An evaluation of potential interferences in a fluorimetric assay for the rapid detection of thermotolerant coliforms in sewage

C.M. DAVIES and S.C. APTE.2000. A 1-h fluorimetric assay of β- d -galactosidase activity was evaluated for determining thermotolerant coliforms (TTC) in sewage samples. Above TTC concentrations of 2·3 × 10³ colony-forming units (cfu) 100 ml⁻¹, the assay response was related to TTC concentration. However, below this concentration, a large background signal was observed which was independent of TTC concentration. A separation scheme involving various filtration treatments and additions of a β- d -galactosidase inhibitor was devised and used to quantify the sources of this anomalous assay response. The interferences encountered were largely due to the presence in sewage of non-specific cell-free enzymes or other cell-free substances that were capable of hydrolysing the fluorogenic substrate. Despite this apparent limitation, the fluorimetric enzyme assay has potential as an 'early warning' indicator of treatment process failure and gross sewage contamination and leakage in situations where TTC concentrations exceed 2·3 × 10³ cfu 100 ml⁻¹     

Purification of a β-galactosidase from rice shoots and its involvement in hydrolysis of the natural substrate in cell walls

Several glycosidase and glycanase activities have been detected in homogenates of rice ( Oryza sativa L. cv. Nipponbare) shoots after successive extraction with K-phosphate (pH 7. 0) and buffer containing 3 M LiCl. The major β-D-galactosidase (EC 3. 2. 1. 23) present in the buffer-soluble protein fraction was purified to electrophoretic homogeneity by a combination of chromatographic techniques including DEAE-Sepharose CL-6B, Sephacryl S-200HR and p -aminophcnyl-β-D-thiogalactopyranoside–Sepharose 4B. Analysis by denaturing gel electrophoresis revealed a single polypeptide chain with an apparent molecular mass of 42 kDa. Similar to the value of 40 kDa estimated for the native protein by gel-permeation. The isoelectric point was pH 6. 0. The K_m and V_max values for p -nitrophenyl (PNP)-β-D-galactopyra-noside were 0. 63 m M and 0. 32 mmol (mg protein)⁻¹ h⁻¹, respectively. Maximum activity in McIlvaine buffer occurred at pH 3. 4, and the activity was inhibited by Ag²⁺, Cu²⁺. Hg²⁺, p -chloromercuribenzoate (PCMB) and D-galactono-l,4-lactone. The enzyme hydrolyzed larchwood arabinogalactan in an exo-fashion, and acted weakly on arabinosyl and galactosyl residue-rich polymer of pectic polysaccharides and cell walls from rice shoots.     

Fluorogenic assay for immediate presumptive detection of Escherichia coli in urine

A rapid and direct fluorogenic assay was used to detect Escherichia coli in urine. Most clinical isolates of E. coli produce β-glucuronidase, whereas almost all other enterobacteria lack the enzyme. Spectrofluorimetric assay of β-glucuronidase, without previous induction, was performed on growing and starved uropathogenic E. coli in artificial urine. The presence of 10³ cfu ml^-1 of E. coli in urine was detected by β-glucuronidase activity in less than 1 h. These results indicate that β-glucuronidase is a rapid, specific and sensitive indicator of the presence of E. coli in urine, and provide additional information on the biological state of the infecting bacterial population.     

Production of xylanolytic enzymes during growth on pulverized grass by Aspergillus ochraceus-42

Xylanase and β-xylosidase with activity of 6.46 U mg^-1 and 0.500 U mg^-1, respectively, were produced extracellularly by Aspergillus ochraceus during growth on pulverized grass in liquid state fermentation, compared to 9.3 U mg^-1 and 0.74 U mg^-1 when pure xylan was used. The culture filtrate was devoid of any cellulase activity. Xylanolytic enzymes were produced optimally in 144 h of incubation on 1% pulverized grass, pH 6.5. About 8.43% (w/w) sugars were liberated from alkali-treated grass in 6 h by the synergistic effect of xylanolytic enzymes. The half-lives for xylanase and β-xylosidase at 50°C were 210 min and 300 min, respectively, and half-life increased with the increase in protein concentration. Both mono- and divalent cations, especially K⁺ and Zn²⁺, exhibited a profound effect on the rate of enzyme saccharification.     

Identification and Characterization of Two DNA Polymerase Activities Present in Trypanosoma brucei Mitochondria

We have identified and partially purified two DNA polymerase activities from purified Trypanosoma brucei mitochondrial extracts. The DNA polymerase activity eluted from the single-stranded DNA agarose column at 0.15 M KCI (polymerase MI) was significantly inhibited by salt concentrations greater than 100 mM, utilized Mg²⁺ in preference to Mn²⁺ as a cofactor on deoxyribonucleotide templates with deoxyribose primers, and in the presence of Mn²⁺ favored a ribonucleotide template with a deoxyribose primer. A 44 kDa peptide in this fraction crossreacted with antisera against the Crithidia fasciculata β-like mitochondrial polymerase. In activity gels the catalytic peptide migrated at an apparent molecular weight of 35 kDa. The DNA polymerase activity present in the 0.3 M KCI DNA agarose fraction (polymerase M2) exhibited optimum activity at 120-180 mM KCI, used both Mg²⁺ and Mn²⁺ as cofactors, and used deoxyribonucleotide templates primed with either deoxyribose or ribose oligomers. Activity gel assays indicate that the native catalytic peptide(s) is ˜ 80 kDa in size. The two polymerases showed different sensitivities to several inhibitors: polymerase MI shows similarities to the Crithidia fasciculata β-like mitochondrial polymerase while polymerase M2 is a novel, salt-activated enzyme of higher molecular weight.     

Characterization of a cell wall β-galactosidase of Cicer arietinum epicotyls involved in cell wall autolysis

β-Galactosidase (EC 3.2.1.23) has been established as the main enzyme involved in the autolytic process. The enzyme extracted from cell walls of epicotyls of Cicer arietinum L. cv. Castellana with 3 M LiCl is a 45 kDa protein composed of a single subunit, having an optimum pH of 4; an optimum temperature of 45°C and K_m and V_max of 1.72 m M and 18.5 nkat (mg protein)^–1 respectively, as evaluated against p -nitrophenyl-β- d -galactopyranoside. The enzyme is inhibited by Hg²⁺, d -galactono-1,4-lactone and galactose, substances that also inhibit the autolytic process. Ca²⁺ and EDTA, which do not affect the activity of the β-gaiactosidase, do however modify the hydrolysis of the cell wall mediated by the enzyme, and they also inhibit the autolytic process. Ca²⁺ decreased both processes, whereas EDTA increased them; and when both substances were added together, their individual effects were neutralized. The effects of both agents is probably due to modifications in the cell wall that prevent access of the enzyme to its substrate.     

Purification and characterization of an extracellular cysteine proteinase produced by Micrococcus sp. INIA 528

Micrococcus sp. INIA 528, a micro-organism isolated from raw ewe's milk Manchego cheese, produced an extracellular proteinase. This enzyme was purified to homogeneity from culture supernatant fluid in two chromatographic steps, with a 29-fold increase of specific activity and a 28% recovery of proteinase activity. The homogeneous protein was characterized biochemically. The molecular weight of the enzyme was determined to be 19.4 kDa by mass spectrometry. The purified enzyme was inhibited by E-64, PMSF and iodoacetamide and activated by cysteine, glutathione, dithiothreitol and β-mercaptoethanol. These results suggest that the enzyme is a cysteine proteinase. Optimal conditions for activity on azocasein were 34°C and a pH of 7.0. The proteinase preferentially degraded β-casein, while after a longer incubation period α^s1-casein was also extensively hydrolysed. The proteinase had a K _m value of 6.12 g 1⁻¹ for casein and 2.20 g 1⁻¹ for azocasein.     

Purification and characterization of an endoamylase from tulip (Tulipa gesneriana) bulbs

Amylase activity extracted from tulip ( Tulipa gesneriana L. cv. Apeldoorn) bulbs that had been stored for 6 weeks at 4°C was resolved to 3 peaks by anion-exchange chromatography on diethylaminoethyl-Sephacel. These 3 amylases exhibited different relative mobilities during non-denaturing polyacrylamide gel electrophoresis (PAGE). The most abundant amylase form (amylase I) was purified to apparent homogeneity using hydrophobic interaction chromatography, gel filtration and chromatofocusing. The apparent molecular mass of the purified amylase was estimated to be 51 kDa by sodium dodecyl sulfate-PAGE and 45 kDa by gel filtration chromatography. The purified amylase was determined to be an endoamylase (EC 3.2.1.1) based on substrate specificity and end-product analysis. The enzyme had a pH optimum of 6.0 and a temperature optimum of 55°C. The apparent K_m value with soluble starch (potato) was 1.28 mg ml⁻¹. The presence of Ca²⁺ increased the activity and thermal stability of the enzyme. The presence of dithiothreitol enhanced the activity, while β -mercaptoethanol and reduced glutathione had no significant effect. When pre-incubated in the absence of the substrate, N-ethylmaleimide and 5,5'-dithiobis-(2-nitrobenzoic acid) partially inhibited the enzyme. α -cyclodextrins or β -cyclodextrins had no effect on enzyme activity up to 10 m M . In addition to CaCl₂, CoCl₂ slightly enhanced activity, while MgCl₂ and MnCl₂ had no significant effect at a concentration of 2 m M . ZnCl₂, CuSO₄, AgNO₃ and EDTA partially inhibited enzyme activity, while AgNO₃ and HgCl₂ completely inhibited it at 2.0 m M .     

An extracellular β-galactosidase secreted from cell suspension cultures of carrot. Its purification and involvement in cell wall-polysaccharide hydrolysis

β-Galactosidase (β-Galase, EC 3.2.1.23) activity has been detected in a culture medium of cell suspension cultures of carrot ( Daucus carota L. cv. Kintoki). The extracellular β-Galase (β-Galase-II) was purified to electrophoretic homogeneity from the concentrated medium using ammonium sulfate precipitation, chromatography on CM-Sephadex C-50. DEAE-Sepharose CL-6B and Sephacryl S-200HR, and preparative PAGE. The molecular mass of the purified enzyme was estimated to be 65 kDa by Sephacryl S-200HR gel-permeation, and 60 kDa by SDS-PAGE after treatment with SDS and 2-mercaptoethanol. The pI was 6.5. The K_m and V_max values for p -nitrophenyl (PNP)-β-D-galactopyranoside were 0.17 m M and 31.9 μmol (mg protein)^-1, h^-1, respectively. The optimal activity in McIlvaine's buffer occurred at pH 4.0–4.4. The enzyme activity was inhibited by Co²⁴, Cu²⁺, Hg^2-, p -chloromercuribenzoate (PCMB) and D-galactono-1,4-lactone. The enzyme acted on citrus galactan and larchwood arabinogalactan in an exo-fashion, and was slightly involved in the hydrolysis of an acidic pectic polymer containing arabinosyl and galactosyl residues and in the breakdown of cell walls isolated from carrot cell cultures.     

Characteristics of β-galactosidase purified from cell suspension cultures of carrot

Five glycosidase activities from cell homogenate of carrot ( Daucus carota L. cv. Kintoki) cell cultures were assayed after extraction successively by phosphate buffer (pH 7.0) and the buffer plus 2 M NaCl. A β-galactosidase (EC 3.2.1.23) was isolated in a highly purified state from the buffer-soluble protein fraction by ammonium sulfate fractionation and chromatography on CM-Sephadex C-50, DEAE-Sephadex A-50 and Sephadex G-200. The molecular weight of this enzyme was ca 104 000 and the isoelectric point was pH 7.8. The optimal activity occurred at pH 4.4 with McIlvaine buffer. The K_m and V_max values were 1.67 m M and 201 units (mg protein)⁻¹, respectively, for p -nitrophenyl β- d -galactopyranoside. The enzyme activity was strongly inhibited by Zn²⁺, Cu²⁺, Hg²⁺ and d -galactono-1,4-lactone. The enzyme acted on the β-1,4-linked galactan prepared from citrus pectin in an exo-fashion. Furthermore, the enzyme was slightly involved in the hydrolysis of the pectic polymer and cell walls purified from carrot cell cultures.     

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.     

Purification and biochemical characterization of β-xylosidase from Humicola grisea var. thermoidea

Abstract β-d-Xylosidase production was maximal for Humicola grisea var. thermoidea grown on xylan as the sole carbon source. The main β-d-xylosidase activity was localised in the periplasm. β-Xylosidase was purified from crude extracts by heat treatment, ammonium sulfate precipitation and chromatography on DEAE-cellulose and Sephadex G-100. The purified enzyme was a monomer of molecular mass estimated to be 43 kDa by SDS-PAGE and gel filtration. Optima of pH and temperature were 6.0 and 50 °C, respectively. The enzyme activity was stimulated by Ca²⁺, Fe²⁺, and Mg²⁺. The purified β-xylosidase did not exhibit xylanase, carboxymethylcelullase, galactosidase, glucosidase, fucosidase or arabinosidase activities. The purified β-xylosidase hydrolysed xylobiose and xylo-oligosaccharides of up to five monosaccharide units. The enzyme had a K _m of 0.49 mM for p -nitrophenyl- β -d-xylopyranoside and was not inhibited by its product, xylose.     

A β-N-acetylhexosaminidase from Penicillium oxalicum implicated in its cell-wall degradation

High β- N -acetylhexosaminidase (EC.3.2.1.52) activity was detected during autolysis of Penicillium oxalicum . Purification of the enzyme to homogeneity yielded an enzyme with a molecular weight of 132 000 Da by gel filtration and 71 900 Da by SDS polyacrylamide gel electrophoresis, suggesting a dimeric structure. The enzyme is an acidic protein with a pl of 5.0. Optimal activity was at pH 4.0 and 40°C, with a K _m of 0.80 mmol 1^-1 for p -nitrophenyl-β- N -acetylglucosaminide and 1.03 mmol 1^-1 for p -nitrophenyl-β- N -acetylgalactosaminide. The K _i with the competitive inhibitor O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino- N -phenylcarbamate was 1 μmol 1^-1. Hg²⁺, Ag⁺ and Fe³⁺ were effective inhibitors. β- N -acetylhexosaminidase hydrolysed chitobiose, chitotriose, chitotetrose and chitopentose to monomer to an extent of 92, 74, 44 and 17% respectively in 40 min. This enzyme, in conjunction with a purified endochitinase from P. oxalicum , hydrolysed a cell-wall chitin fraction isolated from this fungus, with the production of N -acetylglucosamine.     

A β-(1→4)-xylosyltransferase involved in the synthesis of arabinoxylans in developing barley endosperms

A β-(1→4)-xylosyltransferase (XylTase; EC 2.4.2.24) participating in the synthesis of arabinoxylans was investigated using microsomal membranes prepared from developing barley ( Hordeum vulgare L.) endosperms. The microsomal fraction transferred Xyl from uridine 5'-diphosphoxylose (UDP-Xyl) into exogenous β-(1→4)-xylooligosaccharides derivatized at their reducing ends with 2-aminopyridine. HPLC analysis showed chain elongation of pyridylaminated β-(1→4)-xylotriose (Xyl₃-PA) by repeated attachment of one to five single xylosyl residues depending on the reaction time, leading to the formation of Xyl₄₋₈-PA. Methylation analysis and enzymatic digestions with β-xylosidase (EC 3.2.1.37) and endo -β-(1→4)-xylanase (EC 3.2.1.8) confirmed that the transfer of xylosyl residues into the newly synthesized products occurred through β-(1→4)-linkages. The activity of the XylTase was maximal at pH 6.8 and 20°C and most enhanced in the presence of 0.5% Triton X-100 and 5 m M MnCl₂. The apparent Michaelis constant and maximal velocity of the enzyme for Xyl₃-PA were 2.1 m M and 25 400 pmol min⁻¹ mg protein⁻¹, respectively. The enzyme also transferred [¹⁴C]Xyl from UDP-[¹⁴C]Xyl into higher β-(1→4)-xylooligosaccharides and birchwood xylans through β-(1→4)-linkages. The enzyme activity varied according to the stage of development (7–35 days after flowering) of the endosperms. Maximal activity occurred at 13–16 days; no activity was detectable in mature seeds. A comparison of endosperms from 10 different cultivars of barley harvested 11–22 days after flowering showed no correlation between enzyme activity and the amount of Xyl in the cell walls.     

Subject index volume 144

Abstract β-xylosidase (EC 3.2.1.37) has been purified from Aspergillus nidulans mycelium grown on oat-spelt xylan as sole carbon source. Its pH optimum for activity was found to be 5.0 and the optimum temperature was 50 °C. Its molecular mass was estimated by gel filtration to be 180000. Using p-nitrophenyl-β-d-xylopyranoside as substrate, the K _m and V _max values have been found to be 1.1 mM and 25.6 μmol min⁻¹(mg protein)⁻¹, respectively. Enzyme activity was inhibited by Hg²⁺, Ag²⁺, and Cu²⁺ at a concentration of 1 × 10⁻³ M. The synthesis of β-xylosidase in A. nidulans is strongly induced by arabinose and xylose and is subject to carbon catabolite repression mediated by the cre A gene product.     

Site-directed mutagenesis of the aspartokinase gene lysC and its characterization in Brevibacterium flavum

Using overlap extension polymerase chain reaction (PCR), five transformants of Escherichia coli containing site-directed mutagenized lys Cβ gene were generated and analysed. Exchange of C to A and C to T at nucleotide 1118 of the mutated lys Cβ gene causes a substitution of serine³⁰¹ in the wild-type enzyme for tyrosine³⁰¹ and phenylalanine³⁰¹ in the mutant enzymes, respectively. Enzyme assays showed that Brevibacterium flavum cells harbouring pSUMN18 with mutated lys Cβ genes exhibited 16–20 fold lower specific activities of aspartokinase as compared to that of host containing wild-type lys C gene. The mutation introduced into lys Cβ of B. flavum CCRC18271 resulted in partial feedback-resistant aspartokinase activity.     

Role of G Protein βγ Subunits in Muscarinic Receptor-Induced Stimulation and Inhibition of Adenylyl Cyclase Activity in Rat Olfactory Bulb

Abstract: In the olfactory bulb, muscarinic receptors exert a bimodal control on cyclic AMP, enhancing basal and G_s-stimulated adenylyl cyclase activities and inhibiting the Ca²⁺/calmodulin- and forskolin-stimulated enzyme activities. In the present study, we investigated the involvement of G protein βγ subunits by examining whether the muscarinic responses were reproduced by the addition of βγ subunits of transducin (βγ_t) and blocked by putative βγ scavengers. Membrane incubation with βγ_t caused a stimulation of basal adenylyl cyclase activity that was not additive with that produced by carbachol. Like carbachol, βγ_t potentiated the enzyme stimulations elicited by vasoactive intestinal peptide and corticotropin-releasing hormone. RT-PCR analysis revealed the expression of mRNAs encoding both type II and type IV adenylyl cyclase, two isoforms stimulated by βγ synergistically with activated G_s. In addition, βγ_t inhibited the Ca²⁺/calmodulin- and forskolin-stimulated enzyme activities, and this effect was not additive with that elicited by carbachol. Membrane incubation with either one of two βγ scavengers, the GDP-bound form of the α subunit of transducin and the QEHA fragment of type II adenylyl cyclase, reduced both the stimulatory and inhibitory effects of carbachol. These data provide evidence that in rat olfactory bulb the dual regulation of cyclic AMP by muscarinic receptors is mediated by βγ subunits likely acting on distinct isoforms of adenylyl cyclase.     

Properties and localization of the homoglutathione synthetase from Phaseolus coccineus leaves

The synthesis of homoglutathione (hGSH) by several plants of the tribe Phaseoleae is shown to be catalysed by a β-alanine-specific hGSH synthetase, Properties of the enzyme from Phaseolus coccineus L. cv. Preisgewinner were studied, using ammonium sulfate precipitates of primary leaf extracts. The hGSH synthetase showed a broad pH optimum at pH 8–9, an absolute requirement for Mg²⁺, a stimulation by K⁺, and a high affinity for γ-glutamylcysteine [K_m(app.) 73 μ M ]. The enzyme exhibited a high specificity for β-alanine [K_m(app.) 1.34 m M ] compared to glycine [K_m(app.) 98 m M ]. Chloroplasts, isolated from the leaves of Phaseolus coccineus , contained about 17% of the hGSH synthetase activity in the leaf cells.     

Isoform-Specific Up-Regulation by Ouabain of Na+,K+-ATPase in Cultured Rat Astrocytes

Abstract: There are two α-subunit isoforms (α1 and α2) and two β-subunit isoforms (β1 and β2) of Na⁺,K⁺-ATPase in astrocytes, but the functional heterodimer composition is not known. Ouabain (0.5–1.0 m M ) increased the levels of α1 and β1 mRNAs, whereas it decreased those of α2 and β2 mRNAs in cultured rat astrocytes. The increases in α1 and β1 mRNAs were observed at 6–48 h after addition of the inhibitor. Immunochemical analyses showed that ouabain increased α1 and β1, but not α2 and β2, proteins, and that the isoforms in control and ouabain-treated cultures were of glial origin. Low extracellular K⁺ and monensin (20 µ M ) mimicked the effect of ouabain on α1 mRNA. The ouabain-induced increase in α1 mRNA was blocked by the protein synthesis inhibitor cycloheximide (10 µ M ), the intracellular Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane- N,N,N',N' -tetraacetic acid tetraacetoxymethyl ester (30 µ M ), and the calcineurin inhibitor FK506 (1 n M ). These findings indicate that chronic inhibition of Na⁺,K⁺-ATPase up-regulates the α1 and β1, but not α2 and β2, isoforms in astrocytes, suggesting a functional coupling of α1β1 complex. They also suggest that intracellular Na⁺, Ca²⁺, and calcineurin may be involved in ouabain-induced up-regulation of the enzyme in astrocytes.     

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.