Purification of glycomacropeptide from non-dialyzable fraction of sweet whey by hydrophobic interaction chromatography on phenyl-agarose

Non-dialyzable fraction of sweet whey was chromatographed on a column of phenyl-agarose equilibrated with 0.01 M sodium phosphate buffer, pH 6.8 containing 5 M NaCl. Most whey proteins were adsorbed on the column, while the glycomacropeptide (GMP) was not. Amino acid analysis of the GMP fraction showed presence of traces (each < 1 residue/peptide) of arginine, histidine and phenylalanine which are not found in GMP. The estimated yield of GMP fraction was approximately 1.6 g l^-1 of sweet whey.     

Production of extracellular lactase fromFusarium moniliforme

Summary A strain ofFusarium moniliforme, previously used for microbial protein production, excreted lactase (-D-galactosidase, EC.3.2.1 23) when cultivated either in a whey liquid medium or on a wheat bran solid medium. The enzyme produced in both media had pH and temperature optima of 4–5 and 50–60°C respectively and was particularly suitable for processing acid whey.In the whey culture, maximum lactase yield was observed after 95 h of growth at 30°C and whey lactose concentration of 9%. The addition of ammonium, potassium and sodium ions to the growth medium considerably enhanced lactase production. A maximum enzyme yield corresponding to hydrolysis of 3 nmoles o-nitrophenyl--D-galactopyranoside sec^–1 ml^–1 of growth medium, at pH 5 and 60°C, was obtained.In the wheat bran culture, the maximum enzyme yield was obtained after 140 h of growth at 28–30°C. A marked increase in the enzyme production was observed when nitrate or phosphate was added to the growth medium. Also, the addition of certain agricultural by-products (molasses, whey) enhanced lactase production. The observed maximum yield corresponding to the hydrolysis of 182 nmoles of ONPG sec^–1 g^–1 of wheat bran, at pH 5 and 60°C, is comparable to that reported for certain microorganisms used commercially for lactase production.     

Purification of kappa-casien glycomacropeptide from sweet whey with undetectable level of phenylalanine

Glycomacropeptide (GMP) found in sweet whey is a biologically active compound released from kappa-casein by the action of chymosin during cheese making. This study was undertaken to purify GMP from sweet whey as a research chemical on a laboratory scale. Glycomacropeptide was isolated from proteins and other non-GMP compounds by deproteinization with trichloroacetic acid and gel chromatography on Sephacryl S-200. The purified GMP accounted for 0.12% of dry sweet whey powder and contained 107.0, 50.9, 61.2 and 4.3 microg, respectively, of sialic acid, galactose, galactosamine and phosphorus per mg dry weight. The GMP was of high purity, with its amino acid composition showing undetectable levels of phenylalanine, tyrosine and arginine, the amino acids that do not occur in bovine GMP. On gel electrophoresis, the GMP showed a single broad band with an average mobility faster than that of carbonic anhydrase (molecular weight = 31 kDa). The purified GMP may be used as a standard glycopeptide in chromatography and electrophoresis and may also be used to test various known or unknown properties and biological activities of this compound.     

Purification of glycomacropeptide from non-dialyzable fraction of sweet whey by anion-exchange chromatography

Glycomacropeptide (GMP) was purified from non-dialyzable fraction of sweet whey by anion-exchange chromatography on DEAE-Sephacel at two pHs 6.4 and 3.0. Chromatography at pH 3.0 (but not pH 6.4) gave a GMP fraction of high purity with its yield (1 g from every litre of whey) being approximately 100 times higher than that shown in the previous report. It was concluded that DEAE-Sephacel chromatography at pH 3.0 is a simple useful method to separate GMP from most whey proteins. It may be applicable to a large scale production of GMP.     

Affinity purification and characterization of a yeast epoxide hydrolase

Purification of the membrane-associated epoxide hydrolase from the yeast Rhodosporidium toruloides CBS 0349 to electrophoretic homogeneity was achieved in a single chromatographic step employing the affinity ligand adsorbent Mimetic Green. More than 68% of the total epoxide hydrolase activity present in the whole cells was recovered from the membrane fraction. The enzyme was purified 26-fold with respect to the solubilized membrane proteins and was obtained in a 90% yield. The purified epoxide hydrolase has an apparent monomeric molecular weight of 54 kDa, and a pI of 7.3. The enzyme was optimally active at 30–40 °C, and pH 7.3–8.5. The enzyme is highly glycosylated with a carbohydrate content >42%. The specific activity of the purified enzyme for (±)-1,2-epoxyoctane is 172 mol min^–1 mg protein^–1. The amino acid composition of the protein was determined. This is the first report of a yeast epoxide hydrolase purified to homogeneity in milligram amounts.     

Production of propionic acid by mixed cultures ofPropionibacterium shermanii andLactobacillus casei in autoclave-sterilized whey

Summary Pure cultures ofPropionibacterium freudenreichii ss.shermanii did not grow in autoclave-sterilized cheese whey (121°C, 15 psi, 20 min) at whey concentrations greater than 2% (w/v) spray-dried sweet dairy whey. Propionic acid was produced from autoclave-sterilized whey by growingP. shermanii in mixed culture withLactobacillus casei. In medium containing 5–12% autoclaved whey solids and 1% yeast extract, the mixed culture produced 1.3–3.0% propionic acid, 0.5–1.0% acetic acid, and 0.05–0.80% lactic acid. All the lactose was consumed. Using pH-controlled fermentors (pH=7.0), mixed cultures produced at least 30% more propionic acid than cultures in which pH was not controlled.     

The production of lactic acid from whey permeate byLactobacillus helveticus

Summary The effect of pH on growth and lactic acid production ofLactobacillus helveticus was investigated in a continuous culture using supplemented whey ultrafiltrate. Maximum lactate productivity of 5 gl^–1h^–1 occurred at pH 5.5. Whey permeates concentrated up to four times were fermented using batch cultures. Maximum lactic acid concentration of 95 gl^–1 was attained, but residual sugars indicated a possible limitation in growth factors.Nomenclature D Dilution rate [h^–1] - X Biomass [gl^–1] - Glu Glucose consentration [gl^–1] - Gal Galactose consentration [gl^–1] - S Substrate, Lactose consentration [gl^–1] - P Product, Lactate consentration [gl^–1] - Y_p/s Yield, defined as P/S [gg^–1] - r_i Rate of synthesis or consumption of i [gl^–1h^–1]     

Synthesis and glycosylation of fibronectin in hepatocytes from vitamin A-deficient rats

Summary Recent work from our laboratory (Kim and Wolf, J Biol Chem 262: 365–371, 1987) has shown increased uptake of labeled amino acids into fibronectin (FN), increased net synthesis of FN and increased levels of FN-mRNA in primary cultures of hepatocytes from vitamin A-deficient rats compared to controls. We now find, surprisingly, decreased uptake of labeled sugars into the oligosaccharide chains of FN from vitamin A-deficient hepatocytes. This decrease could be reversed by added retinoic acid at physiological concentration. At the same time, FN from deficient hepatocytes (–A.FN) was more susceptible to proteolytic degradation. Decreased uptake of the core sugar mannose into –A.FN was similar to that of glucosamine, yet the percent of label in sialic acid was the same as in +A.FN, suggesting a smaller number of oligosaccharide chains per molecule of –A.FN. Upon enzymatic removal of oligosaccharide and labeling with sodium borotritide, it was found that both –A.FN and +A.FN had biantennary oligosaccharide structures. Selective enzymatic removal of sialic acid showed that +A.FN had both sialic acids in an 23 linkage, whereas –A.FN apparently had one 23 and one 26-linked sialic acid. The borotritide experiments allowed us to calculate that +A.FN appeared to have 5 oligosaccharide chains per FN monomer, whereas the –A. FN showed only 4 chains. These results would account for the decreased glycosylation and increased susceptibility to proteolysis of the –A. FN. We conclude that vitamin A controls both the rate of synthesis of the polypeptide chain of FN via its mRNA, as well as the rate of its glycosylation.Abbreviations FN Fibronectin - ELISA Enzyme-linked Immunosorbent Assay - DOC Deoxycholate - TCA Trichloroacetic Acid - PMSF Phenylmethylsulfonyl Fluoride - PBS Phosphate-buffered Saline - BSA Bovine Serum Albumin - AGP Alpha-1 acid Glycoprotein - SDS-PAGE Sodium Dodecylsulfate-Polyacrylamide Gel Electrophoresis     

Effect of cultural conditions on production of eicosapentaenoic acid byPythium irregulare

Summary The effect of culture conditions upon lipid content and fatty acid composition of mycelia ofPythium irregulare was investigated with particular attention to increasing the yield of 5,8,11,14,17-eicosapentaenoic acid (205; –3) (EPA). All experiments were done by shake flask culture using a yeast extract + malt extract medium. The maximum growth rate was obtained at 25°C, but maximum EPA production was obtained at 12°C. The highest EPA production was 76.5 g EPA/ml 13 days fermentation at 12°C. Addition of glucose during fermentation increased the yield considerably. The highest yield was 112 g/ml, obtained at 13 days fermentation with spiking on day 11. Fermentation time could be shortened by initial incubation at 25°C for 2 days, followed by incubation at 12°C for 6 days. The culture also produced arachidonic acid and other -6 polyunsaturated fatty acids. EPA production was also obtained with lactose or sweet whey permeate, a by-product of cheese manufacture that contains lactose as the main carbohydrate.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

Qualitative Analysis of the Carbohydrate Composition of Apolipoprotein H

The specific binding of digoxigenin-labeled lectins to carbohydrate moieties is used to characterize the carbohydrate chains bound to apolipoprotein H. Our results show that apolipoprotein H is rich in sialic acid linked (2–6) to galactose or N-acetylgalactosamine. Sialic acid is not (2–3)-linked to galactose. Galactose is (1–4)-linked to N-acetylglucosamine and (1–3)-linked to N-acetylgalactosamine. High-mannose N-glycan chains are barely detectable. After N-glycosidase F treatment the molecular weight is substantially reduced. The main band is 32,500 daltons. Carbohydrate O-linked chains, which are mainly represented by sialic acid, are (2–6)-linked to galactose or N-acetylgalactosamine. Galactose is also organized in O-linked chains and it is (1–4)-linked to N-acetylglucosamine and (1–3)-linked to acetylgalactosamine. Biochemical analysis of carbohydrate structures reveals that no specific carbohydrate complex is bound to a single isoform.     

Surface charge of eosinophils. Binding of cationic particles and measurement of cellular electrophoretic mobility

Summary The surface charge of eosinophils, isolated from the peritoneal exudate of rats by the use of a Metrizamide gradient, was analysed by ultrastructural cytochemistry and cellular electrophoretic mobility. Binding of colloidal iron hydroxide and of cationized ferritin particles at pH 1.8 and 7.2 respectively, was observed on the surface of the eosinophils. An electrophoretic mobility of –1.08 and –1.39 m·s^–1·V^–1·cm was determined for living and glutaraldehyde-fixed eosinophils, respectively. Treatment of the cells with neuraminidase reduced the electrophoretic mobility to –0.64 m·s^–1·V^–1·cm (glutaraldehyde-fixed), reduced significantly and abolished completely the binding of both colloidal iron hydroxide and cationized ferritin particles to the surface of the cells. These results indicate that sialic acid exists on the surface of eosinophils, where it accounts for part of the negative surface charge.     

Isoenzymes of human liver α-L-fucosidase: Chemical relationship,kinetic studies,and immunochemical characterization

Summary The chemical relationship of the seven forms of human liver -L fucosidase has been studied by isoelectric focusing of neuraminidase- and sialyltransferase-treated preparations of -L-fucosidase. Neuraminidase treatment leads to a decrease in the activity of the more acidic forms (IV–VII) and a concomitant increase in the activity of the more neutral forms (I–II). Incubation of the neuraminidase-treated fucosidase (forms I–III) with radiolabelled cytidine monophosphate-N-³H-acetylneuraminic acid and an enriched preparation of sialyltransferase devoid of fucosidase activity led to regeneration of the more acidic fucosidase isoenzymes (IV–VII) with the same isoelectric points and in nearly the same proportion as before neuraminidase treatment. These experiments suggest that the isoenzymes of human liver -L-fucosidase are related, at least in part, by sialic acid residues.The seven isoenzymes of purified human liver -L-fucosidase have been separated by preparative isoelectric focusing and characterized kinetically and immunochemically. Differences in Michaelis constants (Km's) and pH optimum curves were found for some of the isoenzymes. All seven isoenzymes were immunoprecipitated using the IgG fraction of anti--L-fucosidase antiserum suggesting that the presence of sialic acid residues does not affect the antigenicity of the forms of -L-fucosidase.     

Growth and lactic acid production ofPediococcus pentosaceus at different gas environments,temperatures, pH values and nitrite concentrations

Summary In order to obtain a better understanding of the behaviour ofPediococcus pentosaceus in food products as well to facilitate the designing of industrial production processes for the organism, the growth and lactic acid production ofPediococcus pentosaceus in a complex glucose medium was followed in batch cultures at different gas environments (CO₂, air, N₂ and static cultures without gasflow), temperatures (10–50°C), pH (4.3–7.3) and nitrite concentrations (0–700 ppm). Optimal growth was obtained in CO₂ at 40°C and pH 6.3 and resulted in a maximum specific growth rate ( _max) of 1.27 h^–1. In static culture at 40°C and pH 6.3 the _max was 1.21 h^–1. The _max was, compared with static culture, reduced in air (12%) and nitrogen (26%). At 10°C the _max was reduced by 99% and at 50°C by 88%. The reduction at pH 4.3 and 7.3 was 65% and 57%, respectively. Nitrite did not affect the _max at any pH but increased the lag phase at pH 4.3 by a factor of 12. The lactic acid production was linked to the growth. The total amount of lactic acid produced was the same in all the tested gases and nitrite concentrations and also within the wide temperature range (15–45°C) and pH range (5.3–7.3). Mainly L(+)-lactic acid was produced during the exponential growth phase, but after this growth declined about 30% of the L(+)-lactic acid was converted to D(–)-lactic acid. The lactic acid product yield and the cellmass yied were both affected by the temperature but not by the pH.     

Biological and physicochemical characterization of recombinant human erythropoietins fractionated by Mono Q column chromatography and their modification with sialytransferase

Ten erythropoietin (EPO) fractions differing in sialic acid content, ranging from 9.5 to 13.8 mol mol^–1 of EPO, were obtained from baby hamster kidney cell-derived recombinant human EPO by Mono Q column chromatography. The mean pI values of the EPO fractions determined by IEF-gel electrophoresis systematically shifted from 4.11 to 3.31, coinciding with the sialic acid content, without a change in the constitution of asialo N-linked oligosaccharides of each fraction. Although a linear relationship between thein vivo bioactivity and the sialic acid content of the fractionated, samples was observed until 12.1 mol mol^–1 of EPO, there was no further increase in their activity over 12.4 mol mol^–1 of EPO. On the other hand, an inverse relationship between thein vitro bioactivity and sialic acid content of EPO was observed. Also, we showed that thein vivo bioactivity of some fractions with low sialic acid contents was increased after treatment with 2,6-sialyltransferase, but thein vivo bioactivity of the other fractions with high sialic acid contents was either decreased or not affected.Abbreviations EPO erythropoietin - rHuEPO recombinant human erythropoietin - hCG human chorionic gonadotropin - BHK baby hamster kidney - CHO Chinese hamster ovary - NeuAc N-acetyl neuraminic acid - Gal galactose - HRCs hemolyser-resistant cells - WST-1 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium Na - IEF isoelectric focusing - pI isoelectric point     

Production of fungal chitosan by solid substrate fermentation followed by enzymatic extraction

An improved method is described for the production of chitosan from mycelia of the fungus Gongronella butleri, grown by solid substrate fermentation on sweet potato. The chitosan was extracted subsequently by 11 M NaOH at 45 °C, and 0.35 M acetic acid at 95 °C. The resulting extract was clarified using a heat-stable, commercial -amylase. The yield (4–6 g/100 g mycelia) and relative number average molecular weight (44–54 kDa) of the chitosan increased with increasing duration of fungal growth up to the sixth day.     

Batch propagation of Lactobacillus helveticus for production of lactic acid from lactose concentrated cheese whey with microaeration and nutrient supplementation

Continuous mix batch bioreactors were used to study the kinetic parameters of lactic acid fermentation in microaerated-nutrient supplemented, lactose concentrated cheese whey using Lactobacillus helveticus. Four initial lactose concentrations ranging from 50 to 150 g l^–1 were first used with no microaeration and no yeast extract added to establish the substrate concentration above which inhibition will occur and then the effects of microaeration and yeast extract on the process kinetic parameters were investigated. The experiments were conducted under controlled pH (5.5) and temperature (42 °C) conditions. The results indicated that higher concentrations of lactose had an inhibitory effect as they increased the lag period and the fermentation time; and decreased the specific growth rate, the maximum cell number, the lactose utilization rate, and the lactic acid production rate. The maximum lactic acid conversion efficiency (75.8%) was achieved with the 75 g l^–1 initial lactose concentration. The optimum lactose concentration for lactic acid production was 75 g l^–1 although Lactobacillus helveticus appeared to tolerate up to 100 g l^–1 lactose concentration. Since the lactic acid productivity is of a minor importance compared to lactic acid concentration when considering the economic feasibility of lactic acid production from cheese whey using Lactobacillus helveticus, a lactose concentration of up to 100 g l^–1 is recommended. Using yeast extract and/or microaeration increased the cell number, specific growth rate, cell yield, lactose consumption, lactic acid utilization rate, lactic acid concentration and lactic acid yield; and reduced the lag period, fermentation time and residual lactose. Combined yeast extract and microaeration produced better results than each one alone. From the results it appears that the energy uncoupling of anabolism and catabolism is the major bottleneck of the process. Besides lactic acid production, lactose may also be hydrolysed into glucose and galactose. The -galactosidase activity in the medium is caused by cell lysis during the exponential growth phase. The metabolic activities of Lactobacillus helveticus in the presence of these three sugars need further investigation.     

Amylolytic enzymes produced by a color variant strain ofAureobasidium pullulans

A color variant strain ofAureobasidium pullulans (NRRL Y-12974) produced amylase and -glucosidase activities when grown at 28°C for 4 days in liquid culture on a wide variety of carbon sources such as starch, pullulan, glucose, maltose, cyclodextrins, sucrose, xylose, and xylan. An -glucosidase was separated by Q-Sepharose adsorption from the cell-free culture broth and partially purified by hydroxylapatite and octyl-Sepharose chromatography. After ammonium sulfate treatment of the culture supernatant (obtained after Q-Sepharose adsorption), the amylase fraction was separated into three active fractions by hydroxylapatite column chromatography, which were identified as -amylase, glucoamylase A, and glucoamylase B. The glucoamylase A was further purified by octyl-Sepharose column chromatography. The pH optima for the action of -amylase, glucoamylase A, glucoamylase B, and -glucosidase were 5.0, 4.5, 4.0–4.5, and 4.5, respectively. The -amylase and glucoamylase B were fully stable at pH 3.0–6.0, glucoamylase A at pH 4.5–5.5, and -glucosidase at pH 3.5–7.0 for 1 h at 50°C. The optimum temperatures for the action of these enzymes were 55°, 50°–60°, 65°, and 65°C, respectively. The -amylase, glucoamylase A, and glucoamylase B were adsorbed onto raw corn starch and degraded it. Glucoamylase B readily cleaved pullulan. The -glucosidase was not adsorbed onto raw starch and did not degrade it at all. It hydrolyzed both -1,4 and -1,6 linkages in oligosaccharides. All four enzymes did not require any metal ion for activity and were inhibited by cyclodextrins (-and -, 10mm).     

Partial purification and biochemical characterization of alkaline 5'-phosphodiesterase from barley malt sprouts

An alkaline 5-phosphodiesterase (5-PDE) from barley (Hordeum distichum) malt sprouts was partially purified by thermal treatment and acetone precipitation to diminish phosphomonoesterase (PME) activity. 5-PDE was purified 40-fold to a specific activity of 30 U mg^–1 protein with a final yield of about 32%. With synthetic substrate, the enzyme had an optimum pH of 8.9, maximum activity at 70 °C over 10 min, and a Km of 0.26 mM. The partially purified enzyme was activated by 10 mM Mg²⁺ up to 168% of the original activity, while Zn²⁺, Mn²⁺ and Cu²⁺ ions, chelating agent (EDTA) and NaN₃ (1–10 mM), and 5-ribonucleotides (1–5 mM) were inhibitory. Final enzyme preparation was stable over 8 d at 4 °C), at 70 °C for up to 120 min and without loss of activity over 90 d at –18 °C.     

Enzymatic transfer of sialic acids modified at C-5 employing four different sialyltransferases

We present kinetic studies on the enzymatic transfer of several synthetic sialic acid analogues, modified at C-5, to distinct glycoprotein glycans by sialytransferases differing in acceptor- and linkage-specificity. Biochemical properties of sialic acids were modified by introducing formyl-, trifluoroacetyl-, benzyloxycarbonyl-, and aminoacetyl-groups to the amino group at C-5 of neuraminic acid. The latter substitution renders the corresponding -glyocoside resistant towards sialidases. The respective CMP-sialic acid analogues were prepared by CMP-sialic acid synthase with a yield of 13–55%.The kinetic parameters of several sialyltransferases for the 5-substituted CMP-glycosides differed significantly. Relative to parent CMP-NeuAc, reaction rates of human- and rat liver Gal1, 4GlcNAc 2,6-sialyl-transferases ranged from 50 to 170%, of GalNAc 2,6-sialyltransferases from 40–140%, and of Gal1,3Gal-NAc 2,3-sialyltransferase from 20–50%. Resialylation of asialo-₁-acid glycoprotein by 5-N-formyl- and 5-N-aminoacetyl-neuraminic acid employing rat liver Gal1,4GlcNAc 2,6-sialyltransferase proceeded to about 80% of galactose sites which is identical to the extent achieved with parent NeuAc.According to our data, neosialoglycoconjugates which carry sialic acids modified at theN-acetyl group can be prepared for structure-function analysis, as this position seems crucial for recognition of adhesion proteins and influenza viruses.     

Production of inulooligosaccharides from inulin by a novel endoinulinase from Xanthomonas sp.

A novel inulinolytic microorganism, Xanthomonas sp. produced an endoinulinase, to be used for inulooligosaccharide (IOS) formation from inulin, at an activity of 11 units ml^–1 (1.2 mg protein ml^–1). The endoinulinase was optimally active at 45°C and pH 6.0. Batchwise production of IOS was carried out by the partially purified endoinulinase with a maximum yield of about 86% on a total sugar basis with 10 g inulin l^–1. The major IOS components were DP (degree of polymerization) 5 and 6 with trace amount of smaller oligosaccharides.