Microbial transformations in a cyclodextrin medium. Part 3. Cholesterol oxidation by Rhodococcus erythropolis

An intensive and systematic investigation of the oxidation of cholesterol (CL) to cholest-4-en-3-one (CN) by Rhodococcus erythropolis was undertaken in the presence of natural and chemically modified cyclodextrins (CDs) in a stirred bioreactor. The biotransformation was found to be strongly affected by the mode of addition of the natural CDs. While simultaneous addition of CL with either - or -CD led to a limited enhancement effect, the microbial oxidation of - and -CD complexes of CL was totally inhibited. In contrast, the alkylated CDs- dimethyl-, trimethyl- and hydroxypropyl--CD exhibited a remarkable enhancement of the microbial oxidation, irrespective of their mode of addition. The performance of the alkylated CDs was interpreted in the light of the measured phase solubility diagrams of CL and CN. It was thus shown that unlike the low solubilising power of hydroxypropyl--CD, dimethyl- and trimethyl--CD at 90 mm each, dissolved 9.3 and 8.7 g/l of CL and CN, respectively. Further investigation focused on the formation of CD complexes with CL and CN, analysed by X-ray powder diffractometry, differential scanning calorimetry and ¹H-nuclear magnetic resonance. It was thus shown that -CD forms a 2:1 CD:CL and CD:CN water-insoluble complexes. A mechanism of the biotransformation in homogeneous and heterogeneous CD media was presented while suggesting a direct interaction of the CD-substrate complex with microbial cells. Correspondence to: R. Bar     

Glyoxylate decarboxylation during glycollate oxidation by pea leaf extracts: significance of glyoxylate and extract concentrations

Hydrogen peroxide-dependent glyoxylate decarboxylation occurring during glycollate oxidation by pea leaf extracts (Pisum sativum L.) has been studied in relation to the effects of glyoxylate and extract concentration. With a saturating concentration of glycollate, decarboxylation was greatly stimulated by raising the glyoxylate concentration; at 30°C and with approx. 0.04 nkat of glycollate oxidase (as leaf extract) in the reaction mixture, CO₂ release in the presence of 5 mM glycollate and 5 mM glyoxylate was equal to about 45% of glycollate oxidation. However, CO₂ release at these substrate concentrations was not linearly proportional to the amount of extract supplied and was equal to a diminishing proportion of glycollate oxidation as the amount of extract was increased. This was shown to be due to the low affinity of catalase for H₂O₂, so that the endogenous catalase was able to destroy a larger proportion of the H₂O₂ generated at higher extract concentrations. It is argued that although at high glycoxylate concentrations (5–10 mM) in vitro, glyoxylate decarboxylation can be made to equal more than a third of the glycollate oxidised, less than 10% of the glyoxylate generated in vivo is likely to be decarboxylated in peroxisomes where high concentrations of glycollate oxidase and catalase are localised and where high concentrations of glyoxylate are unlikely to be maintained.Abbreviation PHMS pyrid-2-yl--hydroxymethane sulphonic acid     

The effect of 2-chloro, 6-(trichloromethyl) pyridine on the chemoautotrophic metabolism of nitrifying bacteria

Studies were conducted to elucidate the mechanism of action of 2-chloro-6-(trichloromethyl)pyridine or Technical N-SERVE on the nitrification process brought about byNitrosomonas europaea. The growth ofNitrosomonas was completely inhibited in the presence of 0.2 ppm N-SERVE while 1.0 ppm of the chemical was effective in the complete inhibition of ammonia oxidation by fresh cell suspensions. Cells stored at 4 C for a period of three days required somewhat higher concentrations (1.5 ppm) of N-SERVE for the complete inhibition of their ammonia oxidizing ability while the cytochrome oxidase of these cells was inhibited to the extent of 65 to 70 percent in the presence of a corresponding amount of N-SERVE. A 45 – 70 percent reversal of the inhibition of ammonia oxidation caused by N-SERVE was obtained by the addition of 6×10^–4 M Cu⁺⁺. An equivalent concentration of Cu⁺⁺ was also effective for the complete reversal of the inhibition of cytochrome oxidase present in whole cells.Hydroxylamine oxidation by intactNitrosomonas cells was not affected by levels of N-SERVE ranging from 1 – 3 ppm. The cytochrome oxidase effective in hydroxylamine oxidation and present in cell-free extracts was not inhibited by even 100 ppm N-SERVE. Likewise, the hydroxylamine activating enzyme hydroxylamine cytochromec reductase was also not inhibited by such levels of the chemical. Raising the concentration to 170 ppm N-SERVE, however, caused a 90 percent inhibition of the enzyme.Although a 5×10^–6 M concentration of allylthiourea completely inhibited ammonia oxidation byNitrosomonas cells, concentrations up to 10^–3 M of this compound did not affect the cytochrome oxidase activity of whole cells or cell-free extracts. The inhibition of ammonia oxidation caused by 5×10^–6 M allythiourea, unlike the inhibition by N-SERVE, could not be reversed by the addition of 6×10^–4 M Cu⁺⁺.Evidence is presented that the action of N-SERVE is on that component of cytochrome oxidase which is involved in ammonia oxidation.     

Morphological quantification of pellets in Streptomyces hygroscopicus var. geldanus fermentation broths using a flatbed scanner

An image analysis technique has been developed to allow high throughput morphological characterisation of microbial fermentation broths containing spherical pellets greater than 100 m in diameter. Images of stained Streptomyces hygroscopicus var. geldanus culture samples at three different inoculum levels were captured using a flatbed scanner, at a resolution of 21 m per pixel (1200 dots per inch) and subsequently analysed leading to the generation of a morphological profile of each sample. The time taken for image capture and analysis of a prepared sample, containing approx. 2000 particles, was 3 min 6 s.     

Transformation of Nε-CBZ-l-lysine to CBZ-l-oxylysine using l-amino acid oxidase from Providencia alcalifaciens and l-2-hydroxy-isocaproate dehydrogenase from Lactobacillus confusus

Summary Biotransformations were developed to oxidize N-carbobenzoxy(CBZ)-l-lysine and to reduce the product keto acid to l-CBZ-oxylysine. Lysyl oxidase (l-lysine: O₂ oxidoreductase, EC 1.4.3.14) from Trichoderma viride was relatively specific for l-lysine and had very low activity with N-substituted derivatives. l-Amino acid oxidase (l-amino acid: O₂ oxidoreductase [deaminating], EC 1.4.3.2) from Crotalus adamanteus venom had low activity with l-lysine but high activity with N-formyl-, t-butyoxycarbonyl(BOC)-, acetyl-, trifluoroacetyl-, or CBZ-l-lysine. l-2-Hydroxyisocaproate dehydrogenase (EC 1.1.1.-) from Lactobacillus confusus catalyzed the reduction by NADH of the keto acids from N-acetyl-, trifluoroacetyl-, formyl- and CBZ-l-lysine but was inactive with the products from oxidation of l-lysine, l-lysine methyl ester, l-lysine ethyl ester or N-t-BOC-l-lysine. Providencia alcalifaciens (SC9036, ATCC 13159) was a good microbial substitute for the snake venom oxidase and also provided catalase (H₂O₂:H₂O₂ oxidoreductase EC 1.11.1.6). N-CBZ-l-Lysine was converted to CBZ-l-oxylysine in 95% yield with 98.5% optical purity by oxidation using P. alcalifaciens cells followed by reduction of the keto acid using l-2-hydroxyisocaproate dehydrogenase. NADH was regenerated using formate dehydrogenase (formate: NAD oxidoreductase, EC 1.2.1.2) from Candida boidinii. The Providencia oxidase was localized in the particulate fraction and catalase activity was predominantly in the soluble fraction of sonicated cells. The pH optima and kinetic constants were determined for the reactions. Correspondence to: R. L. Hanson     

Microbial activity and enzymic decomposition processes following peatland water table drawdown

Microbial activity and enzymic decomposition processes were followed during a field-based experimental lowering of the water table in a Welsh peatland. Respiration was not significantly affected by the treatment. However, the enzymes sulphatase, -glucosidase and phosphatase were stimulated by between 31 and 67% upon water table drawdown. A further enzyme, phenol oxidase, was not significantly affected. The observation of elevated enzyme activities without an associated increase in microbial respiratory activity suggests that drought conditions influence peatland mineralisation rates through a direct stimulation of existing enzymes, rather than through a generalised stimulation of microbial metabolism (with associated de-novo enzyme synthesis). Hydrochemical data suggest that the stimulation may have been caused by a reduction in the inhibitory action of iron and phenolics in the peat pore waters. Overall, the findings support the recent hypothesis that drier conditions associated with climate change could stimulate mineralisation within wetlands. ei]R Merckx     

Extracellular Glucose Oxidase of Penicillium funiculosum 46.1

A method for isolating extracellular glucose oxidase from the fungus Penicillium funiculosum 46.1 using ultrafiltration membranes was developed. Two samples of the enzyme with a specific activity of 914–956 IU were obtained. The enzyme exhibited a high catalytic activity at pH above 6.0. The effective rate constant of glucose oxidase inactivation at pH 2.6 and 16°C was 2.74 × 10^–6 s^–1. This constant decreased significantly as the pH of the medium increased (4.0–10.0). The temperature optimum for glucose oxidase–catalyzed -D-glucose oxidation was in the range 30–65°C. At temperatures below 30°C, the activation energy for -D-glucose oxidation was 6.42 kcal/mol; at higher temperatures, this parameter was equal to 0.61 kcal/mol. Kinetic parameters of glucose oxidase–catalyzed -D-glucose oxidation depended on the initial concentration of the enzyme in the solution. Glucose oxidase also catalyzed the oxidation of 2-deoxy-D-glucose, maltose, and galactose.     

Enzymatic Improvement of Food Flavor IV. Oxidation of Aldehydes in Soybean Extracts by Aldehyde Oxidase

Aldehyde oxidase (aldehyde: oxygen oxidoreductase, EC 1.2.3.1) was partially purified from bovine liver. The enzyme irreversibly oxidized various aldehydes to the corresponding acids by using dissolved oxygen as an electron acceptor. Although the Km value for n-hexanal was low (6 µm), that for acetaldehyde was high (20 mm).

Medium-chain aldehydes such as hexanal and pentanal appear to be mainly responsible for green beany odor of soybean products. A great reduction in the beany odor was observed after the soybean extract was incubated with aldehyde oxidase under aerobic conditions. Dissolved oxygen was utilized as the electron acceptor throughout the enzyme-catalyzed oxidation of aldehydes and none of other cofactors were found to be required.

It has been shown that bovine liver mitochondrial aldehyde dehydrogenase oxidizes the soybean protein-bound aldehyde with a rate comparable to that for free n-hexanal (Agric. Biol. Chem., 43, in press). Comparative studies of aldehyde oxidase and aldehyde dehydrogenase with respect to oxidation-rates of free aldehydes and the soybean protein-bound aldehydes indicated that aldehyde oxidase acted on the bound aldehyde with a much slower rate.     

Oxygen consumption by Campylobacter sputorum subspecies Bubulus with formate as substrate

The kinetics of oxygen utilization by the microaerophile Campylobacter sputorum subspecies bubulus was studied. With formate as substrate two enzyme systems were found to be responsible for electron transfer between formate and oxygen. In the case of lactate oxidation one enzyme system could account for the activity measured. One of the formateoxidizing systems possessed a high affinity for oxygen [K _m(O₂)=approx. 4M O₂]. From inhibitor studies it was concluded that a respiratory chain was involved in its activity. Respiration by this system must be responsible for proton translocation and electron transport-linked phosphorylation at formate oxidation. The other enzyme system had an extremely low affinity for oxygen [K _m (O₂)=approx. 1 mM O₂]. It was tentatively identified as the H₂O₂-producing formate oxidase previously found in C. sputorum. The H₂O₂ production by this enzyme is implicated in an explanation of the microaerophilic nature of C. sputorum. Sensitivity of formate dehydrogenase to H₂O₂ was demonstrated. The influence of the formate concentration on aerobic formate oxidation was determined. The pH- and temperature dependencies of oxygen uptake with formate as substrate were examined at airsaturation and at a low dissolved oxygen tension.Abbreviations TL medium tryptose-lactate medium - TF medium tryptose-formate medium - HQNO 2-n-heptyl-4-hydroxyquinoline N-oxide - SHAM salicylhydroxamic acid - DCPIP 2,6-dichlorophenolindophenol     

Characterization of a Phenol Oxidase from Cryptococcus neoformans var. neoformans

In Cryptococcus neoformans, enzymic oxidation of various catechols leads to melanin, a proposed virulence factor. A phenol oxidase enzyme of Cryptococcus neoformans var. neoformans produced at 25 C has been purified from an ultracentrifugal supernatant of an extract of broken cells. Hydrophobic interaction chromatography followed by anion-exchange column chromatography allowed purification of the phenol oxidase. The molecular weight of the enzyme estimated by gel filtration was about 80,000 and a dimeric species (Mw = 160,000) was suggested. The isoelectric point of the protein was approximately 4.1. An NH₂-terminal 31 amino acid sequence was determined using phenol oxidase electroblotted onto a PVDF membrane after nondenaturing gel electrophoresis. Upon searching the Peptide Institute (Osaka) data base, no proteins with high degrees of homology were found.     

Changes in neck muscles in Swedish reindeer bucks during rutting season

Summary Three neck muscles in Swedish reindeer bucks have been studied before and during the rutting season. These were M. splenius, M. sternocephalicus and M. brachiocephalicus. For comparison, M. longissimus dorsi was chosen.Fibre composition and fibre size were studied in the four muscles as also was the metabolic potential of three enzymes, representing respiratory chain (cytochrome oxidase), -oxidation of fatty acids (3-hydroxyacyl-CoA dehydrogenase) and anaerobic glycolysis (lactate dehydrogenase).The extreme increase in size of certain muscles in the neck in connection with the rutting season (e.g. sternocephalicus, which increases from 250 g to 1,500 g) was to a great extent due to an increase in fibre size. In splenius, all three fibre types studied increased (I, IIA, IIB); in brachiocephalicus, mainly IIA and IIB; and in sternocephalicus, only the IIB. No corresponding fibre increase could be found in longissimus dorsi. In splenius and sternocephalicus from bucks older than 54 months, 60–70% of the fibres were of type I, and in brachiocephalicus, only about 40%.In all muscles but one, oxidative capacity (cytochrome oxidase) and -oxidation of fatty acids (3-hydroxyacyl-CoA dehydrogenase) decreased significantly during the rutting season. This indicates that purposes other than the enhancement of energy production by fatty acid oxidation must account for the enlargement of the neck muscles.Abbreviations Cytox cytochrome oxidase - HAD 3-hydroxyacyl-CoA dehydrogenase - LDH lactate dehydrogenase     

Bioproduction of D-Psicose from Allitol with Enterobacter aerogenes IK7: A New Frontier in Rare Ketose Production

D-Psicose, a new alternative sweetener, was produced from allitol by microbial oxidation of the newly isolated strain Enterobacter aerogenes IK7. Cells grown in tryptic soy broth medium (TSB) supplemented with D-mannitol at 37 °C were found to have the best oxidation potential. The cells, owing to broad substrate specificity, oxidized various polyols (tetritol, pentitol, and hexitol) to corresponding rare ketoses. By a resting cell reaction, 10% of allitol was completely transformed to the product D-psicose, which thus becomes economically feasible for the mass production of D-psicose. Finally, the product was crystallized and confirmed to be D-psicose by analytical methods.     

Revealing interaction between sulfobutylether‐β‐cyclodextrin and reserpine by chemiluminescence and site‐directed molecular docking

The host–guest interaction between sulfobutylether‐β‐cyclodextrin (SBE‐β‐CD) and reserpine (RSP) is described using flow injection‐chemiluminescence (FI‐CL) and site‐directed molecular docking methods. It was found that RSP could inhibit the CL intensity produced by a luminol/SBE‐β‐CD system. The decrease in CL intensity was logarithmic over an RSP concentration range of 0.03 to 700.0 nM, giving a regression equation of ?I = 107.1lgC_RES + 186.1 with a detection limit of 10 pM (3σ). The CL assay was successfully applied in the determination of RSP in injection, saliva and urine samples with recoveries in the range 93.5–106.1%. Using the proposed CL model, the binding constant (K_CD‐R) and the stoichiometric ratio of SBE‐β‐CD/RSP were calculated to be 7.4 × 10⁶ M^‐1 and 1 : 1, respectively. Using molecular docking, it was confirmed that luminol binds to the small cavity of SBE‐β‐CD with a nonpolar interaction, while RSP targeted the larger cavity of SBE‐β‐CD and formed a 1 : 1 complex with hydrogen bonds. The proposed new CL method has the potential to become a powerful tool for revealing the host–guest interaction between CDs and drugs, as well as monitoring drugs with high sensitivity. Copyright © 2013 John Wiley & Sons, Ltd.     

A quantum model for controlled enzymic reactions

A quantum model for the general enzymic reaction,E+S ⇌ ES → P, is presented, starting with the assumptions that any chemical substanceS, which may be a substrate for a particularE _(S)-enzyme is a microphysical system and any enzymeE-molecule, capable of interacting with anS-substrate is a “measuring system” which will “measure” one or more of theS-observables. According to the above assumptions a stochastic model of the reaction is constructed and a computer simulation of the steady state performed. The results thus obtained predicted fluctuations in the enzymic reaction rate, function of the substrate “perturbation”. On an experimental basis it is demonstrated that the irradiation of an enzymic substrate with low energies results in the inducement of a dose-dependent oscillatory behavior in the corresponding enzymic reaction rate. In the reaction type, the oscillations thus induced in theE-activity by the corresponding substrates are out-of-phase, realizing a biochemical discriminating net. Likewise, in an reaction type, the oscillations induced by the irradiatedS-substrate in the activities of the respective enzyme, realize a biochemical switching net.     

Oxidation of Methanol and Formaldehyde by a System Containing Alcohol Oxidase and Catalase Purified from Candida sp. N-16

The oxidation of methanol and formaldehyde was investigated by using some combination systems of alcohol oxidase, catalase, which were purified from Candida N-16, and hydrogen peroxide. The activity of alcohol oxidase was irreversibly inhibited when the enzyme was incubated with 2.5 mm hydrogen peroxide for 15 min. However, the oxidation of methanol to formaldehyde by alcohol oxidase in the presence of catalase was extremely promoted by the addition of 30 mm hydrogen peroxide. Alcohol oxidase could oxidize not only methanol but also formaldehyde as follows: HCHO + 0₂ + H₂O→HCOOH + H₂O₂. The formaldehyde oxidizing activity was inhibited by hydrogen peroxide. The system containing alcohol oxidase and catalase appears to be the entity of the oxygen-dependent oxidation system of formaldehyde previously found in the cell-free extract of the yeast.     

S-Adenosyl-l-methionine: Columbamine-O-methyl transferase,a compartmentalized enzyme in protoberberine biosynthesis

Suspension cultures of Berberis wilsoniae var. subcaulialata and B. aggregata are a useful source for the detection, partial purification and characterization of a new enzyme which specifically transfers the methyl group from (S)-adenosyl-L-methionine to the 2-OH-position of columbamine, thus producing palmatine. The enzyme was enriched approx. 30-fold; it is situated in a vesicle with the density =1.14, has a pH-optimum of 8.9, a molecular weight of 52 kD and shows a high degree of substrate specificity.Abbreviations SAH S-adenosyl-L-homocysteine - SAM S-Adenosyl-L-methionine - STOX (S)-Tetrahydroprotoberberine oxidase     

Partial purification and some properties of biopterin synthase and dihydropterin oxidase from Drosophila melanogaster

An enzyme which has been named biopterin synthase has been discovered in Drosophila melanogaster. This enzyme, which has been purified 200-fold from extracts of Drosophila, catalyzes the conversion of sepiapterin to dihydrobiopterin, or oxidized sepiapterin to biopterin. The K _m values for the two substrates are 63 µm for sepiapterin and 10 µm for oxidized sepiapterin. NADPH is required in this enzymatic reaction. An analysis of enzyme activity during development in Drosophila indicates a correlation between enzyme activity and biopterin content at various development stages. Another enzyme, called dihydropterin oxidase, was also discovered and partially purified. This enzyme catalyzes the oxidation of dihydropterin compounds to the corresponding pterin compounds. For example, sepiapterin (a dihydropterin) is oxidized to oxidized sepiapterin in the presence of this enzyme. The only dihydropterin that has been tested that is not a substrate for this enzyme is dihydroneopterin triphosphate, the compound thought to be a precursor for all naturally occurring pterins and dihydropterins. Since the action of dihydropterin oxidase is reduced significantly when the concentration of oxygen is very low, it is likely that this enzyme uses molecular oxygen as the oxidizing agent during the oxidation of dihydropterins. Neither NAD⁺ or NADP⁺ is required. In the presence of the two enzymes dihydropterin oxidase and biopterin synthase, sepiapterin is converted to biopterin. However, in the presence of biopterin synthase alone, sepiapterin is converted to dihydrobiopterin.This work was supported by research grants from the National Institutes of Health (AMO3442) and the National Science Foundation (PCM75-19513 AO2).