An immunoadsorbent process for removing hepatitis antigen from blood and plasma

An immunoadsorbent process has been devised for removing serum hepatitis antigen (HAA), from blood, blood plasma, and plasma products. The immunoadsorbent complexes specifically with HAA and the complex is removed from the plasma by filtration. The complex is dissociated with 0.23M NH₄OH. The immunoadsorbant is regenerated for further processing, while the purified HAA by-product my be used to produce more antibody in animals. The rate of complexing is such that HAA is reduced one log cycle each 2hr. Since available tests can only detect HAA to about 10⁹ particles per ml, it is proposed that HAA negative plasma and plasma products can be processed to reduce HAA to a probability of less than one HAA particle per plasma pool. A gibbon injected with infectious commercial Factor IX that was subjected to the immunoadsorbent process showed no sign of infection after 8 months. However 14 weeks after injection with unprocessed Factor IX, the gibbon showed signs of infection.     

Ion exchange membrane bioreactor for selective removal of nitrate from drinking water: control of ion fluxes and process performance

An ion exchange membrane bioreactor (IEMB), consisting of a monoanion permselective membrane dialyzer coupled to a stirred anoxic vessel with an enriched mixed denitrifying culture, has been studied for nitrate removal from drinking water. The influence of nitrate and chloride concentrations on the selectivity of nitrate transport in the IEMB process was investigated. With appropriate dosing of chloride ions to the IEMB biocompartment, it was possible to regulate the net bicarbonate flux in the system, thus maintaining the bicarbonate concentration in the treated water at the desired level. The latter was not possible to achieve in Donnan dialysis, operated as a single process in which, besides the lower nitrate removal efficiency found, bicarbonate was co-extracted together with nitrate from the polluted water stream. Residual carbon source (ethanol) and nitrite were not detected in the treated water produced in the IEMB system. With a concentration of nitrate in the polluted water three times higher than the maximum contaminant level of 50 mg L(-1) allowed, the IEMB process was successfully operated for a period of 1 month before exceeding this limit.     

Comparison of the action of glucoamylase and glucosyltransferase on D-glucose, maltose, and malto-oligosaccharides.

The action patterns of glucoamylase (amyloglucosidase) and glucosyltransferase (transglucosylase) on D-[1-14C]glucose, [1-14C]maltose, and [1-14C]malto-oligosaccharides (labeled at position 1 of the D-glucose group at the reducing end) have been investigated by paper-chromatographic and oligosaccharide-mapping techniques. Under the conditions of the experiments, the extent of conversion of D-glucose and of maltose into new oligosaccharides was 2.2 and 1.9% with glucoamylase, and 5.7 and 33% with glucosyltransferase. The major oligosaccharides produced by both enzymes were isomaltose (6-O-alpha-D-glucopyranosyl-alpha-D-glucose), panose (O-alpha-D-glucopyranosyl (1 leads to 6)-O-alpha-D-glucopyranosyl-(1 leads to 4)-alpha-D-glucose), and nigerose (3-O-alpha-D-glucopyranosyl-alpha-D-glucose). The glucosyltransferase also synthesized oligosaccharides from malto-oligosaccharides of higher molecular weight to yield compounds having alpha-(1 leads to 6)-linked D-glucosyl groups at the non-reducing ends. Glucoamylase exhibited little, if any, such activity on malto-oligosaccharides.     

抗狂犬病血清热原处理工艺的试验研究

作者对抗狂犬病血清精制工艺中明矾吸附步骤进行了试验研究。结果表明,采用合理的吸附剂浓度、适当的吸附时间,对制品中热原质的去除效果明显。改进工艺应用于大批量生产,取得满意效果。     

Iridoid-specific glucosyltransferase from Gardenia jasminoides

Iridoids are one of the most widely distributed secondary metabolites in higher plants. They are pharmacologically active principles in various medicinal plants and key intermediates in the biosynthesis of monoterpenoid indole alkaloids as well as quinoline alkaloids. Although most iridoids are present as 1-O-glucosides, the glucosylation step in the biosynthetic pathway has remained obscure. We isolated a cDNA coding for UDP-glucose:iridoid glucosyltransferase (UGT85A24) from Gardenia jasminoides. UGT85A24 preferentially glucosylated the 1-O-hydroxyl group of 7-deoxyloganetin and genipin but exhibited only weak activity toward loganetin and no activity toward 7-deoxyloganetic acid. This suggests that, in the biosynthetic pathway of geniposide, a major iridoid compound in G. jasminoides, glucosylation occurs after methylation of 7-deoxyloganetic acid. UGT85A24 showed negligible activity toward any acceptor substrates other than iridoid aglycones. Thus, UGT85A24 has a remarkable specificity for iridoid aglycones. The mRNA level of UGT85A24 overlaps with the marked increase in genipin glucosylation activity in the methyl jasmonate-treated cell cultures of G. jasminoides and is related to iridoid accumulation in G. jasminoides fruits.     

Ion exchange purification of estrogens

With the aim of developing a method for the determination of the whole estrogen profile in biological materials, including the labile estrogens, several alternative ion exchange Chromatographie purifications have been assessed. A two-step anion exchange chromatography, first on a DEAE-Sephadex A-25 gel in the acetate form and then on the same gel in the hydroxyl form, gave the best results. The use of ascorbic acid and small columns (in Pasteur pipettes) combined with silanization of all glassware were essential for the preservation of the labile estrogens. The recovery of estrogen standards varied from 85 to 95% and no significant interconversions were detected. When estrogens were added to hydrolysed urine the recoveries were 50–90% and the extracts were pure enough for gas chromatography on capillary columns. These results can be improved by adding a further purification procedure prior to the ion exchange chromatography on DEAE-Sephadex A-25 in the hydroxyl form.     

Thermophilic glucoamylase from Talaromyces flavus

The heat-resistant mold, Talaromyces flavus , was found to produce a thermophilic glucoamylase that exhibited the highest activity at 50°C and in the pH range of 4.0–4.8. The K _m and V _max values of the crude enzyme for amylopectin were 0.21% and 16.7 mg glucose 1^-1, min^-1, respectively. The molecular weight of the enzyme as estimated by the gel filtration method was 42 kDa.     

Characteristics of glucoamylase from Aspergillus terreus

Glucose was the only product of starch hydrolysis liberated by glucoamylase. The enzyme was a glycoprotein with an isoelectric point at pH 3·4 and was optimally active at pH 4·0 and 60°C. It was remarkably stable over a wide range of pH and at elevated temperatures. Divalent Mg²⁺'and Ca²⁺ slightly stimulated glucoamylase activity. The enzyme exhibited specificity for substrates containing α(1 → 4) glucosidic linkages and the Km for starch hydrolysis was 4·0 g/l.     

Ion exchange chromatography of antibody fragments

Effects of pH and conductivity on the ion exchange chromatographic purification of an antigen-binding antibody fragment (Fab) of pI 8.0 were investigated. Normal sulfopropyl (SP) group modified agarose particles (SP Sepharosetrade mark Fast Flow) and dextran modified particles (SP Sepharose XL) were studied. Chromatographic measurements including adsorption isotherms and dynamic breakthrough binding capacities, were complemented with laser scanning confocal microscopy. As expected static equilibrium and dynamic binding capacities were generally reduced by increasing mobile phase conductivity (1-25 mS/cm). However at pH 4 on SP Sepharose XL, Fab dynamic binding capacity increased from 130 to 160 (mg/mL media) as mobile phase conductivity changed from 1 to 5 mS/cm. Decreasing protein net charge by increasing pH from 4 to 5 at 1.3 mS/cm caused dynamic binding capacity to increase from 130 to 180 mg/mL. Confocal scanning laser microscopy studies indicate such increases were due to faster intra-particle mass transport and hence greater utilization of the media's available binding capacity. Such results are in agreement with recent studies related to ion exchange of whole antibody molecules under similar conditions.     

Ion exchange immobilization of changed beta-galactosidase fusions for lactose hydrolysis

The use of charged peptides fused to enzymes for immobilization onto ion-exchange membranes was explored for the enzyme x-galactosidase. The additional charged peptides, containing 1, 5, 11, and 16 aspartates, fused to x-galactosidase, for the most part did not interfere with the kinetic behavior for lactose hydrolysis. There was a 2-fold decline in V(m) for the 16-aspartate fusion, but the others were quite similar to the wild type enzyme (BGWT). BGWT and the fusions all retained approximately 50% of their activities when adsorbed onto ion-exchange membranes. In contrast to BGWT, the enhanced binding strength of the 11 aspartate fusion provided the ability to hydrolyze whey permeate at 0.3 M ionic strength without enzyme leakage, and to immobilize the enzyme directly from diluted cell extract with 83% purity. (c) 1994 John Wiley & Sons, Inc.     

Purification of supercoiled plasmids from crude cell lysates using high performance anion exchange chromatography

High performance liquid chromatography is of increasing importance in the purification of nucleic acids. Recently, a new anion exchange column called Gen-Pak FAX has been introduced for this purpose. Previously, it has been used in the purification of restriction fragments and oligonucleotides. In this paper we present the use of the Gen-Pak FAX column for the purification of plasmids from crude E. coli lysates. The different conformational forms of the plasmid can be well separated and collected with high recoveries of both mass and activity. Up to 50 micrograms of supercoiled plasmid can be purified in a single 30 min run with up to 98% purity.