Conversion of 1,3-Butanediol or Acetaldol to β-Hydroxybutyric Acid in Chicks

Enzymic oxidizing system of 1,3-butanediol in chicks was studied in comparison with that in rats. 1,3-Butanediol was oxidized with NAD⁺ as a cofactor in the cytosol fraction of chick liver, kidney, and rat liver. NADP⁺ was about 40% as effective as NAD⁺ in chick liver, kidney, whereas the former was ineffective in rat liver. Inhibitory effect of pyrazole on 1,3-butanediol dehydrogenase activity was recognized distinctly both in chicks and rats, but the effect was much higher in rat liver than that in chick liver and kidney. Both in chicks and rats, the conversion of 1,3-butanediol to β-hydroxybutyric acid was detected clearly, and the rate of β-hydroxybutyrate production increased remarkably by employing NAD⁺-regenerating system, i.e., lactic dehydrogenase and pyruvic acid. β-Hydroxybutyric acid was also formed from acetaldol, the most probable intermediate of 1,3-butanediol, with NAD⁺ both in chicks and rats. From the observations for coenzyme specificity, inhibitory effect of pyrazole, and so on, it is concluded that appreciable difference may be present in 1,3-butanediol oxidizing system between chicks and rats.     

Availability of Energy in Ester and Ether Derivatives of Glycols by Growing Chicks

Biological availability of 33 esters, 17 ethers and 2 acetals of ethanediol, 1,2-propanediol, 1,3-butanediol and 1,4-butanediol was compared by mini-test with chicks. Chicks can utilize esters of ethanediol, 1,2-propanediol and 1,3-butanediol with acetic acid and fatty acids of carbon chain length from 5 to 12 with more improved palatability than that of free acids, while availability of esters of these glycols with propionic and butyric acids was low. Esters of 1,4-butanediol and ether derivatives of these glycols was not available, except ethyl ether of di-ethanediol which was partially available. Acetacetal of ethanediol was partially available but n-butyracetal was not.     

Evaluation of Available Energy of Aliphatic Chemicals by Rats

Bioassy procedure to evaluate biologically available energy of chemicals applicable to rats was established, and available energy of 36 chemicals was determined and compared with that estimated by chicks previously. Rats can utilize energy of propionic and butyric acids and n-hexyl propionate and butyrate well, while chicks cannot. Succinic acid, lauryl alcohol and dilauryl succinate at 5% dietary level were available by rats, though at 10% level lauryl alcohol was toxic. Ethyl lactate, octyl and decyl acetates and 1,2-propanediol dilaurate were available by both rats and chicks. Availability of other 6 esters including ethyl succinate and citrate was low. Availability and digestibility of aldehydes by rats were also low.     

Separation of Racemic frommeso-2,3-Butanediol

2,3-Butanediol containing less than 3% of themesoform has been obtained from samples containing up to 50% of themesoform. The diacetate was obtained by esterification with acetic anhydride in the presence of traces of sulfuric acid as a catalyst and was then purified. When the diacetate was held at 4°C, crystals of racemic 2,3-butanediol diacetate formed, and these were separated by filtration. The diacetate was then transformed back to 2,3-butanediol by transesterification with methanol in the presence of sodium methylate as a catalyst. The resulting 2,3-butanediol contained less than 3% of themesoform. For an original batch of 2,3-butanediol containing 50%dland 50%meso,this method can isolate up to 70% of the racemate content. If the original 2,3-butanediol contains too muchmesoform, racemic 2,3-butanediol diacetate does not crystallize, but 2,3-butanediol containing up to 60% of themesoform can be enriched up to 70% racemate by distillation.     

Availability of Energy in Esters of Aliphatic Acids and Alcohols by Growing Chicks

Biological availability of 106 esters of alcohols and aliphatic mono-, di- and tri-carboxylic acids and diethylene glycol succinate was compared by the mini-test with chicks. Chicks can utilize methyl esters of saturated fatty acids of carbon chain from 10 to 14, ethyl esters of those from 9 to 12, propyl caprate, n-butyl esters of those from 8 to 12, n-amyl esters of those from 6 to 12, n-hexyl n-butyrate and i-vaterate, and n-octyl and n-decyl acetates. Only 3 dicarboxylates, i.e. di-octyl and di-lauryl succinates and di-methyl cis-cyclopropane-l,2-dicarboxylate, were available among the dicarboxylates tested. Availability of ethyl esters of succinic, fumaric and citric, acid was unexpectedly low.     

Lipase-catalyzed diacylation of 1,3-butanediol

Summary A kinetic resolution of 1,3-butanediol was accomplished by lipase-catalyzed enantioselective diacylations in organic solvent. Diacylation of 1,3-butanediol was carried out using immobilized lipase SP382 (from Candida sp.) to produce (R) -1,3-diacetoxybutane with 85.8% e.e.. And then, this optically active product was chemically hydrolyzed to diol, and re-acylated with lipase SP382 to (R) -1,3-diacetoxybutane with over 98% e.e..     

Inactivation of <Emphasis Type="Italic">dhaD</Emphasis> and <Emphasis Type="Italic">dhaK</Emphasis> abolishes by-product accumulation during 1,3-propanediol production in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

1,3-Propanediol (1,3-PD) can be used for the industrial synthesis of a variety of compounds, including polyesters, polyethers, and polyurethanes. 1,3-PD is generated from petrochemical and microbial sources. 1,3-Propanediol is a typical product of glycerol fermentation, while acetate, lactate, 2,3-butanediol, and ethanol also accumulate during the process. Substrate and product inhibition limit the final concentration of 1,3-propanediol in the fermentation broth. It is impossible to increase the yield of 1,3-propanediol by using the traditional whole-cell fermentation process. In this study, dhaD and dhaK, the genes for glycerol dehydrogenase and dihydroxyacetone kinase, respectively, were inactivated by homologous recombination in Klebsiella pneumoniae. The dhaD/dhaK double mutant (designated TC100), selected from 5,000 single or double cross homologous recombination mutants, was confirmed as a double cross by using polymerase chain reaction. Analysis of the cell-free supernatant with high-performance liquid chromatography revealed elimination of lactate and 2,3-butanediol, as well as ethanol accumulation in TC100, compared with the wild-type strain. Furthermore, 1,3-propanediol productivity was increased in the TC100 strain expressing glycerol dehydratase and 1,3-PDO dehydrogenase regulated by the arabinose P_BAD promoter. The genetic engineering and medium formulation approaches used here should aid in the separation of 1,3-propanediol from lactate, 2,3-butanediol, and ethanol and lead to increased production of 1,3-propanediol in Klebsiella pneumoniae.     

Butanediol and lipid metabolism.

Young growing rats, chicks and pigs were fed diets containing graded levels of 1,3-butanediol (BD). Replacement of up to 20% of the dietary carbohydrate energy with BD did not affect body weight gain or food efficiency in these species. Blood beta-hydroxybutyrate levels were markedly elevated when BD was added to the diet. Plasma triglyceride response varied with species. In the rat, plasma triglyceride levels were decreased when BD was added to a high-carbohydrate diet. Plasma triglyceride levels were increased when BD-containing diets were fed to pigs and unchanged when chicks consumed diets containing BD. The hepatic lactate:pyruvate ratio was increased in rats fed BD and decreased in chicks fed BD. Hepatic long-chain acyl CoA levels were increased in rats, but not in chicks, fed BD. Addition of BD to a high-carbohydrate diet markedly decreased the rate of fatty acid synthesis, as measured in vitro or in vivo, in rat liver but not in rat or pig adipose tissue. Hepatic fatty acid synthesis in the chick was not affected by replacement of up to 18% of the dietary carbohydrate with BD. We propose that the hepatic conversion of BD to beta-hydroxybutyrate in the rat shifts the cytoplasmic redox state, reduces the glycolytic rate, and reduces substrate availability for fatty acid synthesis. Further, the concomitant shift in the mitochondrial redox state allows long-chain acyl CoA levels to increase. The overall effect is a decrease in the rate of fatty acid synthesis in livers of rats fed BD.     

Continuous production of chiral 1,3-butanediol using immobilized biocatalysts in a packed bed reactor: promising biocatalysis method with an asymmetric hydrogen-transfer bioreduction

An asymmetric hydrogen-transfer biocatalyst consisting of mutated Rhodococcus phenylacetaldehyde reductase (PAR) or Leifsonia alcohol dehydrogenase (LSADH) was applied for some water-soluble ketone substrates. Among them, 4-hydroxy-2-butanone was reduced to (S)/(R)-1,3-butanediol, a useful intermediate for pharmaceuticals, with a high yield and stereoselectivity. Intact Escherichia coli cells overexpressing mutated PAR (Sar268) or LSADH were directly immobilized with polyethyleneimine or 1,6-diaminehexane and glutaraldehyde and evaluated in a batch reaction. This system produced (S)-1,3-butanediol [87% enantiomeric excess (e.e.)] with a space time yield (STY) of 12.5 mg h⁻¹ ml⁻¹ catalyst or (R)-1,3-butanediol (99% e.e.) with an STY of 60.3 mg h⁻¹ ml⁻¹ catalyst, respectively. The immobilized cells in a packed bed reactor continuously produced (R)-1,3-butanediol with a yield of 99% (about 49.5 g/l) from 5% (w/v) 4-hydroxy-2-butanoate over 500 h.     

Epicuticular wax of Cirsium arvense

Epicuticular wax of Cirsium arvense contains hydrocarbons (12%), esters (35%), free acids (3%), free alcohols (10%), triterpene acetates (8%) and 1,3-ditetradecanoyl-2-hexanoylglycerol (8%) as major components. Minor components are triterpene alcohols (3%) and nonacosan-10-ol (2%). The esters contain triterpene alcohol esters (19%) as well as esters of alkanols.     

Analysis of the labial gland secretions of the male bumblebee Bombus griseocollis (Hymenoptera: Apidae)

The labial gland secretions from males of the bumblebee Bombus (Separatobombus) griseocollis De Geer, a bumblebee exhibiting perching behaviour, were analysed by gas chromatography/mass spectrometry (GC/MS) in the electron impact and positive ion chemical ionization mode. The major compound of the complex mixture of alkenols, acetates, hydrocarbons, wax type esters and steroids is tetradecyl acetate, considerable amounts of hexadecyl, geranyllinaloyl, geranylgeranyl, docosyl, tetracosenyl and hexacosenyl acetate were also found. 1,3-Tetradecanediol diacetate, detected as a minor component, has not yet been identified in male bumblebee labial gland secretions. Besides small amounts of primary alcohols (tetradecanol and hexadecanol) the tertiary alcohol geranyllinalool (3,7,11,15-tetramethyl-hexadeca-1,6,10,14-tetraene-3-ol) was also present. The primary alcohols were also present as esters of butanoic, dodecanoic, tetradecanoic, and hexadecanoic acid. Besides the usual mixture of un- and mono-unsaturated straight chain hydrocarbons, the labial gland contains the isoprenoid hydrocarbons beta-springene [(6E, 10E)-7,11,15-trimethyl-3-methylene-hexadeca-1,6,10,14-tetraene] and two isomers of a-springene [(3Z,6E,10E)- and (3E,6E,10E)-3,7,11,15-tetramethyl-hexadeca-1,3,6,10,14-pentaene]. The close relationship in chemical composition in male bumblebees with perching and flight pass behaviour is discussed.     

Asymmetric synthesis of (<Emphasis Type="Italic">R</Emphasis>)-1,3-butanediol from 4-hydroxy-2-butanone by a newly isolated strain <Emphasis Type="Italic">Candida krusei</Emphasis> ZJB-09162

Biocatalytic asymmetric preparation of (R)-1,3-butanediol has been attracting much attention in pharmaceuticals industry. A new ideal strain, ZJB-09162, which exhibited high reduction activity and excellent (R)-stereospecificity towards 4-hydroxy-2-butanone, has been successfully isolated from soil samples. Based on morphology, physiological tests (API 20 C AUX), and 5.8S-ITS sequence, the isolate was identified as Candida krusei. Kinetic characterization demonstrated that carbonyl reductase from C. krusei ZJB-09162 preferred NADH to NADPH as cofactor, indicating it might be a new carbonyl reductase. (R)-1,3-Butanediol was produced in 19.8 g/L, 96.6% conversion, and 99.0% ee at optimal pH 8.5, 35 °C with a 2:1 molar ratio of glucose to 4H2B. In order to achieve higher product titer, the substrate loading was optimized in fixed catalysts and fixed substrate/catalysts ratio mode. The bioreduction of 4-hydroxy-2-butanone at a concentration of 45.0 g/L gave (R)-1,3-butanediol in 38.7 g/L and 83.9% conversion. Therefore, C. krusei ZJB-09162 was, for the first time, proven to be a promising biocatalyst for enzymatic preparation of (R)-1,3-butanediol.     

Fermentation of glycerol to 1,3-propanediol and 2,3-butanediol by Klebsiella pneumoniae

Klebsiella pneumoniae was shown to convert glycerol to 1,3-propanediol, 2,3-butanediol and ethanol under conditions of uncontrolled pH. Formation of 2,3-butanediol starts with some hours' delay and is accompanied by a reuse of the acetate that was formed in the first period. The fermentation was demonstrated in the type strain of K. pneumoniae, but growth was better with the more acid-tolerant strain GT1, which was isolated from nature. In continuous cultures in which the pH was lowered stepwise from 7.3 to 5.4, 2,3-butanediol formation started at pH 6.6 and reached a maximum yield at pH 5.5, whereas formation of acetate and ethanol declined in this pH range. 2,3-Butanediol and acetoin were also found among the products in chemostat cultures grown at pH 7 under conditions of glycerol excess but only with low yields. At any of the pH values tested, excess glycerol in the culture enhanced the butanediol yield. Both effects are seen as a consequence of product inhibition, the undissociated acid being a stronger trigger than the less toxic diols and acid anions. The possibilities for using the fermentation type described to produce 1,3-propanediol and 2,3-butanediol almost without by-products are discussed. Received: 4 February 1998 / Received revision: 30 March 1998 / Accepted: 13 April 1998     

Studies on purification of 1,3-propanediol by molecular distillation

The separation of 1,3-propanediol using molecular distillation has been studied. The effects of operating temperature and feed flow rate through a sequential distillation strategy were investigated. The optimal experimental temperature was at 70°C for separating 1,3-propanediol and the by-product 2,3-butanediol. Meanwhile, the volume flow rate was 10 mL/min. As a result, the recovery of 1,3-propanediol and 2,3-butanediol were 87.6 and 87.5%, respectively. Furthermore, the integrated separation characteristic of 1,3-propanediol was evaluated through macrolevel and micro-level models. The separation factors of 1,3-propanediol versus 2,3-butanediol and glycerol were 0.11 and 1.07, respectively, affirming that the separation of 1,3-propanediol by molecular distillation was feasible.     

Selective monoacetylation of diol compounds by Aspergillus niger lipase

Summary The primary monoesters of diol compounds were formed exclusively in the reaction with vinyl acetate and Aspergillus niger lipase for 24~72 h. Various diol compounds which included 1,3-butanediol, 1,4-butanediol, 1,5-hexanediol, 1-phenyl-1,2-ethanediol, 1-phenyl-1,3-propanediol, 2, 3, or 4-hydroxybenzyl alcohol, methyl 2, 3-O-acetyl-D-glycopyranosides and phenyl 1-thio--D-xylopyranoside have been examined and showed nearly 100% regioselectivity.     

Site-Specific and Asymmetric Hydrolysis of Prochiral 2-Phenyl-1,3-propanediol Diacetate by a Bacterial Esterase from an Isolated Strain

Bacillus cereus 809A and Burkholderia sp. 711C were isolated from soil. These strains demonstrate hydrolysis activity towards prochiral 2-phenyl-1,3-propanediol diacetate and accumulated the corresponding chiral monoacetates into the reaction mixture. When 2-phenyl 1,3-propanediol diacetate was used as a substrate, the produced monoacetates with Burkholderia sp. 711C were obtained in a racemic form but that produced by Bacillus cereus 809A showed an excess of the (S)-form. The resting cell reaction revealed that for Bacillus cereus 809A, there was an enrichment of one of the enantiomers of the monoacetate such that the enantiomeric excess (e.e.) of the (S)-form was over 95%. The purified enzyme from Bacillus cereus 809A hydrolyzed diacetate to monoacetate, and the e.e. value of the (S)-form increased by prolonged reaction in a way similar to the resting cell reaction. From N-terminal amino acids, this esterase is conserved in some strains of Bacillus for which the genomic sequences have been reported.     

Glass-forming tendency and stability of the amorphous state in the aqueous solutions of linear polyalcohols with four carbons. I. Binary systems water-polyalcohol

All the aqueous solutions of linear saturated polyalcohols with four carbons have been investigated at low temperature. Only ice has been observed in the solutions of 1,3-butanediol and 1,2,3- and 1,2,4-butanetriol. For same solute concentration, the glass-forming tendency on cooling is highest with 2,3-butanediol, where it is comparable to that with 1,2-propanediol, the best solute reported to date. However, the quantity of ice and hydrate crystallized is particularly high on slow cooling or on subsequent rewarming. The highest stability of the amorphous state is observed on rewarming the 1,2-butanediol and 1,3-butanediol solutions. With respect to this property, these compounds come just after 1,2-propanediol and before all the other compounds studied so far. They are followed by dimethylsulfoxide and 1,2,3-butanetriol. The glass-forming tendency of the 1,3-butanediol solutions is also very high; it is third only to that of 1,2-propanediol and 2,3-butanediol. The glass-forming tendency is a little smaller with 1,2-butanediol, but it is cubic instead of ordinary hexagonal ice which crystallizes on cooling rapidly with 35% 1,2-butanediol. Cubic ice is thought to be innocuous. A gigantic glass transition is observed with 45% of this strange solute. 1,4-Butanediol, 45% also favors cubic ice greatly. Therefore, 1,2- and 1,3-butanediol with comparable physical properties are perhaps as interesting as 1,2-propanediol for cryopreservation of cells or organs by complete vitrification. Together with 1,2-propanediol, 1,2- and 1,3-butanetriol, 1,2,3-butanetriol, and perhaps 2,3-butanediol provide an interesting battery of solutions for cryopreservation by vitrification.     

Short chain flavour esters synthesis by microbial lipases

Summary The peparative synthesis of 35 short chain flavour esters by lipases fromMucor miehi, Aspergillus sp.,Candida rugosa andRhizopus arrhizus was investigated in organic media. Acetic, propionic, butyric, valeric and caproic acids, as well as methanol, ethanol, butanol, i-pentanol, hexanol, citronellol and geraniol were used as substrates. Most of the esters were synthesized in good yield by at least one of the lipase preparations tested. Different conversion yields were observed according to the lipase specificity toward the acid or the alcohol moiety of the ester. Methyl- and ethyl acetates were also produced by changing the organic solvent. Enzymatic catalysis in organic solvent is thought to be a valuable method for preparative synthesis of flavour esters.     

Synthesis of All Four Possible Stereoisomers of 1-Phenyl-2,3-butanediol and Both Enantiomers of 3-Hydroxy-4-phenyl-2-butanone to Determine the Absolute Configuration of the Natural Constituents

Enantioselective syntheses of all the possible stereoisomers of 1-phenyl-2,3-butanediol (erythro isomer 1 and threo isomer 2) and of 3-hydroxy-4-phenyl-2-butanone 3, the odor components of wisteria flowers, was accomplished via Sharpless asymmetric epoxydation. The absolute configurations of 1–3 were determined by an HPLC analysis of the corresponding MTPA esters of synthetic samples.     

Effects of over-expression of glycerol dehydrogenase and 1,3-propanediol oxidoreductase on bioconversion of glycerol into 1,3-propandediol by <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> under micro-aerobic conditions

Glycerol dehydrogenase (GDH) and 1,3-propanediol (1,3-PD) oxidoreductase had been proved two key enzymes for 1,3-PD production by Klebsiella pneumoniae. Fed-batch fermentations of the recombinant K. pneumoniae strains, over-expressing the two enzymes individually, were carried out under micro-aerobic conditions, and the behaviors of the recombinants were investigated. Results showed that over-expression of 1,3-PD oxidoreductase did not affect the concentration of 1,3-PD. However, it enhanced the molar yield from 50.6 to 64.0% and reduced the concentration of by-products. Among them, the concentrations of lactic acid, ethanol and succinic acid were decreased by 51.8, 50.6 and 47.4%, respectively. Moreover, in the recombinant the maximal concentration of 3-hydroxypropionaldehyde decreased by 73.6%. Over-expression of GDH decreased the yield of ethanol and 2,3-butanediol, meanwhile it increased the concentration of acetic acid. No significant changes were observed both in 1,3-PD yield and glycerol flux distributed to oxidative branch.