex showed the highest thermostability among the characterized dextranases, and may be a suitable enzyme for use in sugar manufacture without decreased temperature.     

Molecular cloning and nucleotide sequencing of the Arthrobacter dextranase gene and its expression in Escherichia coli and Streptococcus sanguis

A bacterial strain, which assimilated dextran and water-insoluble glucan produced by Streptococcus mutans, was isolated from soil. The bacterium produced and secreted potent dextranase activity, which was identified as Arthrobacter sp. and named CB-8. The dextranase was purified and some enzymatic properties were characterized. The enzyme efficiently decomposed the water-insoluble glucan as well as dextran. A gene library from the bacteria was constructed with Escherichia coli, using plasmid pUC19, and clones producing dextranase activity were selected. Based on the result of nucleotide sequencing analysis, it was deduced that the dextranase was synthesized in CB-8 cells as a polypeptide precursor consisting of 640 amino acid residues, including 49 N-terminal amino acid residues which could be regarded as a signal peptide. In the E. coli transformant, the dextranase activity was detected mostly in the periplasmic space. The gene for the dextranase was introduced into Streptococcus sanguis, using an E. coli-S. sanguis shuttle vector that contained the promoter sequence of a gene for glucosyltransferase derived from a strain of S. mutans. The active dextranase was also expressed and accumulated in S. sanguis cells.     

Molecular cloning of the extracellular endodextranase of Streptococcus salivarius.

We report the cloning in Escherichia coli of the gene encoding an extracellular endodextranase (alpha-1,6-glucanhydrolase, EC 3.2.1.11) from Streptococcus salivarius PC-1. Recombinants from a S. salivarius PC-1-Lambda ZAP II genomic library specifying dextranase activity were identified as plaques surrounded by zones of clearing on blue dextran agar. One such clone, PD1, had a 6.3-kb EcoRI fragment insert which encoded a 190-kDa protein with dextranase activity. The recombinant strain also produced two lower-molecular-mass polypeptides (90 and 70 kDa) that had dextranase activity. Native dextranase was recovered from concentrated culture fluids of S. salivarius as a single 110-kDa polypeptide. PD1 phage lysate and PC-1 culture supernatant fluid extract were used to measure substrate specificity of the recombinant and native forms of dextranase, respectively. Analysis of these reaction products by thin-layer chromatography revealed the expected isomaltosaccharide products yielded by the recombinant-specified enzyme but was unable to resolve the larger polysaccharide products of the native enzyme. Furthermore, S. salivarius utilized neither the substrates nor the products of dextran hydrolysis for growth.     

Action patterns of immobilized dextranase

Dextranase, isolated from Penicillium funiculosum and P. lilacinum, was immobilized on porous, silanized-silica beads and a phenol-formaldehyde resin. A commercial dextran of relatively low molecular weight (～2 × 10⁶) was degraded by immobilized dextranase, with the formation of reducing sugars, but with little decrease in viscosity. In contrast, soluble dextranase caused rapid loss of viscosity, but only a slight increase in reducing sugar. Native dextran of high molecular weight, from Leuconostoc mesenteroides NRRL B-512 (F), was attacked very slowly by immobilized dextranase, with the release of oligosaccharides of low molecular weight.     

Purification and immobilization of dextranase

An enzymic characteristic of Novo dextranase was presented. In addition to a high dextranolytic activity (7,200 U/ml), the crude enzyme also contained small amounts of protease, glucoamylase, polygalacturonase, carboxymethylcellulase, laminarinase and chitinase. A highly purified dextranase was then simply separated from a commercial preparation by column chromatographies on DEAE-Sepharose, CM-Sepharose, and by chromatofocussing on Polybuffer Exchanger PBE-94. The enzyme was recovered with an over 200-fold increase in specific activity and a yield of 84%. The final preparation was homogeneous, as observed during high performance liquid chromatography (HPLC). Size-exclusion HPLC indicated that dextranase had a molecular mass of 35 kDa and its isoelectric point, established by chromatofocussing, was 4.85. Analysis of the dextran break-down products indicated that purified dextranase represents an endolytic mode of action, and isomaltose and isomaltotriose were identified as the main reducing sugars of dextran hydrolysis. The enzyme was then covalently coupled to the silanized porous glass beads modified by glutaraldehyde (Carrier I) or carbodiimide (Carrier II). It was shown that immobilization of dextranase gave optimum pH and temperature ranges from 5.4 to 5.7 and from 50°C to 60°C, respectively. The affinity of the enzyme to the substrate decreased by a factor of more than 13 for dextranase immobilized on Carrier I and increased slightly (about 1.4-times) for the enzyme bound to Carrier II.     

一株产右旋糖酐酶青霉的分离及酶的纯化和性质

[目的]从土壤中筛选到一株新的产右旋糖酐酶的真菌F1001,为酶法制备药用级右旋糖酐提供新的右旋糖酐酶产生菌株.[方法]通过形态特征和ITS rDNA序列分析方法鉴定菌株.利用硫酸铵盐析、Sepharose 6B凝胶柱纯化,得到纯度较高的酶蛋白.以右旋糖酐70 kDa为底物,对右旋糖酐酶酶学性质及催化机理进行研究.[结...     

右旋糖酐酶研究进展

右旋糖酐酶是一种将高分子右旋糖酐催化降解为低分子量多糖的水解酶。该酶及其催化产物在医药、食品、化工等工业领域具有重要的应用价值与广泛的工业用途,因此近年来国内外对右旋糖酐酶的研究逐渐增多。结合文献记载及本实验室研究成果,对右旋糖酐酶的研究进展及其工业应用进行综述,并对当前有关该酶研究的热点和重点、国内右旋糖酐酶研究存在的问题以及未来的研究趋势提出了见解。     

Hydrolytic and chromatographic studies on the PEGylation of dextranase from Penicillium sp.

Dextranases catalyze the hydrolysis of the α-l,6-glucosidic bond of the polysaccharide dextran. Dextranases have been isolated from bacteria, yeast and fungi. Purified dextranase enzyme from Penicillium sp. was PEGylated (polyethylene glycol modification) with mPEG (5000 Da) and showed an increase in the dextranase protein molecular weight as estimated by Superose 12 (23 ml) column and this increment in the molecular weight is directly proportional to mPEG (5000 Da) concentration until a complete dextranase enzyme PEGylation (disappearance of dextranase peak). The residual activity of partially PEGylated dextranase (mPEG 5000 of 5.8 mg/ml) was 33.8% and for the completely PEGylated dextranase (mPEG 5000 of 29 mg/ml) it was 25.75%. Dextranase PEGylated with mPEG (30,000 Da) showed a little PEGylation at mPEG concentration of 5.8 mg/ml but at a concentration of 29 mg/ml several PEGylated peaks were produced with a difference in dextranase activity toward dextran T500, retardation in the activity with the increasing in the molecular weight was clearly appeared with Sephadex G75 but for Sephadex G200 a little retardation than Sephadex G75 has been appeared.     

Milligram to gram scale purification and characterization of dextransucrase from Leuconostoc mesenteroides NRRL B-512F

A sequence of dextranase treatment, DEAE-cellulose chromatography, affinity chromatography on Sephadex G-200, and chromatography on DEAE-Trisacryl M has been optimized to give a dextransucrase preparation with low carbohydrate content (1-100 micrograms/mg protein) and high specific activity (90-170 U/mg protein) relative to previous procedures, in 30-50% yield. Levansucrase was absent after DEAE-cellulose chromatography, and dextranase was undetectable after Sephadex G-200 chromatography. The method could be scaled up to produce gram quantities of purified enzyme. The purified dextransucrase had a pH optimum of 5.0-5.5, a Km of 12-16 mM, and produced the same lightly branched dextran as before purification. The purified enzyme was not activated by added dextran, but the rate of dextran synthesis increased abruptly during dextran synthesis at a dextran concentration of approximately 0.1 mg/mL. The enzyme had two major forms, of molecular weight 177,000 and 158,000. The 177,000 form predominated in fresh preparations of culture supernatant or purified enzyme, whereas the amount of the 158,000 form increased at the expense of the 177,000 form during storage of either preparation.     

Selection of a mutant strain of Lipomyces kononenkoae with dextranase synthesis resistant to catabolite repression

A mutant strain of Lipomyces kononenkoae 2896-3 synthesizing dextranase but resistant to catabolite repression was obtained using N-nitroso-N-methylurea treatment. Enzyme biosynthesis in media with dextran and other carbon sources was then characterized. The capacity of the mutant to produce dextranase when grown on hydrolysed corn starch is demonstrated.     

The purification and characterization of a dextranase from Lipomyces starkeyi

Dextranase produced by Lipomyces starkeyi was purified 43-fold, by carboxymethyl-Sepharose chromatography followed by agarose gel-filtration chromatography. The purified enzyme showed four bands by SDS/polyacrylamide gel electrophoresis with estimated mass 74 kDa, 71 kDa, 68 kDa and 65 kDa. This preparation exhibited multiple isoelectric points between 5.6 and 6.1. All the isoelectric forms were active and catalytically similar. The dextranase contained a carbohydrate moiety (8%). The physical properties of the enzyme were pH and temperature optima of 5.0 and 55 degrees C, respectively. This dextranase was stable between pH 2.5 and 7.0 at temperatures below 40 degrees C. Lipomyces dextranase was a typical endodextranase with the final product of dextran hydrolysis being isomalto-oligosaccharides from glucose to isomaltotetrose.     

Dextran production by Leuconostoc mesenteroides in the presence of a dextranase producing yeast, Lipomyces starkeyi

Summary A new process for the production of small size dextran is developed in which dextran is produced by cultures of Leuconostoc mesenteroides in the presence of a partially constitutive mutant of Lipomyces starkeyi producing dextranase. Mixed cultures were examined by scanning electron microscopy with ruthenium to show the effects of the mixed culture on low molecular weight dextran (M.W. of 5,000 – 100,000) formation. The presence of the size variation in dextran was confirmed by gel permeation chromatography.     

Production and Properties of Alkaline Dextranase from a Newly Isolated Brevibacterium

About 500 strains of dextranase producing microorganisms were examined in detail for pH- activity and enzyme stability. A gram positive bacterium identified as belonging to the genus Brevibacterium was found to produce alkaline dextranase. Maximal dextranase synthesis was obtained when grown aerobically at 26°C for 3 days in a medium containing 1 % dextran, 2% ethanol, 1 % polypeptone and 0.05 % yeast extract together with trace amounts of inorganic salts.

Brevibacterium dextranase had an optimum pH of 8.0 for activity at 37°C and an optimal temperature at 53°C at pH 7.5. The enzyme was quite stable over the range of pH 5.0 to 10.5 on 24 hr incubation at 37°C, especially on alkaline pH. The enzyme was also heat stable at 60°C for 10 min.     

Transisomaltosylation Activity of a Bacterial Isomalto-dextranase

A bacterial isomalto-dextranase, described previously as a new type of dextranase different from the known 1,6-α-d-glucan 6-glucanohydrolase [EC 3.2.1.11], was found to be a configuration-retaining exo-glucanase so far as judged from the downward mutarotation shown by products in a dextran digest, and from the lower activity of the enzyme on lesspolymerized isomaltodextrins according to one of the criteria proposed by Reese et al. The dextranase was observed to cause not only transisomaltosylation (isomaltotetraose formation in dextran digests, isomaltotriose formation in dextran digests containing glucose and transisomaltosylation among isomaltodextrins) but also isomaltose condensation to isomaltotetraose in concentrated solutions. These activities shown by the isomalto-dextranase are in keeping with the novel concept that carbohydrases are catalysts of glycosylation (glycosylhydrogen interchange), proposed by Hehre and his coworkers. The relative ease of isomaltose condensation catalyzed by the enzyme appears due to the exergonic nature of the reaction. A free energy change value of ca. ?1200 cal/mole was obtained for the condensation.     

The use of titanium(IV) oxide coated with diazotised 1,3-diaminobenzene for the immobilisation of carbohydrate-directed enzymes

The utility of porous titanium(IV) oxide particles as a matrix for the immobilisation of enzymes on column packings has been extended. On coating the particles with diazotised 1,3-diaminobenzene, their capacity for binding dextranase was increased two-fold. The stability of the enzyme-matrix bridge was enhanced by the covalent bond so formed. Excess diazonium groups were reacted with 2-naphthol. Investigations of the effects of change of dextran concentration, pH, temperature, and flow rate upon a continuously operated column of the immobilised dextranase permitted assessment of the kinetic aspects of the enzyme via Lineweaver-Burk plots. The change of reaction rate with temperature showed, according to Arrhenius plots, an abrupt change at 28°. Possible sources of the kinetic characteristics of the immobilised dextranase are discussed.