Purification and characterization of an extracellular feruloyl esterase from the thermophilic anaerobe Clostridium stercorarium.

Feruloyl esterases act as accessory enzymes for the complete saccharification of plant cell wall hemicelluloses. Although many fungal feruloyl esterases have been purified and characterized, few bacterial phenolic acid esterases have been characterized. This study shows the extracellular production of a feruloyl esterase by the thermophilic anaerobe Clostridium stercorarium when grown on birchwood xylan. The feruloyl esterase was purified 500-fold in successive steps involving ultrafiltration, preparative isoelectric focusing and column chromatography by anion exchange, gel filtration and hydrophobic interaction. The purified enzyme released ferulic, rho-coumaric, caffeic and sinapinic acid from the respective methyl esters. The purified enzyme also released ferulic acid from a de-starched wheat bran preparation. At pH 8.0 and 65 degrees C, the Km and Vmax values for the hydrolysis of methyl ferulate were 0.04 mmol l-l and 131 micromol min-1 mg-1, respectively; the respective values for methyl coumarate were 0.86 mmol l-l and 18 micromol min-1 mg-1. The purified feruloyl esterase had an apparent mass of 33 kDa under denaturing conditions and showed optimum activity at pH 8.0 and 65 degrees C. At a concentration of 5 mmol l-l, the ions Ca2+, Cu2+, Co2+ and Mn2+ reduced the activity by 70-80%.

Detection of ferulic acid esterase production by Bacillus spp. and lactobacilli.

The production of feruloyl esterase activity by Bacillus spp. and lactobacilli can be detected in an agarplate assay. The assay involves the substitution of the main carbon source in specific agar with ethyl ferulate. A number of Bacillus spp., predominantly B. subtilis strains, were found to exhibit feruloyl esterase activity by this method. Of the examined lactobacilli, Lb. fermentum (NCFB 1751) showed the highest level of ferulic acid esterase activity. The enzyme was released from harvested cells by sonication and showed pH and temperature optima of 6.5 and 30 degrees C respectively.

Purification and characterization of a feruloyl esterase from the fungus Penicillium expansum.

An extracellular phenolic acid esterase produced by the fungus Penicillium expansum in solid state culture released ferulic and rho-coumaric acid from methyl esters of the acids, and from the phenolic-carbohydrate esters O-[5-O-(trans-feruloyl)-alpha-L-arabinofuranosyl]-(1-->3)-O-beta- D-xylopyranosyl-(1-->4)-D-xylopyranose (FAXX) and O-[5-O-((E)-rho-coumaroyl)-alpha-L-arabinofuranosyl]- (1-->3)-O-beta-D-xylopyranosyl-(1-->4)-D-xylopyranose (PAXX). The esterase was purified 360-fold in successive steps involving ultrafiltration and column chromatography by gel filtration, anion exchange and hydrophobic interaction. These chromatographic methods separated the phenolic acid esterase from alpha-L-arabinofuranosidase, pectate and pectin lyase, polygalacturonase, xylanase and beta-D-xylosidase activities. The phenolic acid esterase had an apparent mass of 65 kDa under non-denaturing conditions and a mass of 57.5 kDa under denaturing conditions. Optimal pH and temperature were 5.6 and 37 degrees C, respectively and the metal ions Cu2+ and Fe3+ at concentrations of 5 mmol 1-1 inhibited feruloyl esterase activity by 95% and 44%, respectively, at the optimum pH and temperature. The apparent Km and Vmax of the purified feruloyl esterase for methyl ferulate at pH 5.6 and 37 degrees C were 2.6 mmol 1-1 and 27.1 mumol min-1 mg-1. The corresponding constants of rho-coumaroyl esterase for methyl coumarate were 2.9 mmol 1-1 and 18.6 mumol min-1 mg-1.

Production of feruloyl/rho-coumaroyl esterase activity by Penicillium expansum, Penicillium brevicompactum and Aspergillus niger.

Extracellular esterase production by Penicillium expansum, Penicillium brevicompactum and Aspergillus niger was determined in both liquid and solid-state culture. Methyl ferulate was used as the main carbon source in liquid culture whereas wheat bran and sugar beet pulp were used in solid-state culture. Extracted enzyme for each fungus showed activity in the presence of ONP butyrate, methyl ferulate, methyl coumarate and two 'natural' feruloylated carbohydrate esters. Higher enzyme recoveries were obtained using wheat bran in solid-state culture. Higher levels of feruloyl esterase activity were recovered from P. expansum on all feruloylated substrates than from P. brevicompactum or A. niger. Using ONP butyrate as substrate the pH and temperature optima for the esterases of both Penicillium spp. were 6.0 and 25-30 degrees C. Aspergillus niger esterase activity showed a broader temperature range with an optimum at 40 degrees C.

A comparison of the effects of exogenous and endogenous prostaglandins on fast and slow contractions of field-stimulated guinea-pig vas deferens.

1. This study has compared the effects of exogenous and endogenous prostaglandins on the two phases of contraction of the guinea-pig vas deferens produced by electrical field stimulation. Prostaglandin E2 (PGE2), sulprostone and arachidonic acid dose-dependently and completely inhibited the first (fast) phase of contraction, with IC50s of 2.6 nM, 0.65 nM and 2.2 microM, respectively. 2. Following desensitization of the receptor for adenosine triphosphate (ATP) with alpha, beta-methylene ATP, PGE2, sulprostone and arachidonic acid dose-dependently inhibited the second (slow) phase of contraction of the guinea-pig vas deferens produced by electrical field stimulation, but the inhibition was incomplete (up to only 30%). Indomethacin (2.8 microM) reduced the effect of arachidonic acid. On its own, indomethacin (0.3 to 6.0 microM) had no consistent effect although, on some tissues, a slight potentiation of the contractions was seen. 3. Cicaprost (a PGI2 analogue) at low concentrations (0.5 to 30 nM) potentiated the first phase of contraction but even at high concentrations, had no consistent effect on the second phase of contraction of the guinea-pig vas deferens produced by electrical field stimulation. 4. PGE2, sulprostone and cicaprost potentiated contractions of the guinea-pig vas deferens produced by exogenous ATP. PGE2 and sulprostone also potentiated contractions produced by exogenous noradrenaline, whereas cicaprost had no consistent effect on the response to noradrenaline. 5. These findings indicate that prostaglandins of the E-series inhibit the second phase of contraction as well as the first phase of contraction of the guinea-pig vas deferens produced by electrical field stimulation. However, the extent of the inhibition is much less for the second phase than for the first phase. The reasons for this differential action of PGE are not clear. 6. Cicaprost potentiates the first phase but not the second phase of contraction. Since cicaprost potentiates the contractions produced by exogenous ATP, but not by exogenous noradrenaline, by an action presumably on post-junctional IP receptors, the potentiating action of cicaprost on the first phase of contraction produced by electrical field stimulation would appear to be satisfactorily explained through the action of cicaprost on these post-junctional IP receptors. 7. Exogenous arachidonic acid is apparently converted predominantly to PGE2 by the vas deferens, since the action of arachidonic acid mimicked that of PGE2 and was reduced by indomethacin. However, there was little evidence that sufficient PGE2 is generated during a short period (15 s) of sympathetic nerve stimulation for it to have any significant inhibitory effect on the size of the contractions produced.

Citric acid production by Aspergillus niger in surface culture on inulin.

Aspergillus niger produces citric acid during surface fermentation on inulin, a reserve carbohydrate of plant tubers. Citric acid yields can be improved by airflow over the surface of the fermentation but yields from inulin are 20-30% lower than from sucrose, the traditional commercial substrate.

Measurement of feruloyl/p-coumaroyl esterase by capillary zone electrophoresis.

Ferulic andp-coumaric acid can be separated from their corresponding aliphatic methyl esters by capillary zone electrophoresis, which allows the convenient determination of feruloyl andp-coumaroyl esterase activities using synthetic esters as substrates. A feruloyl-containing sugar ester from wheat bran was also efficiently separated and used as substrate for the enzyme assays.Penicillium expansum was shown to produce feruloyl/p-coumaroyl esterase activity when grown on wheat bran in solid-state culture.

Novel screening assay for the detection of phenolic acid esterases.

A novel plate assay method, developed for the screening of microorganisms or enzyme preparations for phenolic acid esterases, involves incorporating ethyl cinnamate into an agar medium. After inoculation and incubation, the plate is flooded with a pH-sensitive dye to reveal yellow zones around positive cultures against a blue background. A number of yeasts (Rhodotorula spp. and Candida spp.) and fungi (Penicillium sp. and Aspergillus sp.) gave positive results, while a number of commercial enzymes, particularly pectinases, also exhibited good phenolic acid esterase.

Hydrolysis of vicine and convicine from fababeans by microbial beta-glucosidase enzymes.

The toxic glycosides vicine and convicine which are present in fababeans have been implicated in favism, an anaemic disease of humans. Vicine and convicine concentrations are reduced by growth of Lactobacillus plantarum on fababean suspensions. The glycosides are eliminated from the fababean substrate by the growth of the filamentous fungus Fusarium graminearum. Incubation of fababean suspension with concentrated culture filtrate of Aspergillus oryzae, induced for extracellular beta-glucosidase production, results in complete degradation of the glycosides. This study suggests a potential use of micro-organisms or microbial enzymes for detoxification of fababeans.

Growth of fermentative and non-fermentative yeasts in natural yoghurt, stored in polystyrene cartons.

Permeation of oxygen through polystyrene packaging is a factor in the growth of yeasts in natural yoghurt. Diffusion of oxygen through the packaging material can permit the growth of non-fermentative yeasts in yoghurt stored at refrigeration temperatures. Yarrowia lipolytica, a non-fermentative yeasts which does not utilize lactose was isolated from yoghurt. The growth in natural yoghurt of Yarrowia lipolytica and the lactose-fermenting yeast Kluyveromyces marxianus was investigated. Both yeasts grew in yoghurt with reduced fat content. Storage of yoghurt in an anaerobic atmosphere eliminated growth of Yarrowia lipolytica but permitted fermentative growth of Kluyveromyces marxianus.

Hemodynamics versus biopsy findings during cardiac transplant rejection.

Before the use of cyclosporine as the major component for immunosuppression after cardiac transplantation, rejection was accompanied by catastrophic hemodynamic decompensation. However, the hemodynamic changes that occur during rejection after cardiac transplantation in patients treated with cyclosporine have not been clearly described. Between July 1986 and October 1989, 89 adults underwent orthotopic heart transplantation at the University of Michigan Medical Center. All patients received triple-drug therapy immunosuppression consisting of steroids, cyclosporine, and azathioprine. Cardiac hemodynamics were measured and correlated with the histologic assessment of rejection. There have been ten deaths among these 89 patients for an overall survival of 89%. There were no deaths from rejection. One hundred fifty-three of the biopsy specimens were read as grade 0, 31 were grade 1, 75 were grade 2, 103 were grade 3, and 9 patients had grade 4 biopsy specimens. No hemodynamic differences were noted in patients with increasing grade of rejection. Five patients (5/9, 55%) with severe rejection (grade 4) had symptoms of congestive heart failure at the time of biopsy. These symptomatic grade 4 patients differed from asymptomatic grade 4 patients only in cardiac output (2.9 versus 5.2 L/min). Overall hemodynamic decompensation was not evident as rejection grade increased. Routine serial endomyocardial biopsies remain the procedure of choice in the diagnosis of rejection in the asymptomatic patient after cardiac transplantation as hemodynamics do not predict degree of rejection.

Isolation of a polymorphic DNA segment unique to human chromosome 7 by molecular cloning of hybrid cell DNA.

DNA isolated from a rodent-human hybrid cell line containing human chromosomes 3, 7, 9, 10, 14 and 22 was cloned in the plasmid vector pAT153. Recombinant plasmids containing inserts of human origin were identified by colony hybridization to 32P-labelled human DNA under conditions in which only repetitive sequences interact. Single- and low-copy sequences were liberated from these plasmids by restriction endonuclease digestion and used as hybridization probes against human DNA and DNA isolated from a panel of Chinese hamster-human hybrids. One single-copy probe was shown to react with a genomic sequence unique to human chromosome 7 and to recognize an apparent restriction fragment size polymorphism in human DNA.

Oxygen-pulse curves in rat liver mitochondrial suspensions. Some observations and deductions.

1. The inference, implicit in the chemiosmotic hypothesis, that protons move into the bulk phase during ATP synthesis was investigated. 2. Incubation of rat liver mitochondria in the presence of the cation exchanger CM-Sephadex C-50 caused alkalinization in the medium, though total ATP synthesis remained unchanged. The addition of N-ethylmaleimide prevented the alkalinization, but there was still no indication of protons passing into the medium. The expected proton movement [Mitchell & Moyle (1967) Biochem. J. 105, 1147--1162] was readily detected when as an equivalent acid pulse. 3. Analysis of delta H+ decay curves after O2 pulses (3 micrograms-atoms of O/g of protein) indicated the presence of fast and slow components of decay, with first-order rate constants (k) of 0.24s-1 and 0.032s-1. The fast decay was finite and was eliminated in the presence of N-ethylmaleimide. 4. These observations are interpreted as evidence for the development of unmasking of fixed charges on the outer surface of the mitochondrial inner membrane during energization and for the existence of proton-retentive electrical fields (rho-zones) on this surface. The charge concentration is calculated as about 1 charge/10nm2. 5. A cycle of changes in a single fixed-charge molecule is proposed which mediates both Ca2+ uptake and the first step in the utilization of the rho-zone protonmotive force, delta p rho.