The linamarase of Mucor circinelloides LU M40 and its detoxifying activity on cassava

Mucor circinelloides LU M40 produced 12·2 mU ml⁻¹ of linamarase activity when grown in a 3 l fermenter in the following optimized medium (g l⁻¹ deionized water): pectin, 10·0; (NH₄)₂SO₄,
1·0; KH₂PO₄, 2·0; Na₂HPO₄, 0·7; MgSO₄.7H₂O, 0·5; yeast extract, 1·0; Tween-80,
1·0, added after 48 h of fermentation. The purified linamarase was a dimeric protein with a molecular mass of 210 kDa; the enzyme showed optimum catalytic activity at pH 5·5 and 40 °C and had a wide range (3·0–7·0) of pH stability. The enzyme substrate specificity on plant cyanogenic glycosides was wide; the K_m value for linamarin was 2·93 mmol l⁻¹. The addition, before processing, of the fungal crude enzyme to cassava roots facilitated and shortened detoxification; after 24 h of fermentation, all cyanogenic glycosides were hydrolysed.     

Glucosyltransferase activity of commercial juice-processing enzyme preparations

Of various commercial enzyme preparations examined, Cytolase M102 was found to contain the highest glucosyltransferase activity (55 U ml⁻¹). It rapidly converted maltose to panose (Glcα1 → 6Glcα1 → 4Glc) with a V _max value of 5·8 mmol l⁻¹ min⁻¹ at 50°C in 0·05 mol l⁻¹ sodium acetate buffer (pH 4·4). The K _m value of the enzyme for maltose was 750 mmol l⁻¹. Yields of panose and glucose after 45 min of reaction, for example, were 47·2% and 52·8%, respectively, on the basis of the amount of maltose consumed.     

THE PRESENCE OF AN α-GALACTOSIDASE IN BRAIN AND ITS ROLE IN THE DEGRADATION OF THE DIGALACTOSYL DIGLYCERIDE OF BRAIN

Abstract— The presence of α-galactosidase activity has been demonstrated in rat brain. This enzyme, located mainly in the crude mitochondrial fraction, actively hydrolysed the substrates p -nitrophenyl-α-galactoside and melibiose, and also catalysed the hydrolysis of digalactosyl diglyceride of both animal and plant origin. The hydrolysis of p -nitrophenyl-α-galactoside, as catalysed by the α-galactosidase, occurred optimally at pH 4·9, showed an approximate K _m of 1·0 × 10⁻³ m , and was markedly inhibited by melibiose, galactose and the mercuric ion.     

Effect of chlorination on β-D-galactosidase activity of sewage bacteria and Escherichia coli

The effect of chlorine on β- D- galactosidase activity of sewage bacteria and Escherichia coli was studied. β- D- galactosidase activity of sewage was more resistant to chlorine than faecal coliform cultivability. At low initial dosage (0·05 mg Cl₂ l⁻¹) neither cultivability (colony-forming units (cfu)), nor enzyme activity of E. coli suspensions were severely impaired. When initial chlorine concentration was increased to 0·1 mg Cl₂ l⁻¹, the cfu number decreased whereas enzyme activity remained high, i.e. the enzyme activity calculated cfu⁻¹ increased. At higher chlorine doses both cfu and enzyme activity were reduced, but non-cultivable cells retained assayable activity after chlorination. Mean values of the enzyme activity calculated cfu⁻¹ decreased when the chlorine dosage was increased from 0·1 to 0·5 mg Cl₂ l⁻¹, but were not significantly different ( P > 0·05) for dosages of 0·2–0·7 mg Cl₂ l⁻¹. After chlorination, β- D- galactosidase activity of E. coli was less reduced than cfu and direct viable count numbers, but more reduced than 5-cyano-2-3, ditolyl tetrazolium chloride and total cell counts, and the enzyme activity represented an alternative activity parameter of chlorinated samples.     

The Toxicity of Sulphur Dioxide towards Certain Lactic Acid Bacteria from Fermented Apple Juice

This paper describes a method for testing the effect of various concentrations of SO₂ on lactic acid bacteria from ciders. The media and methods were devised to minimize loss of SO₂ due to oxidation or binding with carbonyl compounds. Exposure of laboratory or freshly isolated strains to various concentrations of free SO₂ at pH 4·0 did not readily kill them even at high concentrations of free SO₂ ( c. 150 p/m or 0·97 p/m molecular SO₂) yet they were suppressed at low concentrations ( c. 5 p/m or 0·032 p/m molecular SO₂). Reducing the pH to 3·4 reaffirmed how much more effective SO₂ is against lactic acid bacteria at lower pH levels because more is present as molecular SO₂. As a result of this the idea of quoting SO₂ values as p/m molecular SO₂ is advocated. Addition of hydrogen peroxide or acetaldehyde to a test system containing 142 p/m free SO₂ showed that they had a similar effect in nullifying its antimicrobial properties and allowing the test bacteria to grow. There was no indication that acetaldehyde bisulphite was toxic to the test bacteria.     

CHOLINESTERASE ACTIVITY OF THE MOTOR ENDPLATE IN ISOLATED MUSCLE MEMBRANE

Abstract— The cholinesterase activity of motor endplates in tibialis anterior muscle of rats accounted for about 20 per cent of the total cholinesterase activity of the muscle. In the isolated muscle membrane preparation of rat intercostal muscle, the cholinesterase activity was localized solely in the motor endplate, as shown by cholinesterase staining. The cholinesterase activity of the membrane per unit of nitrogen was 26·9 times that of the muscle homogenate. The membrane (endplate) cholinesterase had an optimal pH of 8, K_m value of 3·1 m m , and was stable at 4° for at least 13 days. Cholinesterase of a motor endplate hydrolysed 2·69 × 10⁸ acetylcholine molecules in 1 msec. Since it is estimated that 10⁸ cholinesterase active sites are present in a motor endplate, the turnover time (time necessary for one enzyme site to hydrolyse one acetylcholine molecule) is calculated to be 372 μ sec, and the turnover number (molecules of acetylcholine hydrolysed by one enzyme site/min) to be 1·61 × 10⁵. From studies with cholinesterase inhibitors, cholinesterase activity was estimated to be due mostly to acetylcholinesterase, and only a minor part to pseudocholinesterase. The muscle membrane preparation seems to be useful for the study of other properties of the motor endplate.     

Research note : Unsuitability of the MRS medium for the screening of hydrogen peroxide-producing lactic acid bacteria

The effect of MRS broth on the stability of hydrogen peroxide (H₂O₂) has been studied. Known concentrations (1–100 μg ml⁻¹) of H₂O₂ were prepared in distilled water, phosphate buffer (pH 7·0) and MRS broth (pH 6·2 and 3·9). H₂O₂ was very stable in aqueous and buffer solutions but it was rapidly degraded in MRS broth (pH 3·9). The presence of H₂O₂ in MRS broth (pH 6·2) could not be detected.     

Purification and characterization of an intracellular β-glucosidase from a new strain of Leuconostoc mesenteroides isolated from cassava

The lactic acid bacterium, Leuconostoc mesenteroides, when grown on an arbutin-containing medium, was found to produce an intracellular β-glucosidase. The enzyme was purified by chromatofocusing, ion-exchange chromatography and gel filtration. The molecular mass of the purified intracellular β-glucosidase, as estimated by gel filtration, was 360 kDa. The tetrameric structure of the β-glucosidase was determined following treatment of the purified enzyme with dodecyl sulphate (SDS). The intracellular β-glucosidase exhibited optimum catalytic activity at 50°C and pH 6 with citrate–phosphate buffer, and 5·5 with phosphate buffer. The enzyme was active against glycosides with (1→4)-β, (1→4)-α and (1→6)-α linkage configuration. From Lineweaver–Burk plots, K _m values of 0·07 mmol l⁻¹ and 3·7 mmol l⁻¹ were found for p -nitrophenyl-β- D -glucopyranoside and linamarin, respectively. The β-glucosidase was competitively inhibited by glucose and by D -gluconic acid–lactone and a glucosyl transferase activity was observed in the presence of ethanol. The β-glucosidase of Leuconostoc mesenteroides, with cyanogenic activity, could be of potential interest in cassava detoxification, by hydrolysing the cyanogenic glucosides present in cassava pulp.     

Isolation and properties of free and immobilized β-galactosidase from the psychrotrophic enterobacterium Buttiauxella agrestis (strain NC4)

A study of the β-galactosidase produced by the psychrotrophic bacterium Buttiauxella agrestis has been carried out. This micro-organism was isolated from raw milk and the enzyme isolated using standard methods. Molecular mass was estimated to be 515 kDa. The isoelectric point was close to 4·45. Optimum pH was 7·25. Maximal activity was observed at 50°C and activation energy was estimated to be 39·1 kJ mol^-1. Lactose enhanced thermal stability. Using α-nitrophenyl-β-D-galactopyranoside as the substrate, the K _m was 11 μmol 1^-1 and V _max was 85 U mg^-1 protein. β-Mercaptoethanol and ethanol were inhibitors; glycerol acted as a complex effector. The enzyme required divalent cations for activity while it was inhibited by EDTA. When the enzyme was immobilized in diethyl aminoethylcellulose the optimum pH of activity was 8. K _m was 47 μmol 1^-1 and V _max was 96 U mg^-1 protein.     

Increased ELISA sensitivity using a modified extraction buffer for detection of Xanthomonas campestris pv. vesicatoria in leaf tissue

In vitro and in planta sensitivity of an indirect enzyme-linked immunoassaytechnique, using a monoclonal antibody specific for the lipopolysaccharide (LPS) of Xanthomonas campestris pv. vesicatoria , was increased 10-foldby using a newextraction buffer (gl of : KH₂PO₄, 2; NaHPO₄, 11·5; EDTAdisodium, 0·14; thimerosal, 0·02; and lysozyme, 0·2). The procedure improvedsensitivity without increasing background levels. In vitro , the limit of detection wasbetween 1×10⁷ and 1×10⁸ cells ml⁻¹ with the conventionalextraction buffer phosphate-buffered saline (PBS) and less than 1×10⁶ cells ml⁻¹ when lysozyme extraction buffer was substituted for PBS. In comparing 22 X. c.vesicatoria strains, absorbance readings were increased close to three-fold with the lysozymeextraction buffer as opposed to PBS. When leaf tissue extract was spiked with the bacterium, thelimit of detection was 1×10⁷ cfu ml⁻¹ and 1×10⁸ cfu ml⁻¹ with the lysozyme solution and PBS, respectively, as the extraction buffers. Whenusing the lysozyme extraction buffer in combination with a commercial amplification system, thelimit of detection was decreased to less than 1×10⁵ cfu ml⁻¹ in leaftissue. The addition of the lysozyme and EDTA to the phosphate buffer resulted in release of asignificant quantity of LPS and concomitant dramatic increase in sensitivity. The new procedure,termed lysozyme ELISA (L-ELISA), should increase sensitivity of ELISA reactions where LPS isthe reacting epitope.     

Aldehyde Dehydrogenases in Rat Brain. Subcellular Distribution and Properties

Abstract: Kinetic studies suggested the presence of several forms of NAD-dependent aldehyde dehydrogenase (ALDH) in rat brain. A subcellular distribution study showed that low- and high- K _m activities with acetaldehyde as well as the substrate-specific enzyme succinate semialdehyde dehydrogenase were located mainly in the mitochondrial compartment. The low- K _m activity was also present in the cytosol (<20%). The low- K _m activity in the homogenate was only 10–15% of the total activity with acetaldehyde as the substrate. Two K _m values were obtained with both acetaldehyde (0.2 and 2000 μ m ) and 3,4-dihydroxyphenylacetaldehyde (DOPAL) (0.3 and 31 μ m ), and one K _m value with succinate semialdehyde (5 μ m ). The main part of the aldehyde dehydrogenase activities with acetaldehyde, DOPAL, and succinate semialdehyde, but only little activity of the marker enzyme for the outer membrane (monoamine oxidase, MAO), was released from a purified mitochondrial fraction subjected to sonication. Only small amounts of the ALDH activities were released from mitochondria subjected to swelling in a hypotonic buffer, whereas the main part of the marker enzyme for the intermembrane space (adenylate kinase) was released. These results indicate that the ALDH activities with acetaldehyde, DOPAL and succinate semialdehyde are located in the matrix compartment. The low- K _m activity with acetaldehyde and DOPAL, but not the high- K _m activities and succinate semialdehyde dehydrogenase, was markedly stimulated by Mg²⁺ and Ca²⁺ in phosphate buffer. The low- and high- K _m activities with acetaldehyde showed different pH optima in pyrophosphate buffer.     

Xylanolytic activity of commercial juice-processing enzyme preparations

Of 22 commercial juice-processing enzyme preparations investigated, Clarex ML was found to exhibit the highest xylanase activity. The xylanase from Clarex ML was most active at 50–60°C and pH 5·0–5·5. The K _m and V _max values of the enzyme with oat-spelt xylan as the substrate were 8·6 mg ml⁻¹ and 42 μmol xylose l⁻¹ min⁻¹, respectively. Xylobiose was the main product of enzymatic hydrolysis of xylan.     

INCORPORATION OF PHENYLALANINE AS A SINGLE UNIT INTO RAT BRAIN PROTEIN: RECIPROCAL INHIBITION BY PHENYLALANINE AND TYROSINE OF THEIR RESPECTIVE INCORPORATIONS

Abstract— Of seven amino acids studied, glutamic acid and phenylalanine were incorporated in highest amounts into the hot-TCA-insoluble material of the 100,000 g supernatant fraction of rat brain homogenate. The system for incorporation of phenylalanine was RNase-insensitive and required ATP (apparent K_m = 0.64 m m ), KC1 (apparent K_m = 14 m m ) and MgCl₂ (optimal concentration range 4-15 m m ). The apparent K_m for phenylalanine was 2.9 m m . [¹⁴C]Phenylalanine did not undergo modification before incorporation. Tyrosine and phenylalanine inhibited the incorporation, respectively, of [¹⁴C]phenylalanine and [¹⁴C]tyrosine when incubated simultaneously or successively. The K_m and K_t (3.3 m m ) values for phenylalanine in the incorporation reaction and as inhibitor of the incorporation of [¹⁴C]tyrosine were similar. We suggest that both the enzyme and the acceptor for the incorporation of these two amino acids are the same. [¹⁴C]Phenylalanine and [¹⁴C]tyrosine entered into COOH-terminal positions in the reactions described. Brain exhibited a 25- to 100-fold higher capacity to incorporate phenylalanine than that of liver, kidney or thyroid. The acceptor capacity in rat brain rapidly decreased from day 5 to day 15 of postnatal age and then slowly until age 150 days.     

UPTAKE OF CALCIUM BY SUBCELLULAR FRACTIONS ISOLATED FROM OUABAIN-TREATED CEREBRAL TISSUES

Abstract— Slices of cerebral cortex were incubated in medium containing 0·75 or 2·8 mM ⁴⁵CaCl₂, in the presence or absence of 0·01–0·1 m m -ouabain. Ouabain induced accumulation of calcium by slices to a maximum of 4 μmoles/g of tissue/hr (0·75 m m -CaCl₂ in the medium) and to 8 μmoles/g of tissue/hr (2·8 m m -CaCl₂ in the medium). Accumulation of Ca²⁺ occurred more slowly than loss of K⁺ from the slices and more closely resembled the pattern of Na⁺ uptake.
Mitochondrial fractions isolated from ouabain-treated slices contained significantly more calcium than controls. Inclusion of EDTA in the homogenization medium resulted in decreased amounts of particulate-bound calcium.
The effect of ouabain on accumulation of calcium is discussed with regard to possible relationships to processes of active and passive transport.     

Effect of high and low sulfate concentrations on adenosine 5'-phosphosulfate sulfotransferase activity from Lemna minor

The effect of Na₂SO₄ concentrations from 0 to 17.6 m M in the nutrient solution of Lemna minor L. strain 6580 on adenosine 5'-phosphosulfate sulfotransferase activity was examined. Routinely, the plants were cultivated on 0.88 mA SO₄²⁻. The enzyme activity was increased by 50 to 100% after transfer to 0 or 0.0088 m M SO₄²⁻. Transfer back to 0.88 m M rapidly decreased the enzyme activity to the initial level. Cultivation on 17.6 mM Na₂SO₄ redueed extractable adenosine 5'-phosphosulfate sulfotransferase by 50%. The original level was rapidly re-established on 0,88 m M . In control experiments, a decrease in adenosine 5'-phosphosulfate sulfotransferase activity was also induced by K₂ SO₄, whereas NaCl caused a small increase. This indicates that the observed effects are dependent on the sulfate ion. ATP-sulfurylase activity measured for comparison was only significantly affected by the omission of sulfate, which induced a 20% increase, indicating that this enzyme activity from Lemna minor is less suseeptible to changes in medium sulfate than adenosine 5'-phosphosulfate sulfotransferase. A close relationship between adenosine 5'-phosphosulfate sulfotransferase activity and the content of asparagine, glutamine, non-protein thiols and sulfate in the tissue was detected, indicating a positive control mechanism induced by amides and a negative mechanism induced by thiols and sulfate.     

Lipid content and fatty acid composition of target tissues in wild Perca fluviatilis females in relation to hepatic status and gonad maturation

Variation in liver ultra‐structure and composition in relation to energy mobilization was investigated in female perch Perca fluviatilis from the Meuse River between August 2001 and June 2002. In April, just before spawning, the lipo‐somatic index ( I _F) was 0·3%, the gonado‐somatic index ( I _G) was 28% and the total lipid content of the liver was 2·53%. The average areas of lipid droplets and mitochondria were 0·05 and 0·06 μm², respectively. Glycogen supply reached 7·9% of the total area of the hepatocyte. During the sexual resting period, females accumulated energy in perivisceral fat and in the liver to reach 1·6% I _F and 4·85% of liver lipid content in August with lipid droplets average size of 0·09 μm² and glycogen average area of 15%. Liver cells contained a weakly developed rough endoplasmic reticulum (RER) and a great number of small mitochondria (average size 0·02 μm²). The I _G was 0·6% at this time. During the whole annual cycle, the average lipid content of female liver never exceeded 3·9 ± 1·9%. The concentration of docosahexaenoic (DHA), linolenic and linoleic acids increased in mature gonads while linolenic and linoleic acids decreased in the liver during the same period. Fatty acid composition of muscles of perch was characterized by a high content of DHA.     

Effects of osmolality and ions on the motility of stripped and testicular sperm of freshwater-and seawater-acclimated tilapia, Oreochromis mossambicus

A significantly higher concentration of testicular spermatozoa was obtained from freshwater Oreochromis mossambicus (9·9×10⁹ spermatozoa ml⁻¹) than seawater O. mossambicus (4·6×10⁹ spermatozoa ml⁻¹). The mean osmolality of the urine of freshwater fish (78·5 mOsmol kg⁻¹) was significantly different from that of seawater fish (304·8 mOsmol kg⁻¹). The mean length of the mid-piece of the spermatozoa together with the tail was more variable in freshwater O. mossambicus (8·80±0·23μm) than in seawater specimens (8·27±0·18 μm). Stripped sperm of freshwater O. mossambicus was highly contaminated by urine which was a good activator of sperm motility in O. mossambicus held in both fresh and sea water. The osmolality for initiation of motility in freshwater O. mossambicus spermatozoa was from 0 to 333 mOsmol kg⁻¹ while for seawater O. mossambicus spermatozoa it was from 0 to 1022 mOsmol kg⁻¹. The optimum osmolality for motility was from 70 to 333 mOsmol kg⁻¹ for freshwater O. mossambicus spermatozoa and from 333 to 645 mOsmol kg⁻¹ for seawater fish. In freshwater O. mossambicus spermatozoa, the presence of 20 mM CaCl₂ increased the permissive osmolality of NaCl from 184 to 645 mOsmol kg⁻¹. For seawater O. mossambicus spermatozoa, solutions of NaCl devoid of CaCl₂ were unable initiate motility, but the addition of 1·5 to 30 mM CaCl₂ to the NaCl solution (0–934 mOsmol kg₁) had a full motility initiating effect.     

PROPERTIES OF RAT BRAIN HISTIDINE DECARBOXYLASE

Abstract– The properties of histidine decarboxylase ( l -histidine carboxylyase EC 4.1,1.22) have been studied in a whole rat brain homogenate. Optimum pH depended upon substrate concentration; the variations of K _m and V _max were determined as a function of pH. pH values lower than 6.0 caused a loss of enzymic activity; activity was stable at pH values higher than 6.0. Enzyme activity was proportional to temperature in the range 30-45°C; temperature characteristic (μ) and Q₁₀ were determined and thermal inactivation was studied. Addition of pyridoxal 5'-phosphate increased enzyme activity. Dialysis of homogenates against phosphate buffer caused a partial loss of enzyme activity which could be restored by addition of the coenzyme to the incubation mixture. Enzyme activity was inhibited by α-methylhistidine and benzene and was unaffected by α-methyl DOPA. The properties correspond to those of a 'specific' histidine decarboxylase. However, the brain enzyme differs from the corresponding enzyme in peripheral tissues in the inability to achieve a total inhibition of activity by dialysis.     

Purification and characterization of galacto-oligosaccharide-producing β-galactosidase from Sirobasidium magnum

Galacto-oligosaccharide-producing β-galactosidase from Sirobasidium magnum CBS6803 was purified to homogeneity with a yield of 60% by DEAE–toyopearl, butyl–toyopearl, p -aminobenzyl 1-thio-β- d -galactopyranoside–agarose and concanavalin A–agarose columns, from a solubilized cell wall preparation. The isoelectric point (pI) of purified β-galactosidase was 3·8, and the relative molecular mass was 67 000 as estimated by SDS gel electrophoresis, and 135 000 as estimated by gel filtration. Optimal β-galactosidase activity was observed at a temperature and pH of 65°C and pH 4·5–5·5, respectively. The K _m values for o -nitrophenyl-β- d -galactopyranoside and lactose were 14·3 and 5·5 mmol l⁻¹, respectively, and the V _max values for these substrates were 33·4 and 94·5 μmol min⁻¹ mg of protein⁻¹, respectively. In addition this enzyme possessed a high level of transgalactosylation activity, and 72 mg ml⁻¹ galacto-oligosaccharide was produced from 200 mg ml⁻¹ lactose.