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Mechanistic aspects of the mode of action of the soluble monooxygenase system of Methylosinus trichosporium OB3b have been investigated. The hydroxylation of 4-deuteroanisole in the 4-position was shown to proceed via an NIH shift with a deuterium retention of 66 %, indicative of an epoxide intermediate. Isotopic studies using ethylbenzene and ethylbertzene-d10 showed an isotopic effect, kH/kD = 4.5 for benzylic hydroxylation, and an inverse isotopic effect, kH/kD = 0.75 for arene hydroxylation. It is suggested that a stepwise mechanism (hydrogen abstraction and hydroxylation) could be involed.

Preliminary investigations on the detection of nucleic acid hybridisation by direct electrochemical techniques are described. Initial experiments were performed with calf thymus DNA, while as a model system, plasmid DNA (pEMBL) which can be prepared in single stranded forms from each of two original double stranded plasmids (+19 and -19) was used. Electrochemical analyses have been performed using mercury pool, glassy carbon and screen printed carbon working electrodes.

A bacterium that was able to utilize Emkarate 1550 (E1550), a synthetic lubricant ester, as the sole source of carbon was isolated. The isolate was tentatively identified as Micrococcus roseus. The components of the E1550 ester, octanoate, decanoate, and 1,1,1-tris(hydroxymethyl)propane (TMP), were detected in the culture medium of cells growing on the ester. The TMP tertiary alcohol accumulated during growth and was not utilized by this isolate. The detection of the components of the ester in the supernatant of cultures indicated that one of the first steps in its degradation was cleavage of the ester bonds. Esterase activity was significantly enhanced in cells grown on E1550 compared with esterase activity measured in cells grown on acetate.

Bioassays, including immunoassays, enzyme assays, and assays using enzyme electrodes, and nucleic acid hybridization probes have been the subject of considerable industrial and academic research. New bioassay methods have applications in the medical, chemical, pharmaceutical, and food products industries. Recent US patents and scientific literature on a variety of new bioassay methods are surveyed. A description of these patents and a list of references are given.

Recent results, showing that the ubiquitous methane-utilizing bacteria (methanotrophs) can partially oxidize and, in some cases, extensively metabolize complex organic compounds, call for a reappraisal of their role in the cycling of elements in the biosphere. Possible environmental implications and opportunities for industrial exploitation are discussed.

The distribution of methane mono-oxygenase (MMO) activities between particulate and soluble fractions of cell-free extracts of Methylosinus trichosporium OB3b was dependent upon growth conditions. Particulate activity was associated with the presence of intracytoplasmic membranes observed only under oxygen-limiting conditions in shake flask cultures. Particulate and soluble activities showed substantially different sensitivities to a range of potential inhibitors. The particulate enzyme was inhibited by metal-chelating agents, thiol reagents and amytal, whereas the soluble MMO was not inhibited by these compounds; both activities were sensitive to KCN, ethyne and 8-hydroxyquinoline. NAD(P)H was the only suitable electron donor. The activities were unstable at O C but the soluble enzyme could be partially stabilized by several compounds. The particulate and soluble MMO activities are compared with previously reported particulate and soluble MMO enzymes from this species and other methane oxidizers.

The continuous culture growth conditions for induction of intracytoplasmic membranes in Methylosinus trichosporium OB3b are described. During oxygen-limited, nitrate-excess chemostat culture, organisms have an extensive intracytoplasmic membrane system and particulate, cell-free methane mono-oxygenase (MMO). Under methane limitation fewer intracytoplasmic membranes are seen, while under all other conditions tested, membranes are absent and cell-free MMO is entirely soluble. These findings may be important in relation to the development of oxidative biotransformation processes using this bacterium.

Relief of copper deficiency during growth promotes the proliferation of intracytoplasmic membranes together with synthesis of particulate methane mono-oxgenase (MMC) inMethylosinus trichosporium OB3b. Particulate MMO functionsin vitro (in cell-free extracts and membrane preparations) with either succinate or NADH as electron donor but soluble MMO functions only with NADH.

Orcinol hydroxylase (EC 1.14.13.6), which catalyzes the first reaction of orcinol catabolism in Pseudomonas putida 01, has been purified to homogeneity, and crystallized. Orcinol hydroxylase catalyzes the hydroxylation of orcinol with equimolar consumption of O2 and NADH (or NADPH) to 2, 3, 5-trihydroxytoluene, which is nonenzymically oxidized to a quinone. The visible absorption spectrum of the enzyme shows maxima at 373 and 454 nm and a shoulder at 480 nm. FAD can be dissociated from the protein. Reconstitution of enzymic activity was achieved with FAD, and to a limited extent by FMN. The enzyme has a molecular weight of 63,000 to 68,000 and contains 1 mol of FAD per mol of protein. K-m values for the three substrates orcinol, NADH, and O2 are 0.03, 0.13, and 0.07mM, RESPECTIVELY. The molecular activity of the crystalline enzyme is 1560 min minus 1. In the absence of orcinol, NADH is only slowly oxidized with formation of H2O2. Several analogs of orcinol also serve as substrates for hydroxylation, namely resorcinol, 4-methylresorcinol, and 4-bromoresorcinol. Other analogs, m-cresol, m-ethylphenol, 4-ethylresorcinol, and phloroglucinol, mimic orcinol as effectors, in that they (a) accelerate electron flow from NADH to the flavin and (b) decrease the apparent K-m for NADH but not to the same extent as the substrates that are hydroxylated. The latter compounds are not hydroxylated. Instead H2O2 accumulates as the only product of O2 reduction. The enzyme therefore behaves either as a hydroxylase or an oxidase. The ratio of hydroxylase to oxidase activities of the enzyme is decreased by an increase in the temperature of incubation; at 60 degrees the reaction with orcinol is almost 50% uncoupled from hydroxylation. The apparent K-m values for the effectors are in good agreement with the D-D values obtained for orcinol, resorcinol, and m-cresol. K-D values were obtained by measurement of the effector-induced perturbations of the visible absorption spectrum of the flavoprotein by difference absorption spectroscopy. The circular dichroism spectrum of orcinol hydroxylase is also altered in the presence of orcinol. The participation of the flavin in the over-all reaction is demonstrated by its rapid reduction under anaerobic conditions by NADH in the presence or orcinol, resorcinol, or m-cresol. Subsequent introduction of oxygen restores the oxidized form and yields H2O2 when m-cresol is the effector, but not when orcinol is the effector. Transfer of reducing equivalents from the reduced flavoprotein to free FAD may also occur. Reduction of orcinol hydroxylase by NADH in the absence of an effector is 10-4-fold slower than in the presence of an effector. The minimal structural requirements for effectors appear to be a 1,3-dihydroxy or 1-alkyl-3-hydorxybenzene, but only the former are substrates for hydroxylation.

Cell-free extracts of Methylosinus trichosporium 0B3b (MT 0B3b) containing the soluble, broad specificity methane mono-oxygenase (MMO) have been shown to catalyse yet another type of reaction : O-dealkylation. Several 4-substituted anisoles were investigated as substrates, all showed O-demethylation to varying extents by cell-free extracts of the bacterium. This catalytic ability is common to organisms grown on either methane or methanol as sole carbon source, although the rates of biotransformation are lower for the latter. O-demethylation of anisole itself was inhibited (> 99%) by ethyne, a known MMO inhibitor, strongly indicating that the MMO is the enzyme responsible for this catalysis.

A new experimental procedure and robust equipment have been developed for studying oil degradation at sea and the system has been proved under harsh environmental conditions. The degradation of three weathered crude oils, differing substantially in composition, has been examined under both winter and late spring conditions. The oils were an Athabasca synthetic crude, its parent sand tar and a North Sea Forties crude. Composition had a major effect on the extent and rate of loss of oil from the system, the lighter oils disappearing more rapidly. After 40 d at sea at a mean temperature of 5 °C, only 16% of the synthetic crude remained compared with 47% and 85% of the Forties crude and sand tar respectively. At a mean temperature of 12 °C, average degradation rates were about three times those at 5 °C. At both temperatures, a mixed bacterial population involved in the degradation process was isolated from each oil. For the Forties crude and synthetic crude, the saturate fraction was degraded most rapidly, whilst in the sand tar, this fraction was not degraded as rapidly as the monoaromatic fraction.

SUMMARY: A micro-organism which grows on cyclohexane as sole carbon and energy source has been isolated from estuarine mud flats. It has been tentatively identified as a Nocardia. The organism, which is auxotrophic for biotin, grows on cyclohexane (supplied as a vapour) with a mean generation time of about 10 h and a Y SUB of 59·9 g dry wt (mol cyclohexane)−1. Growth on cyclohexane leads to the production of intracytoplasmic membrane structures which are not present after growth on succinate. Growth, respiration and enzyme studies are consistent with the degradation of cyclohexane via cyclohexanol, cyclohexanone, caprolactone and 6-hydroxycaproate. Extracts of organisms grown on cyclohexane contained cyclohexanol dehydrogenase, cyclohexanone monooxygenase and caprolactone hydrolase; these enzymes are absent from, or at very low activity in, extracts of organisms grown on succinate.

There is now considerable evidence that benzo[a]pyrene hydroxylase (aryl hydrocarbon hydroxylase: AHH) is involved in the metabolism of petroleum hydrocarbons by several species of marine fish (see review by Varanasi & Malins, 1977), as well as by certain species of marine invertebrates, including crabs and lobsters (Payne, 1977). Several workers (Payne & Penrose, 1975; Payne, 1976, 1977; Bend, James & Dansette, 1977) have found increased activities of this enzyme in fish previously exposed to petroleum hydrocarbons and related compounds: similar findings have been made in a study using two species of polychaete (Lee, Furlong & Singer, 1977 a). No evidence of such enhanced activity, however, has been found in experiments with crabs and lobsters (Payne, 1977).

The obligate methylotroph Methylosinus trichosporium OB3b was capable of growth on methanol as sole carbon source at concentrations as high as 4% (v/v), and viability was maintained over many successive transfers. Methane mono-oxygenase, detected by epoxidizing and hydroxylating activity, was retained. The gross morphology of the organism on this substrate was dependent on culture conditions. It varied from organisms containing extensive peripheral membrane systems to those with extensive inclusions, the latter representing a poorly developed membrane system which predominated under most growth conditions.

SUMMARY: In vivo 13C NMR has been used to observe metabolism of exogenously supplied methanol by suspensions of Methylosinus trichosporium OB3b grown under a variety of conditions. Formaldehyde, formate and bicarbonate ions were the only metabolites of methanol to be detected. Accumulation of formaldehyde was observed only with suspensions grown under conditions which yield particulate, membrane-bound, methane mono-oxygenase (MMO). Ethyne abolished MMO activity, partially inhibited methanol oxidation in whole organisms, and prevented growth of the organism on methanol (1%, v/v) in batch culture. Oxidation of ethanol, a substrate of methanol dehydrogenase, was not affected by ethyne. Ethyne caused accumulation of formaldehyde in all suspensions of the organism incubated with methanol, although oxidation of exogenously added formaldehyde was not affected. These observations are consistent with the proposal that in M. trichosporium OB3b both MMO and methanol dehydrogenase oxidize exogenously supplied methanol and suggest that the further oxidation of formaldehyde is stimulated by the consumption of reducing equivalents by MMO.

SUMMARY: Pseudomonas sp. 8/IX isolated from soil and Pseudomonas sp. NCIB 8858 are capable of growth on DL-I-aminopropan-2-ol or aminoacetone as sole sources of carbon, nitrogen and energy. During growth on the amino alcohol, small amounts of aminoacetone initially accumulate in the medium but disappear during the early logarithmic phase. Aminoacetone accumulation is favoured by high pH and high growth-substrate concentrations. Washed suspensions of the pseudomonads grown on DL-I-aminopropan-2-ol accumulate aminoacetone when incubated with the amino alcohol in the presence of metabolic inhibitors, and rapidly utilize aminoacetone in their absence. Aminoacetone formation is optimal at about pH 10 in the presence or absence of the most effective inhibitor iodoacetate. Aminoacetone utilization occurs optimally at pH 7.7, the apparent K m for the amino ketone being about 0.1 mM. At an initial concn. of 1.0 mM, the rate of utilization is proportional to cell density up to at least 0.5 mg. dry wt organisms/ml. and is constant with time over almost the entire course of the reaction. The maximum rate of utilization is approximately 150 mμmoles/mg. dry wt organisms/min. at 30°. The phenyl analogue 2′-aminoacetophenone is utilized at a comparable rate but 5-aminolaevulate is virtually unaffected. Anaerobic conditions and metabolic inhibitors prevent amino ketone utilization. Growth conditions markedly affect the ability of suspensions both to accumulate and utilize aminoacetone. Of the growth substrates tested, only L-threonine enabled Pseudomonas sp. 8/IX to utilize aminoacetone, the rate being approx. 60 % of that found after growth on DL-I-aminopropan-2-ol The patterns of oxidation of possible intermediates of DL-I-aminopropan-2-ol catabolism are similar using washed organisms grown on either the amino alcohol or aminoacetone. Organisms grown on acetate plus glycine oxidize such compounds at relatively low rates. Methylglyoxal is not a substrate for either growth or oxidation. Comparison of the rates of oxygen uptake and aminoacetone utilization, and measurement of the total oxygen consumed, indicate about 70% complete oxidation of the amino ketone by organisms grown on the amino alcohol. Similar maximum crop-sizes are obtained in media in which ammonium sulphate or different relative amounts of D- and L-I-aminopropan-2-ol serve as the sole sources of growth-limiting nitrogen.

1. Experimental conditions have been found in which small amounts of methanol (approximately 2.5mm) accumulated when washed cell suspensions of methane-grown Pseudomonas methanica and Methanomonas methanooxidans were incubated with methane+oxygen mixtures in Warburg flasks. 2. The methanol formed could be separated completely from water by fractional distillation through glass helices followed by gas chromatography using 20% polyethylene glycol 400 on a Celite 545 support. 3. By using 18O-enriched oxygen gas the abundance of 18O in the methanol formed from oxidation of methane was measured with a Perkin–Elmer 270 combined gas chromatograph/mass spectrometer. The results showed that the oxygen in methanol was derived exclusively from gaseous oxygen in both micro-organisms. 4. Control experiments using [18O]water in incubation mixtures confirmed that there was negligible incorporation of the oxygen atom from water into methanol.

SUMMARY: Two pseudomonad strains grown on DL-I-aminopropan-2-o1 as sole source of carbon + nitrogen provided rich sources of L(+)-I-aminopropan-2-o1: NAD+ oxidoreductase. The activity of this enzyme in crude extracts of Pseudomonas sp. strain NCIB 8858 was about 400 mμmoles aminoacetone formed/mg. protein/min., under optimum conditions. Growth on media containing other carbon sources commonly repressed enzyme formation. Enzyme formation was induced by incubating succinate-grown bacteria in media containing DL-I-aminopropan-2-ol, aminoacetone or L-threonine; but induction was inhibited by DL-2-hydroxy-2-phenylethylamine or DL-2-phenylserine. The enzyme was purified twenty-fold by treatment with protamine sulphate and ammonium sulphate followed by gel-filtration. The molecular weight of the enzyme was estimated to be 70 to 80 thousand. The partly purified enzyme was optimally active at pH 9.5. The Km values for DL-I-aminopropan-2-ol and NAD+ were about 0.1 and 0.3 mM, respectively. Activity with NADP+ as the coenzyme was about the same as that with NAD+, the Km being about 0.2 mM. The enzyme was inactive with α-NAD+. Activity was unaffected by a variety of thiols, thiol-alkylating and metal-chelating agents. The enzyme was virtually inactive with twenty-one potential substrates but oxidized DL-I-aminobutan-2-ol, DL-2-hydroxy-2-phenylethylamine and DL-phenylserine at significant rates and DL-I,3-diaminopropan-2-ol and DL-3-hydroxybutyrate slowly. Enzyme activity towards I-amino-propan-2-ol was stereospecific for the L(+)-isomer, which was oxidized about six times more rapidly than the racemic substrate. The D(-)-enantiomorph was a competitive inhibitor of the reaction, Ki = about 0.3 mM, and no dehydrogenase activity towards this isomer was observed with either purified enzyme preparations or crude cell-free extracts. Aminoacetone-dependent oxidation of NADH occurred optimally at pH 5 in acetate buffer; a second peak of activity was found at pH 8.4 in diethanolamine + HCl buffers. Several buffers appeared to inhibit activity at pH 7.5. 2′-Aminoacetophenone was active as a substrate, but 5-aminolaevulate had little activity. NADPH was also active as coenzyme.

Bacteria, isolated from lakewater and capable of growing at the expense of commercial synthetic 2-stroke lubricant oils released 2-ethylhexanol from this substrate by means of esterases. The alcohol accumulated to approximatedly 6mM in these incubations but was itself mineralized by a strain of Pseudomonas putida isolated from the same lakewater by elective culture. The initial steps in the degradative pathway of 2-ethylhexanol were catalysed by alcohol and aldehyde dehydrogenases. The resultant 2-ethylhexanoic acid was further metabolised via β-oxidation. The accumulation of intermediates in culture fluids facilitated by acrylate addition and higher isocitrate lyase activities in cell extracts, suggested that 2-ethylhexanoic acid was cleaved to two butyrate moieties which were then further metabolised to acetate. The pathway shows the organism has an interesting β-oxidation system capable of utilizing branched substrates.

The current trends and future aspects of the research and development of immunosensors are overviewed. A non-labelled immunosensor, whose selectivity depends on immunochemical affinity of an antigen for its corresponding antibody, has been developed as the basis for the potentiometric determination of an antigen, with an antibody-bound membrane or electrode. Non-labelled immunosensors for syphilis antibody, blood typing, human chorionic gonadotropin (HCG), and human serum albumin have been investigated. In contrast with non-labelled immunosensors, labelled immunosensors may be characterized by marked enhancement of sensitivity. Of these labelled immunosensors, enzyme immunosensors that use the chemical amplification of a labelling enzyme for sensitivity are promising. Enzyme immunosensors with an oxygen electrode have been developed to determine AFP, HCG, IgG and toxin. Bioaffinity sensors with a preformed metastable ligand-receptor complex, which are similar to the enzyme immunosensor have been found effective for the determination of thyroxine (T4), biotin, and insulin.

A bioelectrochemical cell is described in which bacterial methanol dehydrogenase is used to effect the efficient transduction of chemical into electrical energy. The cell can also be used as a sensitive detector for a variety of primary alcohols in the range, 10-7 – 5×10-11 moles.

An oil-degrading bacterium identified as Acinetobacter lwoffi was isolated by elective culture on North Sea Forties crude oil from an activated sludge sample. It grew on a wide range of n-alkanes (C12−C28) and 1-phenylalkanes, including 1-phenyldodecane, 1-phenyltridecane and 1-phenyltetradecane. The organism degraded 1-phenyldodecane to phenylacetic acid which was further metabolized via homogentisic acid, whilst 1-phenyltridecane was transformed to trans-cinnamic and 3-phenylpropionic acid which were not further metabolized. Evidence dence is presented for a relationship between aromatic amino acid catabolism and 1-phenyldodecane degradation in this organism.

A biosensor is an analytical device that responds to an analyte in an appropriate sample and interprets its concentration as an electrical signal via a suitable combination of a biological recognition system and an electrochemical transducer. As a result of recent scientific and technological progress, such devices are likely to play an increasingly important role in generating analytical information in all sectors of human endeavour, from medicine to the military. In particular, biosensors will form the basis of cheap, simple devices for acquiring chemical information, bringing sophisticated analytical capabilities to the non-specialist and general public alike. The market opportunities for the rapid exploitation of novel developments in this sector are substantial. Biosensor research is also likely to have a significant impact on the development of modern electronics.

Nocardia salmonicolor, grown on acetate, commercial D,L-lactate or hydrocarbon substrates, has high isocitrate lyase activities compared with those resulting from growth on other carbon sources. This presumably reflects the anaplerotic role of the glyoxylate cycle during growth on the former substrates. Amongst a variety of compounds tested, including glucose, pyruvate and tricarboxylic acid cycle intermediates, only succinate and fumarate prevented an increase in enzyme activity in the presence of acetate. When acetate (equimolar to the initial sugar concentration) was added to cultures growing on glucose, there followed de novo synthesis of isocitrated lyase and isocitrate dehydrogenase, with increases in growth rate and glucose utilization, and both acetate and glucose were metabolized simultaneously. A minute amount of acetate (40 muM) caused isocitrate lyase synthesis (a three-fold increase in activity within 3 min of addition) when added to glucose-limited continuous cultures, but even large amounts added to nitrogen-limited batch cultures were ineffective. Malonate, at a concentration that was not totally growth-inhibitory (1mM) prevented the inhibition of acetate-stimulated isocitrate lyase synthesis by succinate, but fumarate still inhibited in the presence of malonate. Phosphoenolpyruvate is a non-competitive inhibitor of the enzyme (apparent Ki 1-7 mM). The results are consistent with the induction of isocitrate or a closely related metabolite, and catabolite repression by a C-4 acid of the tricarboxylic acid cycle, possibly fumarate.

It has been recognized for many years that obligate methanotrophs, although requiring C1 compounds for growth, are nevertheless capable of effecting the partial oxidation of several simple methane analogues (short chain alkanes and alkenes) to products which accumulate extracellularly (64). Indeed, the work of Foster and his colleagues (29,30) represents one of the early classical examples of cooxidation. It has generally been assumed that these oxidations are a result of the lack of specificity of enzymes involved in the oxidation of methane to carbon dioxide. The findings of Dalton and his colleagues (7,49) that partially purified methane monooxygenase (MMO) preparations oxygenate a wide range of organic compounds, supported this hypothesis. The extraordinary wide range of oxidations catalyzed by this enzyme in some species is, however, surprising; in addition to alkanes and alkenes, aromatic, alicyclic and heterocyclic compounds are oxidized.

ALTHOUGH aminoacetone was first recognized as a microbial metabolite in 1958 (ref. 1), and found to be formed from L-threonine1–5, glycine plus a source of acetyl-CoA1,2,4,5, or isopropanolamine5,6 (1-aminopropan-2-ol) by a variety of micro-organisms, it has only recently been found that the amino ketone is further metabolized by resting cell suspensions of some bacteria7. The rapid utilization of aminoacetone by extracts free from cells has now been demonstrated and information of the enzyme mechanism of aminoacetone metabolism is available.

Studies with purified orcinol hydroxylase suggest that, contrary to previous conclusions, the enzymes of the orcinol pathway cannot transform analogous compounds to common metabolites. The substrate analogues of orcinol uncouple electron flow from reduced nicotinamide adenine dinucleotide to oxygen from the hydroxylation reaction catalyzed by orcinol hydroxylase.

The metabolic production of L-ornithine by an arg mutant of Acinetobacter lwoffi using n-hexadecane as sole carbon source was studied. Time course experiments under optimised conditions showed that L-ornithine production was growth-related, with maximum concentrations (10.5 gl−1) accumulating in the late exponential phase of growth.

Redox proteins catalyse1 the reactions of a wide variety of otherwise intractable substrates, such as dinitrogen, alkalies, arenes, terpenes and steroids. Two major factors impede the utilization of these enzymes—the inefficient electron transfer between the enzyme and electrode, and the properties often, but not inevitably, associated with enzymes, such as instability, complexity, and expense. We have now shown that the former can be overcome and that proteins can be coupled, via electrodes, to a number of energy sources; the latter is2 the subject of much effort elsewhere. We demonstrated previously3–6 that certain redox proteins can be reduced very efficiently electrochemically (Fig. 1a). Light and hydrogen are the two other convenient energy sources that could be used for such reductions, and we now report the reduction of cytochrome c by these means.