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M P Coughlan

Publications and source records attributed to M P Coughlan.

At least 19 recordsLinked to original sources
PubMed1996

Purification and characterization of two arabinofuranosidases from solid-state cultures of the fungus Penicillium capsulatum.

Two arabinofuranosidases, termed Ara I and Ara II, from solid-state cultures of Penicillium capsulatum were purified to apparent homogeneity as judged by electrophoresis and isoelectric focusing. Each enzyme is a single subunit glycoprotein, and they have M(r)s and pIs of 64,500 and 4.15 (Ara I) and 62,700 and 4.54 (Ara II), respectively. Ara I is most active at pH 4.0 and 60 degrees C, while Ara II exhibits optimal activity at pH 4.0 and 55 degrees C. Ara I is the more thermostable, with its half-life at 70 degrees C and pH 4.0 being 17.5 min. By contrast, the half-life of Ara II is only 9 min at 60 degrees C and pH 4.0. Ara I has the lower Km and higher catalytic constant values with p-nitrophenyl-alpha-L-arabinofuranoside being used as the substrate. Arabinose, a competitive inhibitor (Ki, 16.4 mM) of Ara II, has no effect on Ara I activity at concentrations of up to 40 mM. Each enzyme catalyzes the release of arabinose from pectin, araban, and certain arabinose-containing xylans. The last activity is enhanced by pretreatment of the relevant substrates with xylanase, ferulic acid esterase, or combinations of these enzymes. Thus, arabinoxylooligosaccharides in which arabinose is the sole side chain substituent appear to be the preferred substrates. On the basis of the evidence cited above, each enzyme has been classified as an alpha-L-arabinofuranoside arabinofuranohydrolase (EC 3.2.1.79).

Arabinose↗
PubMed1993

The xylan-degrading enzyme system of Talaromyces emersonii: novel enzymes with activity against aryl beta-D-xylosides and unsubstituted xylans.

Talaromyces emersonii, a thermophilic aerobic fungus, produces a complete xylan-degrading enzyme system when grown on appropriate substrates. In this paper we present the physicochemical and catalytic properties of three enzymes, xylosidase (Xyl) I (M(r) 181,000; pI 8.9), II (M(r) 131,000; pI 5.3) and III (M(r) 54,200; pI 4.2). Xyl I and II appear to be dimeric and Xyl III is a single-subunit protein. All three enzymes catalyse the hydrolysis of aryl beta-D-xylosides and xylo-oligosaccharides. Xyl I is a classic beta-xylosidase (1,4-beta-D-xylan xylohydrolase; EC 3.2.1.37), and Xyl II and III are novel xylanases (endo-1,4-beta-D-xylan xylanohydrolase; EC 3.2.1.8) which we believe have not hitherto been reported. In addition to the above substrates, they also catalyse the extensive hydrolysis of unsubstituted xylans, and may have considerable biotechnological potential. The hydrolysis product profiles and bond-cleavage frequencies with various substrates are presented.

Ascomycota↗
PubMed1992

The stereochemical course of reactions catalysed by the cellobiohydrolases produced by Talaromyces emersonii.

Three forms of exocellobiohydrolase (EC 3.2.1.91), CBH IA, CBH IB and CBH II, were isolated to apparent homogeneity from culture filtrates of the aerobic fungus Talaromyces emersonii. CBH IA and CBH II appear to be native forms of these enzymes, while CBH IB may represent a proteolytic degradation product of the CBH IA enzyme. The hydrolysis of beta-cellobiosyl fluoride by each form was monitored by 1H-n.m.r. spectroscopy. The reactions catalysed by CBH IA and CBH IB proceed with retention of the anomeric configuration, whereas that catalysed by CBH II is one of inversion. Thus one may deduce that CBH IA (or CBH IB) and CBH II operate double and single displacement reactions respectively during catalysis of substrate. On the basis of these findings and the observed substrate specificities of the various forms, one may conclude that CBH IA (and CBH IB) is a family C enzyme, while CBH II belongs to family B [Henrissat, Claeyssens, Tomme, Lemesle & Mornon (1989) Gene 81, 83-95].

Ascomycota↗
PubMed1989

Characterization of purine hydroxylase I from Aspergillus nidulans.

Purine hydroxylase I from Aspergillus nidulans was purified 850-fold. The purified preparations exhibited the spectral and catalytic properties, including broad specificity for oxidizing and reducing substrates, typical of molybdenum/flavin/iron-sulphur-containing hydroxylases (oxotransferases).

Aspergillus nidulans↗
PubMed1987

Macromolecular Organization of the Cellulolytic Enzyme Complex of Clostridium thermocellum as Revealed by Electron Microscopy.

Clostridium thermocellum JW20 and YM4 both synthesize cellulolytic enzyme complexes, cellulosomes, when grown on medium containing cellulose. Electron microscopic studies showed that, in the early stages of growth of strain JW20, clusters of tightly packed cellulosomes, i.e., polycellulosomes, were located on the cell surface and were bound to cellulose. The polycellulosome was estimated to have a particle mass of 50 x 10 to 80 x 10 daltons (Da), while that of the cellulosome was estimated to be 2 x 10 to 2.5 x 10 Da and to contain about 35 polypeptides ranging from 20 to 200 kDa. The cellulosome produced by strain YM4 was found to be somewhat larger, with the estimated particle mass being 3.5 x 10 Da, and the number of polypeptides was counted to be 45 to 50, ranging from 20 to 200 kDa. In the early stages of cultivation, the cellulosomes from both species exist as tightly packed complexes (tight cellulosomes). These subsequently decompose to loosely packed complexes (loose cellulosomes) and ultimately to free polypeptides. Examination of the loose cellulosomal particles showed that they contain rows of equidistantly spaced, similarly sized polypeptide subunits, with an apparently identical orientation arranged parallel to the major axis of the cellulosome. It is postulated that on binding of a cellulose chain alongside such a row of subunits a simultaneous multicutting event occurs that leads to the release of cellooligosaccharides of four cellobiose units in length (C(4)). Rows of smaller-sized subunits with lower center-to-center distances, which are also present in the cellulosome, subsequently cleave the C(4) fragments (or cellulose) to C(2) (cellotetraose) or C(1) (cellobiose). In this way the cellulosome can catalyze the complete hydrolysis of cellulose.

Journal Article↗
PubMed1985

The cellulolytic enzyme complex of Clostridium thermocellum is very large.

The cellulolytic enzyme system bound to cellulose during the early stages of growth of C. thermocellum on this substrate was resolved into two major complexes. These complexes, as viewed by electron microscopy, are spherical particles with diameters of 210 A and 610 A and calculated molecular weights of 4.2 million and 102 million daltons, respectively.

Cellulase↗
PubMed1985

Isolation and characterization of the 1,4-beta-D-glucan glucanohydrolases of Talaromyces emersonii.

Culture filtrates of Talaromyces emersonii were found to contain four endocellulases termed I, II, III and IV, the last having the greatest electrophoretic mobility towards the anode in homogeneous 5%-(w/v)-polyacrylamide gels at pH 4.5. All four are glycoproteins, the carbohydrate contents being: I, 27.7%; II, 29.0%; III, 44.7%; IV, 50.8. Each form is eluted as a single peak corresponding to an Mr value of 68000 on gel filtration at pH 3.5 and as a single band corresponding to an Mr value of 35000 on reductive sodium dodecyl sulphate/polyacrylamide-gradient-gel electrophoresis. However, we believe that the latter represents the native Mr value. The pI values for each lie between pH 2.8 and 3.2. Activity in each case is optimal at pH 5.5-5.8 and at 75-80 degrees C. Half-life values at pH5 and 75 degrees C were from 2 to 4h. The specific activity with any individual substrate was much the same for each enzyme, as was the ratio of activity from one substrate to the next. Possible reasons for the observation that plots of velocity versus substrate concentration are sigmoidal are discussed. We believe that the finding of four endocellulases reflects differential glycosylation of a single enzyme form rather than genetically determined differences in primary structure.

Carbohydrates↗
PubMed1984

Purification and properties of purine hydroxylase II from Aspergillus nidulans.

Purine hydroxylase II from Aspergillus nidulans has been purified to near homogeneity. The enzyme has a pI of 5.7, a molecular weight of 300,000, and two subunits with molecular weight of 153,000 each. The enzyme contains 2 FAD, 2 molybdenum atoms, and 4 (2 Fe-2S) iron-sulfur centers per molecule and exhibits broad specificity for reducing and oxidizing substrates. Among the more notable characteristics are the ability to oxidize hypoxanthine and nicotinic acid but not xanthine and virtually complete inactivity with oxygen. Moreover, while the enzyme is inactivated by borate and methanol, it is very resistant to cyanide and arsenite and it not inactivated by allopurinol. At infinite concentrations of reducing and oxidizing substrates, the Km for hypoxanthine was 119 microM, for nicotinic acid was 136 microM, and for NAD+ was 525 microM.

Alcohol Oxidoreductases↗
PubMed1984

Optical and electron paramagnetic resonance spectroscopic studies on purine hydroxylase II from Aspergillus nidulans.

Purine hydroxylase II from Aspergillus nidulans contains a molybdenum cofactor very similar to that found in a number of other molybdenum-containing hydroxylases. (A. nidulans contains two purine hydroxylases, I and II, related to each other by possession of a common cofactor and overlapping substrate specificity.) Addition of reducing substrates effects bleaching of the visible absorption spectrum of the enzyme, the decrease in absorbance at 450 nm being linearly proportional to that at 550 nm. No increase in absorption at longer wavelengths was observed during such titrations. Electron paramagnetic resonance studies of reduced samples of native and modified enzyme species showed the presence of a number of Mo(V) signals (gav = 1.97), exhibiting H hyperfine coupling, comparable to those in the corresponding enzymes from other sources. The enzyme possesses two non-heme-iron-sulfur centers, one (Fe2S2)I with gav less than 2.0 and the other (Fe2S2)II with gav greater than 2.0. The flavin radical signal observed at pH 7.8 had a linewidth of 1.5 mT, indicating it to be the anionic form FAD- . In this respect purine hydroxylase II is unique among all molybdenum-containing hydroxylases studied to date.

Alcohol Oxidoreductases↗
PubMed1983

The role of molybdenum in human biology.

Molybdenum, because of its unique chemistry, is the biological catalyst for reactions in which proton and electron transfer, and possibly oxygen transfer, are coupled. The molybdoenzymes in man are sulphite oxidase, xanthine oxidase/dehydrogenase and aldehyde oxidase. The former is essential for detoxication of the sulphite arising from metabolism of sulphur-containing amino acids, from ingestion of bisulphite preservative and from inhalation of sulphur dioxide, an atmospheric pollutant. Whether, or not, any of the reactions catalysed by xanthine oxidase/dehydrogenase and aldehyde oxidase are necessary for human well-being has yet to be established.

Chemical Phenomena↗
PubMed1982

Properties of the prosthetic groups of rabbit liver aldehyde oxidase: a comparison of molybdenum hydroxylase enzymes.

Rabbit liver aldehyde oxidase (AO), like milk xanthine oxidase (XO) and chicken liver xanthine dehydrogenase (XDH), possesses the following prosthetic groups: FAD, a functional Mo center, and two spectroscopically distinct iron-sulfur centers, one with gav less than 2.0 (termed Fe/S I) and the other with gav greater than 2.0 (termed Fe/S II) in the reduced enzyme. EPR spectra for the Mov species were found to be nearly identical in AO and XO for a number of enzyme complexes, and the midpoint reduction potentials for functional MoVI/MoV (-359 mV) and MoV/MoVI (-351 mV) were nearly the same in all three enzymes (50 mM phosphate, pH 7.8). A strong magnetic interaction between MoV and reduced Fe/S I, previously detected in XO and XDH, was also found in AO. No MoV-Fe/S II interaction could be detected in AO (nor in XO). In contrast, the order of reduction of Fe/S I and Fe/S II, as measured from their midpoint potentials, is reversed in AO (Em = -207 and -310 mV, respectively) as compared to XO (Em = -280 and -245 mV, respectively) in phosphate buffer at pH 7.8. The oxidized-reduced extinction coefficients at 450 and 550 nm for the two centers are also apparently reversed in AO and XO. Although magnetic interaction between FAD and one or both reduced Fe/S centers has been detected in both AO and XO, no magnetic interaction between the two reduced Fe/S centers themselves was found in AO (although such interaction has been seen in XO). The average FAD reduction potential is substantially more positive in AO (Em for FAD/FADH., -258 mV; FADH./FADH2, -212 mV at pH 7.8) than in XO or XDH. It can be concluded that although the properties and immediate environment of the functional Mo center are conserved in the three Mo hydroxylase enzymes, and all three enzymes possess the same set of prosthetic groups, the properties of the groups which transfer electrons from the Mo to the ultimate electron acceptor can vary substantially in AO, XO, and XDH.

Aldehyde Oxidase↗
PubMed1982

A convenient procedure for the estimation of cellulase activity.

The extent of clarification of cellulose-containing agar by "complete' cellulase is a linear function of the logarithm of the enzyme activity. This fact allows a convenient and quantitative estimation of cellulase activity in culture filtrates to be made without the need for tedious dilution. Moreover, such activity can be determined even in the presence of inhibitors such as cellobiose.

Ascomycota↗