Cryoprotection of red blood cells by 1,3-butanediol and 2,3-butanediol

1,3-Butanediol and 2,3-butanediol have been used in buffered solutions with 20, 30, or 35% (w/w) alcohol to cool erythrocytes to -196 degrees C at different cooling rates between 1 to 3500 degrees C/min, followed by slow or rapid rewarming. 1,3-butanediol shows the same shapes of red blood cell survival curves as 1,2-propanediol. Having nearly the same physical properties, they have comparable effects on cell survival. The classical maximum of survival for intermediate cooling rates and an increase for the highest cooling rates are observed. This increase seems to be correlated with the glass-forming tendency of the solution. After the fastest cooling rates, a warming rate of 5000 degrees C/min is sufficient to avoid cell damage, but a warming rate of 100-200 degrees C/min is not. Yet both of these rates would be insufficient to avoid the intracellular ice crystallization on warming. The damage on warming after fast cooling seems once again to be correlated with the transition from cubic to hexagonal ice. For all our results, 1,3-butanediol is like a "second" 1,2-propanediol and could be useful as a cryoprotectant for preservation by total vitrification. 2,3-Butanediol always gives extremely low survival rates, though it presents good physical properties. The crystallization of its hydrate seems to be lethal on cooling or on rewarming.     

Experimental dissection of devitrification in aqueous solutions of 1,3-butanediol

Devitrification is a major problem which must be overcome for successful organ cryopreservation. Devitrification can be initiated on fracture planes and on bubbles, but the focus of attention here is on devitrification by ordinary heterogeneous and homogeneous mechanisms, which are the most relevant for organ preservation by vitrification. The purpose of the present studies was to define the devitrification process: to determine nucleation rates, ice-crystal growth rates, and the distribution of ice-crystal size and to evaluate the applicability of existing quantitative models of these processes which have successfully approximated the behavior of other aqueous systems. The present work was done using differential scanning calorimetry and cryomicroscopy. The amount of ice formed has been estimated for highly concentrated solutions. Kinetic parameters are presented here for isothermal conditions and continuous heating rate experiments. The classical theory based on the Johnson-Avrami equation has been evaluated and the results are compared with the theory of Boutron. The agreement is good for the continuous heating rate conditions, but results differ for the isothermal conditions.     

Nutritional and metabolic studies in humans with 1,3-butanediol.

Three studies of the role of 1,3-butanediol (BD) in human nutrition are described. Isocaloric substitution of BD for starch in the diets of volunteers caused less negative nitrogen balance. Ingestion of urea also decreased negative nitrogen balance, and the effect of BD plus urea in the diets seemed to be additive. No effects were detected on many blood parameters measured during and after the study, except that BD feeding decreased blood glucose significantly. 1,3-Butanediol was shown to be a nontoxic metabolite providing a source of calories for human nutrition. In a second study, ingestion of BD was shown to cause slight increases in serum insulin and growth hormone concentrations in the fasting state. We next studied the effects of prior ingestion of BD on serum insulin, growth hormone, glucose, and lipids during glucose tolerance tests. No significant differences in these parameters were noted when prior ingestion of sucrose or an isocaloric quantity of BD were compared. Possible mechanisms whereby ingestion of BD spared nitrogen and caused decreased blood glucose are discussed.     

Use of 1,3-butanediol for lactation and growth in cattle.

These studies have been designed to test whether 1,3-butanediol (BD) alleviates milk fat depression in lactating cows, to observe physiological changes in blood and rumen constituents when BD is fed to cows or growing cattle, and to test the effects of BD on growth rates and feed efficiency in growing cattle. In trials with lactating cows, milk fat percentage and total fat production were higher for cows fed BD than for controls. Feeding BD to either cows or growing cattle had no consistent effect on rumen pH or relative concentrations of rumen volatile fatty acids. 1,3-Butanediol feeding had little effect on blood glucose concentrations. Feeding more than 4% BD in diets sometimes caused increased concentrations of blood ketones. In trials where growing cattle were fed 4% BD, rates of gain and feed efficiency were at least as good as and often better than those of cattle fed the same diets without BD. Body composition was not significantly affected. 1,3-Butanediol can be utilized effectively as an energy source for cattle and causes no obvious problems with 4% in diets.     

Quantitation of 1,3-butanediol and its acidic metabolites by gas chromatography-mass spectrometry

A number of problems present themselves during the gas chromatographic-mass spectrometric assay of R,S-1,3-butanediol as its bis-tert-butyldimethylsilyl ether. To circumvent these problems, three labeled internal standards were synthesized: (i) R,S-1,3-[3,4-13C2]-butanediol, (ii) R,S-1,3-[1,1,3-2H3]butanediol, and (iii) R,S-1,3-[1,1,3-2H3,3,4-13C2]butanediol. The availability of internal standards with different degrees of labeling allows (i) assaying of either unlabeled or 13C-labeled R,S-1,3-butanediol and (ii) analysis of 1,3-butanediol in either blood or urine samples. Reproducible standard curves were obtained using both electron impact and ammonia chemical ionization modes. The latter provides greater sensitivity and a lower limit of detection (5 microM). We have also designed an indirect assay of S-3-hydroxybutyrate, a catabolite of R,S-1,3-butanediol, which is difficult to analyze by conventional methods. This assay relies on the difference between (i) the concentration of R,S-3-hydroxybutyrate assayed by gas chromatography-mass spectrometry and (ii) the concentration of R-3-hydroxybutyrate assayed enzymatically.     

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     

Industrial production of (R)-1,3-butanediol by new biocatalysts

We have developed the economical and convenient biocatalytic process for the preparation of (R)-1,3-butanediol (BDO) by stereo-specific microbial oxido-reduction on an industrial scale. (R)-1,3-BDO is an important chiral synthon for the synthesis of various optically active compounds such as azetidinone derivatives lead to penem and carbapenem antibiotics.

We studied on two approaches to obtain (R)-1,3-BDO. The first approach was based on enzyme-catalyzed asymmetric reduction of 4-hydroxy-2-butanone; the second approach was based on enantio-selective oxidation of the undesired (S)-1,3-BDO in the racemate. As a result of screening for yeasts, fungi and bacteria, the enzymatic resolution of racemic 1,3-BDO by the Candida parapsilosis IFO 1396, which showed differential rates of oxidation for two enantiomers, was found to be the most practical process to produce (R)-1,3-BDO with high enantiomeric excess and yield.

We characterized the (S)-1,3-BDO dehydrogenase purified from a cell-free extract of C. parapsilosis. This enzyme was found to be a novel secondary alcohol dehydrogenase (CpSADH). We have attempted to clone and characterize the gene encoding CpSADH and express it in Escherichia coli. The CpSADH activity of a recombinant E. coli strain was more than two times higher than that of C. parapsilosis. The production yield of (R)-1,3-BDO from the racemate increased by using the recombinant E. coli strain. Interestingly, we found that the recombinant E. coli strain catalyzed the reduction of ethyl 4-chloro-3-oxo-butanoate to ethyl (R)-4-chloro-3-hyroxy-butanoate with high enantiomeric excess.     

一株转化4-羟基-2-丁酮为1，3-丁二醇微生物菌株的筛选及鉴定

从本实验室保藏的86株酵母菌株中筛选到20株利用4-羟基-2-丁酮（4H2B）生产1,3-丁二醇（1,3-BDo）的菌株,当转化液中4H2B浓度为10g/L时,经初步测定1,3．BDO含量,其中菌株32 1,3-BDO产量最高达6．5g/L,具有较好的1,3-BDO生产潜力。对其进行形态学和常规生理生化鉴定实验,并结合18S rDNA基因分析,比对结果表明,菌株32与Pichia farinosa strain DC 3343相似性达99．8％,为Pichia farinosa的一个亚种。     

Present state and perspective of downstream processing of biologically produced 1,3-propanediol and 2,3-butanediol

1,3-Propanediol and 2,3-butanediol are two promising chemicals which have a wide range of applications and can be biologically produced. The separation of these diols from fermentation broth makes more than 50% of the total costs in their microbial production. This review summarizes the present state of methods studied for the recovery and purification of biologically produced diols, with particular emphasis on 1,3-propoanediol. Previous studies on the separation of 1,3-propanediol primarily include evaporation, distillation, membrane filtration, pervaporation, ion exchange chromatography, liquid–liquid extraction, and reactive extraction. Main methods for the recovery of 2,3-butanediol include steam stripping, pervaporation, and solvent extraction. No single method has proved to be simple and efficient, and improvements are especially needed with regard to yield, purity, and energy consumption. Perspectives for an improved downstream processing of biologically produced diols, especially 1,3-propanediol are discussed based on our own experience and recent work. It is argued that separation technologies such as aqueous two-phase extraction with short chain alcohols, pervaporation, reverse osmosis, and in situ extractive or pervaporative fermentations deserve more attention in the future.     

Influence of 1,3-butanediol on blood glucose concentration and pancreatic insulin content of streptozotocin-diabetic rats.

Male rats were made diabetic by intravenous administration of 75 mg/kg of streptozotocin and were fed, via a pair-feeding regimen, high-fat diets +/- 1,3-butanediol (BD) at 13.5 and 27% of the dietary calories for 30 days and 31 days, respectively. 1,3-Butanediol was added to the diets primarily as a replacement for fat. Food consumption and rat weight were recorded daily. Whole blood glucose concentrations were determined weekly. At sacrifice, liver, pancreas and epididymal fat pads were excised and blood samples were collected. Liver was analyzed for protein and lipid; pancreas was weighed and analyzed for insulin; fat pads were weighed and discarded; and blood was analyzed for glucose and lipid. The 13.5% BD diet increased the beta-hydroxybutyrate, acetoacetate and cholesterol concentrations, decreased the glucose concentration in blood, and increased the insulin content of the pancreas. The BD diets did not affect the concentrations of phospholipid, triglyceride, cholesterol and fatty acid in the liver; fatty acid concentrations in the blood; or the epididymal fat pad weight. The results suggest that BD produced a slight amelioration of the diabetic condition, which may have resulted from an increased capacity of the pancreas to synthesize insulin. In addition, the data provide further evidence suggesting that in the rat BD is oxidized to the ketone bodies, beta-hydroxybutyrate and acetoacetate.     

Engineering Cupriavidus necator H16 for the autotrophic production of (R)-1,3-butanediol

Butanediols are widely used in the synthesis of polymers, specialty chemicals and important chemical intermediates. Optically pure R-form of 1,3-butanediol (1,3-BDO) is required for the synthesis of several industrial compounds and as a key intermediate of β-lactam antibiotic production. The (R)-1,3-BDO can only be produced by application of a biocatalytic process. Cupriavidus necator H16 is an established production host for biosynthesis of biodegradable polymer poly-3-hydroxybutryate (PHB) via acetyl-CoA intermediate. Therefore, the utilisation of acetyl-CoA or its upstream precursors offers a promising strategy for engineering biosynthesis of value-added products such as (R)-1,3-BDO in this bacterium. Notably, C. necator H16 is known for its natural capacity to fix carbon dioxide (CO₂) using hydrogen as an electron donor. Here, we report engineering of this facultative lithoautotrophic bacterium for heterotrophic and autotrophic production of (R)-1,3-BDO. Implementation of (R)-3-hydroxybutyraldehyde-CoA- and pyruvate-dependent biosynthetic pathways in combination with abolishing PHB biosynthesis and reducing flux through the tricarboxylic acid cycle enabled to engineer strain, which produced 2.97 g/L of (R)-1,3-BDO and achieved production rate of nearly 0.4 Cmol Cmol⁻¹ h⁻¹ autotrophically. This is first report of (R)-1,3-BDO production from CO₂.     

Dog model of therapeutic ketosis induced by oral administration of R,S-1,3-butanediol diacetoacetate

A high-fat, almost carbohydrate-free diet is used in children with intractable epilepsy to help control seizures by inducing a permanent state of ketosis. Esters of ketone bodies have been previously studied for their potential as parenteral and enteral nutrients. We tested in conscious dogs whether ketosis could be induced by repeated ingestion of R,S-1,3-butanediol diacetoacetate with or without carbohydrates. This ester is a water-soluble precursor of ketone bodies. Two constraints were imposed on this preclinical study: The rate of ester administration was limited to one half of the daily caloric requirement and to one half of the capacity of the liver to oxidize butanediol derived from ester hydrolysis. Under these conditions, the level of ketosis achieved in this dog model (0.8 mM) was lower than the level measured in children whose seizures were controlled by the ketogenic diet (1-3 mM). However, because humans may have a lower capacity for ketone body utilization than dogs, the doses of R,S-butanediol diacetoacetate used in the present study might induce higher average ketone body concentrations in humans than in dogs.     

High production of 2,3-butanediol from glycerol without 1,3-propanediol formation by Raoultella ornithinolytica B6

Applied Microbiology and Biotechnology - Conversion of crude glycerol derived from biodiesel processes to value-added chemicals has attracted much attention. Herein, Raoultella ornithinolytica B6...     

2,3-丁二醇代谢途径关键酶基因敲除对克雷伯氏菌发酵产1,3-丙二醇的影响

2,3-丁二醇是克雷伯氏菌发酵产1,3-丙二醇的主要副产物,为减少2,3-丁二醇的产生,利用Red重组技术对克雷伯氏菌2,3-丁二醇合成途径关键酶基因budC和budA进行了敲除。突变株发酵性能实验结果表明,所获得的两株突变株生长性能受到不同程度的影响;budC基因的缺失使菌株1,3-丙二醇产量提高了10%,2,3-丁二醇降低为原来的70%,而budA基因缺失则使菌株无2,3-丁二醇和1,3-丙二醇的产生,但乳酸、琥珀酸、乙醇和乙酸的产量较出发菌株都有明显增长。通过进一步对budC基因缺失菌株主要产物分析,推测在该菌中存在2,3-丁二醇回补途径,这一结果为低副产物克雷伯氏菌的改造提供了新依据。     

Lipase-catalyzed enantiotope selective acetylation of 2-acyloxypropane-1,3-diols. Influence of the acyl moiety on the selectivity

Preparation and lipase-catalyzed enantiotope selective acetylation of the prochiral 2-acyloxypropane-1,3-diols (1a–h) including sulfonic ester (1a–c) and carboxylic ester (1d–h) series is described. A strong influence of the acyl moiety in these diols on the enantiotope selectivity of the porcine pancreatic lipase (PPL)-catalyzed reaction with vinyl acetate was observed. The best results were achieved with the 2-(4-methylbenzoyl)oxy- and cyclohexanecarbonyloxypropane-1,3-diols (1g and 1h) resulting in acetylated products (2g) of ≥98% e.e. in 77% yield and (2h) of 95% e.e. in 66% yield, respectively.     

Lipase-catalyzed monoacylation of fructose

Summary In a one-pot-process the lipase-catalyzed monoacylation of fructose with stearic acid in n-hexane was achieved when phenylboronic acid was used as solubilizing agent.     

Lipase-catalyzed synthesis of L-phenylalanyl-D-glucose

Regioselective synthesis of L-phenylalanyl ester of D-glucose with unprotected L-phenylalanine and D-glucose was carried out in organic solvents using lipases from Rhizomucor miehei (RML) and porcine pancreas (PPL). The reaction was investigated in terms of free unprotected L-phenylalanine, D-glucose, RML and PPL concentrations, buffer salts (pH and concentration), enzyme reusability and incubation period. Under the experimental conditions employed, both the enzymes exhibited good esterification potentialities, with RML exhibiting better conversions (maximum yield, 98%) than PPL (maximum yield, 75.6%). Reactions in the presence of buffer salts gave about 10% higher yields than those in their absence. Two-dimensional heteronuclear single quantum coherence transfer (HSQCT) NMR spectral analysis confirmed the formation of five diastereomeric esters: three different L-phenylalanyl-D-glucose monoesters (6-O: 24.1%, 3-O: 23.3% and 2-O: 19.2%) and two different diesters (2,6-di-O: 16.6% and 3,6-di-O: 16.8%) in an esterification yield of 92.4%.     

Lipase-catalyzed polyester synthesis

Summary The effects of residual enzyme activity, stepwise addition of lipase at different reaction times, and enzyme quantity in direct polyesterification of sebacic acid and 1,4-butanediol catalyzed by a lipase from Rhizomucor miehei were investigated. Although the lipase activity dropped sharply in the beginning period of the reaction, the molar mass of the polyester increased rapidly, up to 39,000 g mol^–1 in 72 h. The residual lipase activity (hydrolytic) was only 14 %. Stepwise addition of lipase did not improve polyester synthesis. Highest mass average molar mass of 56,000 g mol^–1 was obtained with 0.125 g of lipase (28.5%, w/w) in 5 days.     

Lipase-catalyzed synthesis of partial glyceride

Summary Mucor miehei (IM 20) and Candida antarctica (SP 382) lipases were used for esterification of free fatty acids in the absence of organic solvent or transesterification of fatty acid methyl esters in hexane with isopropylidene glycerols. Acid catalyzed cleavage of the isopropylidene groups resulted in the formation of monoacyl glycerol (MAG) and diacyl glycerol (DAG). Both oleic (18:1 n-9) and eicosapentaenoic acid, EPA (20:5 n-3) were successfully incorporated into glycerides. Total acyl donor conversion ranged from 46.9 – 96.9% with MAG content of up to 88.5%.     

Lipase-catalyzed synthesis of aromatic polyesters

The enzymatic synthesis of aromatic polyesters by direct polyesterification between a diacid and a diol is described. The effects of the type of substrate, type and quantities of lipase, temperature, vacuum, and reaction time on the synthesis of aromatic polyesters were studied in detail. Among three lipases investigated, only Novozym 435 worked well for aromatic polyester synthesis. Temperature and vacuum played an important role in obtaining a high molar mass of the aromatic polyesters. Furthermore, with isophthalic acid and 1,6-hexanediol as substrates, the mass average molar mass of the polyester obtained increased with an increase in the lipase quantity up to 0.375 g (11.7%, w/w of total reactor contents). The mass average molar mass of the polyester was as high as 50000 g mol⁻¹ in 168 h, with a polydispersity of PD ≈ 1.4. Received 27 January 1998/ Accepted in revised form 19 May 1998