Formate production from methanol by formaldehyde dismutase coupled with a methanol oxidation system

Summary Formaldehyde dismutase was greatly stabilized by immobilization in a urethane prepolymer (PU-6). The immobilized enzyme exhibited stochiometrical dismutation of formaldehyde to methanol and formate in several repeated reactions. Conversion of methanol to formate occurred in a reaction with an immobilized enzyme system consisting of alcohol oxidase, catalase and formaldehyde dismutase, and with an intact cell-mixture of Hansenula polymorpha and Pseudomonas putida. Furthermore, the stability of the cell-mixture during repeated reactions was greatly improved by the immobilization, the 600 mM methanol added periodically being converted to formate in a 75% yield in 12 h. The immobilized cellsystem was also effective for the conversion of several aliphatic alcohols, C₁ to C₄, to the corresponding acids.     

Lipase-catalyzed transesterification of soybean oil for biodiesel production in <Emphasis Type="Italic">tert</Emphasis>-amyl alcohol

Lipase-catalyzed transesterification of soybean oil and methanol for biodiesel production in tert-amyl alcohol was investigated. The effects of different organic medium, molar ratio of substrate, reaction temperature, agitation speed, lipase dosage and water content on the total conversion were systematically analyzed. Under the optimal conditions identified (6 mL tert-amyl alcohol, three molar ratio of methanol to oil, 2% Novozym 435 lipase based on the soybean oil weight, temperature 40°C, 2% water content based on soybean oil weight, 150 rpm and 15 h), the highest biodiesel conversion yield of 97% was obtained. With tert-amyl alcohol as the reaction medium, the negative effects caused by excessive molar ratio of methanol to oil and the by-product glycerol could be reduced. Furthermore, there was no evident loss in the lipase activity even after being repeatedly used for more than 150 runs.     

l-Serine production using a resting cell system of Hyphomicrobium strains

Twenty-six strains of methylotrophic hyphomicrobia were examined for their ability to produce l-serine from methanol and glycine in a resting cell reaction. l-Serine productivity of over 5 mg/ml was observed in 7 strains, and Hyphomicrobium sp. NCIB10099 was found to exhibit the highest productivity. Under optimized conditions using this bacterium, 45 mg/ml l-serine was produced from 100 mg/ml glycine and 88 mg/ml methanol in 3 d. The high l-serine degrading activity of the bacterium was entirely suppressed by adding an appropriate amount of CdCl₂ (ca. 1 mM), resulting in an enhanced conversion ratio of glycine to l-serine (ca. 100% molar conversion).     

A non-extractable triterpenoid of the hopane series in Acetobacter xylinum

Abstract Cells of the bacterium Acetobacter xylinum were analysed for their residual triterpenoid content after exhaustive lipid extraction using chloroform/methanol. Whereas the well known bacteriohopanetetrol mixture was present in the extract, the cells still contained a single bacteriohopanepentol cyclitol ether which could be detected either as the primary alcohol derivative after H₅IO₆ oxidation followed by NaBH₄ reduction of the already extracted cells or as the octa-acetate by direct acetylation followed by solvent extraction. This is the first evidence of a probable selective complexation of a hopanoid via non-covalent linkages to other cell constituents.     

Isolation and characterization of an obligate methanol-utilizing bacteriumMethylomonas M-15

Summary A new obligate methylotrophic bacterium capable of rapid growth on methanol as its sole carbon and energy source was isolated. The organism grows only on methanol and not on methane or methylamine. It is a gram-negative, motile rod (0.5×1.5 m) with a single polar flagellum and was therefore classified as a species ofMethylomonas.The doubling time was about 2 hr at pH 7.0, at a temperature of 30 to 36°C and at a methanol concentration of 1% (v/v).Cell suspensions were able to oxidize methanol, formaldehyde, and formate; therefore it seems that methanol-, formaldehyde-, and formate-oxidizing enzymes are present in the bacterium. While no hydroxy pyruvate reductase activity was found, the cell extract contained high hexulose phosphate-synthetase activity, indicating the assimilation of methanol via the ribulose phosphate-pathway.The protein content of the bacterium is 60% and the amino acid pattern indicates that this strain could serve as a good source of single cell protein.     

Synthesis of Poly(3-Hydroxybutyrate-Co-3-Hydroxyvalerate) from Methanol and n-Amyl Alcohol by the Methylotrophic Bacteria Paracoccus denitrificans and Methylobacterium extorquens

Strains of two types of methylotrophic bacteria, Paracoccus denitrificans and Methylobacterium extorquens, synthesized the copolyester poly(3-hydroxybutyrate-co-3-hydroxyvalerate) when methanol and n-amyl alcohol were added together to nitrogen-limited medium. The composition of the copolyester differed considerably between the two strains: the copolyester from P. denitrificans was comparatively rich in 3-hydroxyvalerate (3HV). The 3HV content of the copolyester synthesized by this strain increased with increasing concentrations of n-amyl alcohol. Its maximum content was 91.5 mol% under the conditions used. In M. extorquens, the maximum 3HV content was limited to 38.2 mol%. Since n-amyl alcohol served as a substrate for a standard methanol dehydrogenase, the enzyme was proposed to oxidize both methanol and n-amyl alcohol in the first step of copolyester synthesis from these substrates by methanol-grown cells.     

Immobilization of the methanogenic bacterium Methanosarcina barkeri

Whole cells of the methanogen Methanosarcina barkeri were immobilized in an alginate network which was crosslinked with Ca²⁺ ions. The rates of methanol conversion to methane of entrapped cells were found to be in the same range as the corresponding rates of free cells. Furthermore, immobilized cells were active for a longer period than free cells. The particle size of the spherical alginate beads (1.2 mm-3.7 mm ?) and thus diffusion had no obvious influence on the turnover of methanol. The half-value period for methanol conversion activity determined in a buffer medium was approximately 4 days at 37°C for entrapped cells. The apparent K_m value K for such cells was nearly 140mM and the V_max value was about 1.2 μmol methanol/min/mg entrapped protein. Therefore the high rates of methanol degradation measured, e.g., 0.5 μmol methanol/min/mg entrapped protein, indicated that the immobilization technique preserved the cellular functions of this methanogenic bacterium.     

Induction of l-lysine ɛ-aminotransferase by l-lysine in Streptomyces clavuligerus, producer of cephalosporins

Abstract Several alcohols were examined as substrates for the polyhydroxyalkanoate synthesis by Paracoccus denitrificans. The bacterium synthesized a homopolyester of poly(3-hydroxybutyrate) from ethanol. When n -pentanol was used as growth substrate, homopolyester poly(3-hydroxyvalerate) was synthesized, whereas copolyester poly(3-hydroxybutyrate-co-3-hydroxyvalerate) accumulated during bacterial growth on n -propanol. When alcohols were automatically fed as growth substrates, ethanol, n -propanol, and n -pentanol gave higher polyester content. Although poly(3-hydroxybutyrate) was synthesized from methanol or n -butanol, its content was very low. Under nitrogen-deficient conditions, polyester;content in cells increased, especially with ethanol, n -propanol, and n -pentanol. Using a mixture of two alcohols P. denitrificans could synthesize polyesters with varying relative ratios of 3-hydroxybutyrate to 3-hydroxyvalerate.     

Effect of support matrix on ratio of product to by-product formation in L-phenylacetyl carbinol synthesis

Production of L-phenylacetyl carbinol (PAC) and benzyl alcohol by biotransformation from pyruvate and benzaldehyde substrates was investigated using yeast cells in different polymeric matrices. Highest concentration of PAC was produced by cells immobilised in the hydrophilic matrix, ENT-4000. Highest concentration of the by-product, benzyl alcohol, was produced in the hydrophobic matrix, ENTP-2000. Ratio of PAC to benzyl alcohol ranged from 10.08 for cells entrapped in a poly(propyleneglycol)-containing polymer, PU-3, to 11.8 for cells entrapped in ENTP-2000 polymer.     

Biofuel production from palm oil with supercritical alcohols: effects of the alcohol to oil molar ratios on the biofuel chemical composition and properties

Biofuel production from palm oil with supercritical methanol (SCM) and supercritical ethanol (SCE) at 400 °C and 15 MPa were evaluated. At the optimal alcohol to oil molar ratios of 12:1 and 18:1 for the SCM and SCE processes, respectively, the biofuel samples were synthesized in a 1.2-L reactor and the resulting biofuel was analyzed for the key properties including those for the diesel and biodiesel standard specifications. Biofuel samples derived from both the SCM and SCE processes could be used as an alternative fuel after slight improvement in their acid value and free glycerol content. The remarkable advantages of this novel process were: the additional fuel yield of approximately of 5% and 10% for SCM and SCE, respectively; the lower energy consumption for alcohol preheating, pumping and recovering than the biodiesel production with supercritical alcohols that use a high alcohol to oil molar ratio of 42:1.     

Comparison of solid-state and submerged-state fermentation for the bioprocessing of switchgrass to ethanol and acetate by Clostridium phytofermentans

The conversion of sustainable energy crops using microbiological fermentation to biofuels and bioproducts typically uses submerged-state processes. Alternatively, solid-state fermentation processes have several advantages when compared to the typical submerged-state processes. This study compares the use of solid-state versus submerged-state fermentation using the mesophilic anaerobic bacterium Clostridium phytofermentans in the conversion of switchgrass to the end products of ethanol, acetate, and hydrogen. A shift in the ratio of metabolic products towards more acetate and hydrogen production than ethanol production was observed when C. phytofermentans was grown under solid-state conditions as compared to submerged-state conditions. Results indicated that the end product concentrations (in millimolar) obtained using solid-state fermentation were higher than using submerged-state fermentation. In contrast, the total fermentation products (in weight of product per weight of carbohydrates consumed) and switchgrass conversion were higher for submerged-state fermentation. The conversion of xylan was greater than glucan conversion under both fermentation conditions. An initial pH of 7 and moisture content of 80 % resulted in maximum end products formation. Scanning electron microscopy study showed the presence of biofilm formed by C. phytofermentans growing on switchgrass under submerged-state fermentation whereas bacterial cells attached to surface and no apparent biofilm was observed when grown under solid-state fermentation. To our knowledge, this is the first study reporting consolidated bioprocessing of a lignocellulosic substrate by a mesophilic anaerobic bacterium under solid-state fermentation conditions.     

The effect of ethanol and acetaldehyde on [14C]pantothenate incorporation into CoA in cultured rat liver parenchymal cells

The effect of ethanol on [¹⁴C]pantothenate incorporation into CoA and on total CoA levels was measured in 3-day-old primary cultures of adult rat liver parenchymal cells. Ethanol decreased the incorporation of radioactivity into CoA a maximum of 67%, 5 mm ethanol was saturating for the inhibitory effect and 0.2 mm ethanol was sufficient for half-saturation. This inhibitory effect did not result from a loss of CoA precursors or from cell death. Ethanol concentrations up to 10 mm did not decrease the ATP content of cells or the total protein content of cells which adhered to the incubation flask. Ethanol (5 mm) had no effect on the cyteine + cystine content of the cells. Intracellular pantothenate concentrations were not affected by 5 mm ethanol, and increasing the pantothenate concentration did not affect ethanol inhibition. Ethanol inhibition of [¹⁴C]pantothenate conversion to CoA could be fully reversed by rinsing the cells free of ethanol. The ethanol inhibition could also be fully reversed by addition of 4-methylpyrazole, indicating that ethanol must be oxidized via alcohol dehydrogenase to exert its inhibitory effect. Acetaldehyde, the immediate product of alcohol dehydrogenase, was also an inhibitor of the incorporation of [¹⁴C]pantothenate into CoA; the maximum inhibition was 63%. Acetaldehyde concentrations maintained between 18 and 103 μm inhibited incorporation by 57%. The inhibition by acetaldehyde did not correlate well with changes in the NADH and NAD⁺ ratio of the cells (as determined by measuring changes in the lactate-to-pyruvate ratio). The ability of glucagon, dibutyryl cAMP + theophylline, or dexamethasone to stimulate [¹⁴C]pantothenate conversion to CoA was not decreased by the addition of ethanol or acetaldehyde, indicating that ethanol inhibition does not occur by reversal of the cAMP-mediated regulatory mechanism for CoA biosynthesis.     

Oxidation of Lignin-Related Aromatic Alcohols by Cell Suspensions of Methylosinus trichosporium

Cell suspensions of Methylosinus trichosporium oxidized the aromatic alcohols benzyl alcohol, vanillyl alcohol, and veratryl alcohol to the corresponding aldehydes, and with the exception of vanillyl alcohol, the aldehydes were further oxidized to the corresponding aromatic acids. No other transformation was observed, and the methoxyl moieties attached to the aromatic nucleus remained intact. More than 70% of the alcohol oxidized could be accounted for by aldehyde and/or acid. Investigation of the inhibitor kinetics of EDTA or p-nitrophenylhydrazine (specific for NAD⁺-independent methanol dehydrogenase in methylotrophs) on aromatic alcohol oxidation revealed noncompetitive inhibition in which the V_max was decreased but the K_m remained unchanged. The pattern of inhibition of aromatic alcohol oxidation matched that of methanol oxidation, and the K_m values for all of the substrates were similar (12 to 16 mM). The results indicate that the initial step in the oxidation of aromatic alcohols was similar to that for methanol, and because oxidation was incomplete (i.e., only the corresponding aldehyde or acid was produced), there may be some biotechnological advantages in using whole cells of methylotrophs to facilitate aromatic biotransformations.     

Regulation of protein synthesis in methylotrophic yeasts: Repression of methanol dissimilating enzymes by nitrogen limitation

The influence of nitrogen limitation on the regulation of the methanol oxidizing enzymes alcohol oxidase, catalase, formaldehyde dehydrogenase and formate dehydrogenase in the two methylotrophic yeastsHansenula polymorpha andKloeckera sp. 2201 was studied in continuous culture. When shifted from carbon-limited growth conditions (with a mixture of glucose and methanol as carbon sources) to a nitrogen-limited environment both cultures were found to go through a transition phase where neither enhanced residual concentrations of the nitrogen source nor of one of the two carbon sources could be detected in the supernatant. As soon as nitrogen became a limiting substrate an immediate reorganisation of the cell composition was initiated: protein content of the cells dropped to approximately 40% of its initial value, glycogen was synthesized and the enzyme composition of the cells was changed. The peroxisomal enzymes alcohol oxidase and catalase in both organisms and the two dehydrogenases for formaldehyde and formate in cells ofKloeckera sp. 2201 were subject to degradation (catabolite inactivation). The measured rates of inactivation indicated that in cells ofH. polymorpha this process might be limited to peroxisomes, whereas inKloeckera sp. 2201 the degradation was found to affect peroxisomal as well as cytoplasmic enzymes. In contrast to methanol dissimilating enzymes the net rate of synthesis of hexokinase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase was not affected by this process but those enzymes were synthesized with increased rates.     

Accessory cell requirements for lymphoma growth in vitro and in irradiated mice.

Growth of the transplantable B-cell lymphoma, PU-5, is markedly diminished in γ-irradiated as compared to normal BALB/c mice. Transfer of bone marrow, but not of lymph node or peritoneal exudate cells, partially restored the ability of irradiated mice to support lymphoma growth. In vitro growth of PU-5 cells is promoted by silica-sensitive, adherent cells, bearing surface Ia antigen and present in peritoneal exudates, spleen and lymph node, but not in bone marrow. Their action on PU-5 growth can be shown only in rocking cultures; the cells do not have to be histocompatible, they act synergistically with 2-mercaptoethanol (2-ME) in the medium. The growth-promoting action in vitro is decreased 24 hr after γ-irradiation of the adherent cells in vitro. Growth of transplantable reticulum cell sarcoma in SJL/J mice has previously also been shown to be inhibited by prior irradiation of the host and to be restored by transfer of lymphoid cells including a phagocytic component, but in the present studies no consistent growth-promoting effect of accessory cells on reticulum cell sarcomas has been shown in vitro. Both lymphomas are stimulated by the presence of 2-ME in stationary cultures. The relationship between the in vivo and in vitro lymphoma growth-promoting activities of macrophage-like cells is discussed.     

Overproduction of methanol dehydrogenase in glucose grown cells of a restricted RuMP type methylotroph

A restricted facultative methylotrophic RuMP type bacterium that can only utilize methanol and glucose has been found to possess a higher specific activity of methanol dehydrogenase during growth on glucose than during growth on methanol. The increased level of methanol dehydrogenase activity in glucose grown cells was the result of overproduction of the enzyme. In methanol grown cells 8% of the soluble protein consisted of methanol dehydrogenase, whereas in glucose grown cells the proportion amounted to 25%. The type of methanol dehydrogenase produced by this methylotroph could be separated from the crude extract and purified close to homogeneity in a one step procedure using cationic ion exchange chromatography. The enzyme is constitutive, and its level is determined by the growth rate.     

Isolation and Characterization of a Catabolite Repression-Insensitive Mutant of a Methanol Yeast, Candida boidinii A5, Producing Alcohol Oxidase in Glucose-Containing Medium

Mutants exhibiting alcohol oxidase (EC 1.1.3.13) activity when grown on glucose in the presence of methanol were found among 2-deoxyglucose-resistant mutants derived from a methanol yeast, Candida boidinii A5. One of these mutants, strain ADU-15, showed the highest alcohol oxidase activity in glucose-containing medium. The growth characteristics and also the induction and degradation of alcohol oxidase were compared with the parent strain and mutant strain ADU-15. In the parent strain, initiation of alcohol oxidase synthesis was delayed by the addition of 0.5% glucose to the methanol medium, whereas it was not delayed in mutant strain ADU-15. This showed that alcohol oxidase underwent repression by glucose. On the other hand, degradation of alcohol oxidase after transfer of the cells from methanol to glucose medium (catabolite inactivation) was observed to proceed at similar rates in parent and mutant strains. The results of immunochemical titration experiments suggest that catabolite inactivation of alcohol oxidase is coupled with a quantitative change in the enzyme. Mutant strain ADU-15 was proved to be a catabolite repression-insensitive mutant and to produce alcohol oxidase in the presence of glucose. However, it was not an overproducer of alcohol oxidase and, in both the parent and mutant strains, alcohol oxidase was completely repressed by ethanol.     

Enhanced stability of alcohol dehydrogenase by non-covalent interaction with polysaccharides

Non-covalent interaction of alcohol dehydrogenase with polysaccharides was studied using three neutral and three anionic polysaccharides. The process of interaction of alcohol dehydrogenase with gum Arabic was optimized with respect to the ratio of enzyme to gum Arabic, pH, and molarity of buffer. Alcohol dehydrogenase–gum Arabic complex formed under optimized conditions showed 93 % retention of original activity with enhanced thermal and pH stability. Lower inactivation rate constant of alcohol dehydrogenase–gum Arabic complex within the temperature range of 45 to 60 °C implied its better stability. Half-life of alcohol dehydrogenase–gum Arabic complex was higher than that of free alcohol dehydrogenase. A slight increment was observed in kinetic constants (K _m and V _max) of gum Arabic-complexed alcohol dehydrogenase which may be due to interference by gum Arabic for the binding of substrate to the enzyme. Helix to turn conversion was observed in complexed alcohol dehydrogenase as compared to free alcohol dehydrogenase which may be responsible for observed stability enhancement.     

Immobilization of yeast microbodies by inclusion with photo-crosslinkable resins.

Yeast microbodies containing FAD-dependent alcohol oxidase, catalase and D-amino acid oxidase were isolated from methanol-grown cells of Kloeckera sp. 2201 and immobilized intact in matrices formed by a short-time illumination of photo-crosslinkable resin oligomers. The relative activities of catalase, alcohol oxidase and D-amino acid oxidase of the gel-entrapped microbodies were 36, 76 and 31% respectively as compared with those of free microbodies. Immobilization enhance d the stability of catalase to a certain degree, but not that of alcohol oxidase. The pH/activity profiles of catalase and alcohol oxidase of the entrapped organelles showed more narrow pH optima than those of the free counterparts. D-Amino acid oxidase in immobilized microbodies showed a somewhat higher Km value for D-alanine than that in free ones. Immobilized microbodies oxidized two moles of methanol to form two moles of formaldehyde with consumption of one mole of molecular oxygen. Addition of 3-amino-1,2,4-triazole, an inhibitor of catalase, reduced the formation of formaldehyde to half the amount without change in the amount of oxygen consumed, indicating the synergic action of alcohol oxidase and catalase in methanol oxidation in the microbodies of living yeast cells.     

Application of Urethane Prepolymers to Immobilization of Biocatalysts: Δ1Dehydrogenation of Hydrocortisone by Arthrobacter simplex Cells Entrapped with Urethane Prepolymers

Acetone-dried cells of Arthrobacter simplex having appreciable steroid Δ¹-dehydrogenase activity were immobilized by mixing the cell suspension with water-miscible urethane prepolymers synthesized from toluene diisocyanate and polyether diols. The entrapped cell activity in the transformation of hydrocortisone to prednisolone was affected by the properties of urethane prepolymers, such as the isocyanate group content in prepolymers, the molecular weight of polyether diols and the ethylene oxide content in diols. The addition of 10% of organic solvents, such as methanol and glycols, to the aqueous reaction mixture enhanced the solubility of the substrate greatly and the reaction rate of the immobilized cells. The activity of immobilized cells remained high even in the system containing 30% of methanol, which drastically inhibited the activity of free cells. The presence of an electron acceptor, phenazine methosulfate or 2,6-dichlorophenolindophenol, significantly stimulated the steroid conversion with entrapped cells, as well as free cells. The stability of the cells over repeated reactions was greatly improved by immobilization.