Efficient Synthesis of Methyl 3-Acetoxypropionate by a Newly Identified Baeyer-Villiger Monooxygenase.

Baeyer-Villiger monooxygenases (BVMOs) are an emerging class of promising biocatalysts for the oxidation of ketones to prepare corresponding esters or lactones. Although many BVMOs have been reported, the development of highly efficient enzymes for use in industrial applications is desirable. In this work, we identified a BVMO from Rhodococcus pyridinivorans (BVMORp) with a high affinity toward aliphatic methyl ketones (Km < 3.0 µM). The enzyme was highly soluble and relatively stable, with a half-life of 23 h at 30°C and pH 7.5. The most effective substrate discovered so far is 2-hexanone (kcat = 2.1 s-1; Km = 1.5 µM). Furthermore, BVMORp exhibited excellent regioselectivity toward most aliphatic ketones, preferentially forming typical (i.e., normal) products. Using the newly identified BVMORp as the catalyst, a high concentration (26.0 g/liter; 200 mM) of methyl levulinate was completely converted to methyl 3-acetoxypropionate after 4 h, with a space-time yield of 5.4 g liter-1 h-1 Thus, BVMORp is a promising biocatalyst for the synthesis of 3-hydroxypropionate from readily available biobased levulinate to replace the conventional fermentation.IMPORTANCE BVMOs are emerging as a green alternative to traditional oxidants in the BV oxidation of ketones. Although many BVMOs are discovered and used in organic synthesis, few are really applied in industry, especially in the case of aliphatic ketones. Herein, a highly soluble and relatively stable monooxygenase from Rhodococcus pyridinivorans (BVMORp) was identified with high activity and excellent regioselectivity toward most aliphatic ketones. BVMORp possesses unusually high substrate loading during the catalysis of the oxidation of biobased methyl levulinate to 3-hydroxypropionic acid derivatives. This study indicates that the synthesis of 3-hydroxypropionate from readily available biobased levulinate by BVMORp-catalyzed oxidation holds great promise to replace traditional fermentation.

Simultaneous production of furfural, lignin and cellulose-rich residue from Eucalyptus urophylla × E. grandis by ChCl/1,2-propanediol/MIBK biphasic system pretreatment.

A facile biphasic system composed of choline chloride (ChCl)-based deep eutectic solvent (DES) and methyl isobutyl ketone (MIBK) was developed to realize the furfural production, lignin separation and preparation of fermentable glucose from Eucalyptus in one-pot. Results showed that the ChCl/1,2-propanediol/MIBK system owned the best property to convert hemicelluloses into furfural. Under the optimal conditions (MRChCl:1,2-propanediol = 1:2, raw materials:DES:MIBK ratio = 1:4:8 g/g/mL, 0.075 mol/L AlCl3·6H2O, 140 °C, and 90 min), the furfural yield and glucose yield reached 65.0 and 92.2 %, respectively. Meanwhile, the lignin with low molecular weight (1250-1930 g/mol), low polydispersity (DM = 1.25-1.53) and high purity (only 0.08-2.59 % carbohydrate content) was regenerated from the biphasic system. With the increase of pretreatment temperature, the ß-O-4, ß-ß and ß-5 linkages in the regenerated lignin were gradually broken, and the content of phenolic hydroxyl groups increased, but the content of aliphatic hydroxyl groups decreased. This research provides a new strategy for the comprehensive utilization of lignocellulose in biorefinery process.

Recovery of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from Ralstonia eutropha cultures with non-halogenated solvents.

Reduced downstream costs, together with high purity recovery of polyhydroxyalkanoate (PHA), will accelerate the commercialization of high quality PHA-based products. In this work, a process was designed for effective recovery of the copolymer poly(hydroxybutyrate-co-hydroxyhexanoate) (P(HB-co-HHx)) containing high levels of HHx (>15 mol%) from Ralstonia eutropha biomass using non-halogenated solvents. Several non-halogenated solvents (methyl isobutyl ketone, methyl ethyl ketone, and butyl acetate and ethyl acetate) were found to effectively dissolve the polymer. Isoamyl alcohol was found to be not suitable for extraction of polymer. All PHA extractions were performed from both dry and wet cells at volumes ranging from 2 mL to 3 L using a PHA to solvent ratio of 2% (w/v). Ethyl acetate showed both high recovery levels and high product purities (up to 99%) when using dry cells as starting material. Recovery from wet cells, however, eliminates a biomass drying step during the downstream process, potentially saving time and cost. When wet cells were used, methyl isobutyl ketone (MIBK) was shown to be the most favorable solvent for PHA recovery. Purities of up to 99% and total recovery yields of up to 84% from wet cells were reached. During polymer recovery with either MIBK or butyl acetate, fractionation of the extracted PHA occurred, based on the HHx content of the polymer. PHA with higher HHx content (17-30 mol%) remained completely in solution, while polymer with a lower HHx content (11-16 mol%) formed a gel-like phase. All PHA in solution could be precipitated by addition of threefold volumes of n-hexane or n-heptane to unfiltered PHA solutions. Effective recycling of the solvents in this system is predicted due to the large differences in the boiling points between solvent and precipitant. Our findings show that two non-halogenated solvents are good candidates to replace halogenated solvents like chloroform for recovery of high quality PHA.

Intermittent trickling bed filter for the removal of methyl ethyl ketone and methyl isobutyl ketone.

Biodegradations of methyl ethyl ketone and methyl isobutyl ketone were performed in intermittent biotrickling filter beds (ITBF) operated at two different trickling periods: 12 h/day (ITBF-12) and 30 min/day (ITBF-0.5). Ralstonia sp. MG1 was able to degrade both ketones as evidenced by growth kinetic experiments. Results show that trickling period is an important parameter to achieve high removal performance and to maintain the robustness of Ralstonia sp. MG1. Overall, ITBF-12 outperformed ITBF-0.5 regardless of the target compound. ITBF-12 had high performance recovery at various inlet gas concentrations. The higher carbon dioxide production rates in ITBF-12 suggest higher microbial activity than in ITBF-0.5. Additionally, lower concentrations of absorbed volatile organic compound (VOC) in trickling solutions of ITBF-12 systems also indicate VOC removal through biodegradation. Pressure drop levels in ITBF-12 were relatively higher than in ITBF-0.5 systems, which can be attributed to the decrease in packed bed porosity as Ralstonia sp. MG1 grew well in ITBF-12. Nonetheless, the obtained pressure drop levels did not have any adverse effect on the performance of ITBF-12. Biokinetic constants were also obtained which indicated that ITBF-12 performed better than ITBF-0.5 and other conventional biotrickling filter systems.

Response of earthworm coelomocytes and catalase to pentanone and hexanone: a revelation of the toxicity of conventional solvents at the cellular and molecular level.

Organic solvents like 2-pentanone and 2-hexanone which are widely used in industrial production make up a large proportion of the source of chemical pollution. What is worrisome is that the cellular and molecular toxicity of 2-pentanone and 2-hexanone has not been reported yet. Based on this, earthworms and catalase (CAT) were chosen as target receptors for the toxicity studies. The cytotoxicity of 2-pentanone and 2-hexanone was revealed by measuring the multiple intracellular indicators of oxidative stress. At the molecular level, changes in the structure and function of CAT were characterized in vitro by the spectroscopy and molecular docking. The results show that 2-pentanone and 2-hexanone that induced the accumulation of reactive oxygen species can eventually reduce coelomocytes viability, accompanying by the regular changes of antioxidant activity and lipid peroxidation level. In addition, the exposure of 2-pentanone and 2-hexanone can shrink the backbone structure of CAT, quench the fluorescence, and misfold the secondary structure. The decrease in enzyme activity should be attributed to the structural changes induced by surface binding. This study discussed the toxicological effects and mechanisms of conventional solvents at the cellular and molecular level, which creatively proposed a joint research method.

Biodegradation kinetics of methyl iso-butyl ketone by acclimated mixed culture.

Methyl iso-butyl ketone (MIBK) is a widely used volatile organic compound (VOC) which is highly toxic in nature and has significant adverse effects on human beings. The present study deals with the removal of MIBK using biodegradation by an acclimated mixed culture developed from activated sludge. The biodegradation of MIBK is studied for an initial MIBK concentration ranging from 200-700 mg l(-1) in a batch mode of operation. The maximum specific growth rate achieved is 0.128 h(-1) at 600 mg l(-1)of initial MIBK concentration. The kinetic parameters are estimated using five growth kinetic models for biodegradation of organic compounds available in the literature. The experimental data found to fit well with the Luong model (R(2) = 0.904) as compared to Haldane model (R(2) = 0.702) and Edward model (R(2) = 0.786). The coefficient of determination (R(2)) obtained for the other two models, Monod and Powell models are 0.497 and 0.533, respectively. The biodegradation rate found to follow the three-half-order kinetics and the resulting kinetic parameters are reported.

Biofiltration for removal of methyl isobutyl ketone (MIBK): experimental studies and kinetic modelling.

The present study deals with the biofiltration of methyl isobutyl ketone (MIBK), which is considered to be a highly toxic volatile organic compound. It is released from the paint and petrochemical industries and is one of the major contributors to air pollution. The biofiltration study was carried out on a lab scale for two months in the presence of acclimated mixed culture. The performance of the biofilter column was evaluated for different inlet loads of MIBK at air flow rates ranging from 0.18 to 0.3 m3 h(-1). The maximum removal efficiency of 93% was obtained after 60 days of biofilter operation for an inlet MIBK concentration of 0.45 g m(-3), and a microbial concentration of 2.36 x 10(8) CFU g(-1) of packing material was obtained. This led to a study of shock loadings for 20 days, by varying the inlet MIBK load and air flow rate after every five days, to observe the behaviour of the biofilter column in removing sudden loads of MIBK. The biokinetic constants r(max) and Ks were obtained using the Michaelis-Menten kinetics and were found to be 1.046 g m(-3) and 0.115 g m(-3) h(-1),respectively, with a coefficient of determination (R2) of 0.993. The obtained experimental results were validated with the Ottengraf and Van den Oever kinetic model. The critical inlet concentration, critical inlet load and biofilm thickness were also estimated using the results obtained from the model predictions.

Removal kinetics of acetone and MIBK from a complex industrial wastewater by an acclimatized activated sludge.

Removal rates of acetone and methyl isobutyl ketone (MIBK) were determined singularly and in combination in batch experiments employing an acclimatized activated sludge from a pilot system treating an organic chemical manufacturer industrial wastewater, as well as during the course of operation of the pilot system. Both acetone and MIBK removals were described by first-order removal kinetics. Acetone, as a single substrate, was biodegraded at a rate of 1.7 day(-1). MIBK was biodegraded, during the single-substrate experiments, at an observed rate of 2.23 day(-1). Relative to the control (no spike) experiments, acetone removal was augmented by a factor of 3 during the acetone spike, with an observed biomass-specific removal rate of 0.0023 L/mg VSSday. MIBK removal during an MIBK spike was also enhanced by a factor of 2, and the greatest biomass-specific removal rate of MIBK was observed during the acetone spike of 0.0019 L/mg VSSday. The observed rates were approximately 3 orders of magnitude greater than the observed rates in the pilot scale system. MIBK exerted a synergistic effect on the acetone-removal rate while acetone exerted an inhibitory impact on the MIBK-removal rate.

Methyl isobutyl ketone exposure-related increases in specific measures of α2u-globulin (α2u) nephropathy in male rats along with in vitro evidence of reversible protein binding.

Chronic exposure to methyl isobutyl ketone (MIBK) resulted in an increase in the incidence of renal tubule adenomas and occurrence of renal tubule carcinomas in male, but not female Fischer 344 rats. Since a number of chemicals have been shown to cause male rat renal tumors through the α2u nephropathy-mediated mode of action, the objective of this study is to evaluate the ability of MIBK to induce measures of α2u nephropathy including renal cell proliferation in male and female F344 rats following exposure to the same inhalation concentrations used in the National Toxicology Program (NTP) cancer bioassay (0, 450, 900, or 1800ppm). Rats were exposed 6h/day for 1 or 4 weeks and kidneys excised approximately 18h post exposure to evaluate hyaline droplet accumulation (HDA), α2u staining of hyaline droplets, renal cell proliferation, and to quantitate renal α2u concentration. There was an exposure-related increase in all measures of α2u nephropathy in male, but not female rat kidneys. The hyaline droplets present in male rat kidney stained positively for α2u. The changes in HDA and α2u concentration were comparable to d-limonene, an acknowledged inducer of α2u nephropathy. In a separate in vitro study using a two-compartment vial equilibration model to assess the interaction between MIBK and α2u, the dissociation constant (Kd) was estimated to be 1.27×10(-5)M. This Kd is within the range of other chemicals known to bind to α2u and cause nephropathy. Together, the exposure-related increase in measures of α2u nephropathy, sustained increase in renal cell proliferation along with an indication of reversible binding of MIBK to α2u, support the inclusion of MIBK in the category of chemicals exerting renal effects through a protein droplet α2u nephropathy-mediated mode of action (MoA).

Pharmacodynamic and metabolic interactions between ethanol and two industrial solvents (methyl n-butyl ketone and methyl isobutyl ketone) and their principal metabolites in mice.

MnBK and MiBK prolong the duration of ketamine-, pentobarbital-, thiopental- and ethanol-induced loss of righting reflex (LRR) in mice. In equimolar doses, (5 mmol/kg i.p.), both isomers were equipotent with respect to the enhancement of ketamine-, pentobarbital-, and thiopental-induced LRR. However, MnBK was significantly more effective (twice as effective) than its isomer with respect to enhancing ethanol-induced LRR. An attempt to explain the difference in effectiveness between the two isomers was carried out. The effects of both ketones and their principal metabolites, (2-hexanol (2-HOL), 2,5-hexanedione (2,5-HD), 4-methyl-2-pentanol (4-MPOL) and 4-hydroxy 4-methyl-2-pentanone (HMP)) on ethanol-induced LRR and ethanol elimination were studied in mice. The ketones and their metabolites were dissolved in corn oil and injected intraperitoneally 30 min before 4 g/kg ethanol for LRR and 2 g/kg for ethanol elimination. Ethanol-induced LRR was significantly prolonged by the following dosages (mmol/kg), MnBK, 5; MiBK, 5; 2-HOL, 2.5; 4-MPOL, 2.5; and HMP, 2.5; 2,5-HD, 2.5, however exerted no effect. Concentrations of ethanol in blood or brain upon return of the righting reflex were similar in solvent-treated and control animals. The mean elimination rate of ethanol was slower in groups pretreated with MnBK or 2-HOL as compared to control animals. Ethanol elimination in animals pretreated with MiBK, HMP, 4-MPOL, or 2,5-HD was similar to that in control animals. These ketones are known to have some central depressant action on their own. This by itself could lead to prolongation of ethanol-induced LRR. However, MnBK, as well as one of its principal metabolites, (2-HOL), markedly reduced ethanol elimination. This could explain the observation that MnBK has a greater potentiating effect on ethanol-induced LRR that its isomer, MiBK, which does not affect ethanol elimination.

Hexacarbon neuropathy: tracking a toxin.

This paper will present the progression of hexacarbon studies which followed the investigation of circumstances surrounding the occurrence of peripheral neuropathy in an occupational setting. The identification of methyl n-butyl ketone as the neurotoxin led to systematic studies of the biotransformation and mechanism of neurotoxicity. A consistent line of evidence showed that the neurotoxic potential of hexacarbons is directly related to the formation of the gamma-diketone metabolite, 2,5-hexanedione. The precise nature of the chemical interaction at the neurofilament is the subject of continuing investigation.

Effect of aeration during cell growth on ketone reactions by immobilized yeast.

The effect of aeration during cell growth on the subsequent reduction of 2-hexanone and 2-octanone by yeast cells entrapped in calcium alginate beads was studied. The reactions were conducted using 2-propanol as a sacrificial substrate to regenerate the cofactor NAD(H), and a mixture of (S)- and (R)-alcohols was produced. The use of strictly aerobic conditions when growing the cells resulted in the highest initial reaction rates, as well as the production of only a single product (i.e., the enantiomeric excess of the (S)-alcohols was 1.0). However, initial reaction rates decreased proportionally with fermentation time regardless of whether the yeast were grown aerobically or under both aerobic and anaerobic conditions. The data also suggest that it is the aerobic (or anaerobic) condition, rather than the cell growth phase, which is responsible for the results seen.

Tissue concentrations of methyl isobutyl ketone, methyl n-butyl ketone and their metabolites after oral or inhalation exposure.

Quantitative relationships between plasma, liver and lung methyl isobutyl ketone (MiBK) and methyl n-butyl ketone (MnBK) concentrations after oral or inhalation exposure were established. Their respective metabolites (4-methyl-2-pentanol, 4-hydroxy-methyl isobutyl ketone, 2-hexanol, and 2,5-hexanedione) were also quantified. Male Sprague-Dawley rats were exposed for 3 days to MiBK or MnBK vapors (4 h/day) or treated orally for 3 days with a MiBK- or MnBK-corn oil solution. Both ketones and their respective metabolites in plasma or tissue concentrations were determined by gas chromatography. MiBK and MnBK plasma and tissue concentrations increased in a dose-related manner with the administered dose irrespective of the route of administration. Metabolite concentrations, however, were influenced by the route of administration.

An investigation into the role of rat skeletal muscle as a site for xenobiotic metabolism using microsomes and isolated cells.

1. The role of skeletal muscle microsomes as a site of extrahepatic xenobiotic metabolism using n-hexane as a model substrate was investigated. The observed cytochrome P450-dependent metabolism was compared with that found with liver, and brain microsomal fractions. 2. Rat skeletal muscle microsomes metabolised n-hexane to 1-, 2- and 3-hexanol at rates 40-300 times lower than observed with rat liver microsomes. 3. Fast-twitch extensor digitorum longus muscle (EDL) microsomes had twice as much n-hexane hydroxylase activity as the slow-twitch soleus and furthermore the EDL microsomes produced 2-hexanol, a bioactivation product of n-hexane, as a major metabolite. 4. Metabolism of hexane to 1-, 2- and 3-hexanol and 2-hexanon was demonstrated in cultured rat myoblasts. 5. Ethoxyresorufin and pentoxyresorufin O-dealkylation were not detected in either muscle microsomes or myoblasts although immunocytochemical localisation studies were suggestive of the presence of cytochrome P-450. 6. In conclusion, rat skeletal muscle has a low level of xenobiotic metabolism activity. The relevance to neuromuscular toxicity of n-hexane is discussed.

Distribution of aged 14C-PCB and 14C-PAH residues in particle-size and humic fractions of an agricultural soil.

Organic matter is considered to be the single most importantfactor limiting availability and mobility of persistent organic pollutants (POPs) in soil. This study aimed to characterize the distribution of 14C-PCB (congeners 28 and 52) and 14C-PAH (fluoranthene and benzo[a]pyrene) residues in an Orthic Luvisol soil obtained from two lysimeter studies initiated in 1990 at the Agrosphere Institute (Forschungszentrum Jülich GmbH, Germany). The lysimeter soils contained a low-density OM fraction, isolated during soil washing, which contained a significant fraction (3-12%) of the total 14C-activity. Soils were also fractionated according to three particle sizes: >20, 20-2, and <2 microm. Relative affinity values of 14C-activity for the different particle sizes varied in the order 20-2 microm > (<2 microm) approximately (>20 microm) for the PCBs. Relative affinity values of 14C-activity for the different particle sizes varied in the order 20-2 microm > (<2 microm) > (>20 microm) for the PAHs. The distribution of 14C-PCB or 14C-PAH residues in the organic and inorganic matrixes of the particle-size fractions was determined using methyl isobutyl ketone (MIBK). 14C-PCB and 14C-PAH-associated activities were primarily located in the humin fraction of the 20-2 and <2 microm particle-size fractions of the soil. A small fraction was associated with the fulvic and humic acid fractions; these were quantitatively more important for the PAHs than the PCBs. There appeared to be a high degree of association of 14C-activity with the mineral fraction following MIBK separation of the humic fractions, ranging between 8 and 52% for 14C-PCBs and 57-80% for 14C-PAHs. The mineral (inorganic) component of the soils apparently played a significant (previously unreported) role in the sequestration of both PCBs 28 and 52 and the PAHs fluoranthene and benzo[a]pyrene.

Protein-bound pyrroles in rat hair following subchronic intraperitoneal injections of 2,5-hexanedione.

Studies were initiated to ascertain whether body hair could be used to develop a biological marker for chronic exposure to industrial neurotoxicants that yield the metabolite 2,5-hexanedione (2,5-HD), that is, n-hexane and methyl n-butyl ketone. Rats were injected daily with a 50 mg/kg ip dose of 2,5-HD for 45 d. At intervals, body hair and individual vibrissae were removed (under general anesthesia) and tested for the presence of pyrrole substances with p-N,N-dimethylaminobenzaldehyde (DMAB, Ehrlich's reagent). Vibrissae and body hair were stained a reddish color that was distinctly different from that observed with the hair taken from control animals. Solubilized body hair protein from the treated animals gave a positive Ehrlich's test, while that from control animals was negative. Spectral analysis of the DMAB-treated hair from experimental animals disclosed a maximum absorbance at 530 nm, which indicated the presence of pyrrole substituents. Serial analysis of individual nose hairs taken during 2,5-HD administration showed a progression with time of the region staining positively for pyrroles, thus indicating that the process can proceed in growing hair. These findings suggest the potential utility of hair as an indicator for chronic exposure to this class of industrial chemicals possessing neurotoxicity potential. This could complement urinary analysis, which is now used to confirm recent exposure.

Biotransformation of n-hexane and methyl n-butyl ketone in guinea pigs and mice.

Peripheral neuropathies caused by exposures to the industrial solvents n-hexane and MBK exhibit strinkingly similar characteristics. In in vivo studies, the metabolites of MBK and n-hexane identified in blood and urine of guinea pigs were 2-hexanol (partly as glucuronide in urine); and 2,5-hexanedione which was detected only in MBK treated groups. Phenobarbital pretreatment increased 2-hexanol urinary excretion in both solvent treatment groups. In in vitro studies, hepatic reduction of MBK required the cytosol fraction to form 2-hexanol; whereas the oxidation of MBK and n-hexane required the microsomal fraction to form 2,5-hexanedione and 2-hexanol, respectively. The in vivo and in vitro biotransformation of MBK and n-hexane to a common metabolite (2-hexanol) suggests that the neurotoxic action of these solvents may be metabolite related.

Toxicity and metabolism of the neurotoxic hexacarbons n-hexane, 2-hexanone, and 2,5-hexanedione.

Human exposure to hexacarbon compounds is quite pervasive, including occupational exposures to industrial solvents as well as unintentional and sometimes deliberate exposures to hexacarbon solvents contained in innumerable commercial products. The exact mechanism of hexacarbon neurotoxicity has not yet been identified, but an interference with neuronal axoplasmic flow seems most likely. Metabolism of n-hexane and 2-hexanone to 2,5-hexanedione is a prominent feature which appears to be causally related to the neuropathologic syndrome, and mixed solvent effects have been noted in regard to potentiation of hexacarbon neurotoxicity. Continued effort in investigating the chemically induced peripheral neuropathy is essential not only to define the precise molecular mechanism, but to advance our basic understanding of other polyneuropathies as well. Ultimately, progress in these areas should yield such benefits as early diagnosis of potential neuropathology, better measures for the prevention of neurotoxicities, and more effective modalities of treatment. Indeed, sustained research efforts are imperative in maintaining human health and safety throughout our current era of advancing global technology.

Metabolites and ketone body production following methyl n-butyl ketone exposure as possible indices of MnBK potentiation of carbon tetrachloride hepatotoxicity.

While the biotransformation of methyl n-butyl ketone (MnBK) in animals is well characterized, little is known about the quantitative relationship between hepatic and plasma MnBK concentrations. This study provides such information and emphasizes the usefulness of MnBK metabolite quantification, as well as MnBK-induced metabolic ketosis for the biological monitoring of MnBK exposure in rats. Elimination of MnBK was followed 24 hr after oral administration (0.95, 1.90, and 5.70 mmol/kg in corn oil) to male Sprague-Dawley rats. Two metabolites [2-hexanol (2HOL), and 2,5-hexanedione (2,5HD)] were also monitored and their kinetics determined. These data were compared to ketone body (KB) concentrations found in plasma and liver during the same period. Plasma concentrations of MnBK and 2,5HD correlated well with those in the liver. This was not the case for 2HOL. MnBK, 2HOL, and 2,5HD were no longer detected in plasma and liver 18 hr after dosing. Meanwhile, a marked ketosis was observed from 12 to 24 hr. This ketotic state was due to an increase in beta-hydroxybutyrate (BOHB), while acetoacetate remained at its basal levels. These data indicate that MnBK can induce ketosis in rats and suggest that the resulting BOHB might be used as an alternative biological monitor of MnBK exposures at high concentrations.