Enzymatic production of glycerol acetate from glycerol

In this study, we report the enzymatic production of glycerol acetate from glycerol and methyl acetate. Lipases are essential for the catalysis of this reaction. To find the optimum conditions for glycerol acetate production, sequential experiments were designed. Type of lipase, lipase concentration, molar ratio of reactants, reaction temperature and solvents were investigated for the optimum conversion of glycerol to glycerol acetate. As the result of lipase screening, Novozym 435 (Immobilized Candida antarctica lipase B) was turned out to be the optimal lipase for the reaction. Under the optimal conditions (2.5 g/L of Novozym 435, 1:40 molar ratio of glycerol to methyl acetate, 40 °C and tert-butanol as the solvent), glycerol acetate production was achieved in 95.00% conversion.     

Potentiometric quantitation of glycerol using immobilized glycerol dehydrogenase

Glycerol dehydrogenase was immobilized in polyacrylamide gel layered over a small platinum screen and used to catalyze the oxidation of glycerol. In the presence of NAD(+) and potassium ferricyanide, the coupling reaction generated a measurable electrical potential which was found to be Nernstian with respect to the glycerol concentration range of 10(-4)M to 10(-1)M. The reproducibility of the measurement and the optimal conditions for glycerol determination were described.     

Distribution of glycerol dehydrogenase and glycerol kinase activity in halophilic archaea

Abstract A constitutive NAD⁺-dependent glycerol dehydrogenase activity was detected in Halobacterium salinarium and Halobacterium cutirubrum . Optimal activity was found at 3 M KCl and pH 8–10. No glycerol dehydrogenase activity could be demonstrated in representatives of the genera Haloferax and Haloarcula , even when grown in the presence of glycerol, or in Halobacterium saccharovorum and Halobacterium sodomense . Glycerol kinase activity was shown to be present constitutively in all halophilic archaea examined. The finding that glycerol dehydrogenase is found only in part of the halophilic arachaea makes dihydroxyacetone an improbable candidate as the precursor for the glycerol moiety of halobacterial lipids.     

Hyperhydration with glycerol solutions

Glycerol was tested as an agent to promote hyperhydration of male and female subjects. Series I experiments involved ingesting 0.5, 1.0, or 1.5 g glycerol/kg body wt and within 40 min drinking 0.1% NaCl, 21.4 ml/kg. In series II, 1.0 g glycerol/kg body wt was ingested at time 0, and 25.7 ml/kg of 0.1% NaCl was ingested over a 3.5-h period. Experiments were of 4-h duration and included controls without glycerol as each subject served as his/her control. Blood samples were taken at 40- or 60-min intervals for hemoglobin (Hb), hematocrit (Hct), plasma osmolality, glycerol, and multiple blood chemistry analyses. Urine was collected at 60-min intervals. Glycerol ingestion increased plasma osmolality for 2 h and reduced the total 4-h urine volume. There were no significant changes in Hb or Hct as a result of the glycerol or excess fluid intake. This study demonstrates that glycerol plus excess fluid intake can produce hyperhydration for at least 4 h.     

Aquaporins and glycerol metabolism

The discovery of aquaporin (AQP) has made a great impact on life sciences. AQPs are a family of homologous water channels widely distributed in plants, unicellular organisms, invertebrates, and vertebrates. So far, 13 AQPs have been identified in human. AQP3, 7, 9, and 10 are subcategorized as aquaglyceroporins which permeabilize glycerol as well as water. Many investigators have demonstrated that AQPs play a crucial role in maintaining water homeostasis, but the physiological significance of some AQPs as a glycerol channel is not fully understood. Adipose tissue is a major source of glycerol and glycerol is one of substrates for gluconeogenesis. This review focuses on recent studies of glycerol metabolism through aquaglyceroporins, and briefly discusses the importance of glycerol channel in adipose tissues and liver.     

Aquaporins and glycerol metabolism

The discovery of aquaporin (AQP) has made a great impact on life sciences. AQPs are a family of homologous water channels widely distributed in plants, unicellular organisms, invertebrates, and vertebrates. So far, 13 AQPs have been identified in human. AQP3, 7, 9, and 10 are subcategorized as aquaglyceroporins which permeabilize glycerol as well as water. Many investigators have demonstrated that AQPs play a crucial role in maintaining water homeostasis, but the physiological significance of some AQPs as a glycerol channel is not fully understood. Adipose tissue is a major source of glycerol and glycerol is one of substrates for gluconeogenesis. This review focuses on recent studies of glycerol metabolism through aquaglyceroporins, and briefly discusses the importance of glycerol channel in adipose tissues and liver.     

Lipase-catalyzed synthesis of glycerol carbonate from renewable glycerol and dimethyl carbonate through transesterification

Glycerol carbonate is a key multifunctional compound employed as solvent, additive, monomer, and chemical intermediate. Enzymatic synthesis of glycerol carbonate from renewable starting materials (glycerol and dimethyl carbonate) was successfully achieved by immobilized lipase from Candida antarctica (CALB, Novozym 435). Addition of molecular sieves as scavenger for the removal of methanol, which was generated from dimethyl carbonate during the reaction, accelerated a reaction rate. After the optimization, the equimolar use of glycerol and dimethyl carbonate in the Novozym 435-catalyzed reaction yielded a glycerol carbonate with almost quantitative yield. The resulting glycerol carbonate from 60 °C reaction has shown the low enantiomeric excess (13% ee) as configuration of (R)-enantiomer.     

Renal glycerol metabolism and the distribution of glycerol kinase in rabbit nephron.

Glycerol and dihydroxyacetone are metabolized by rabbit kidney-cortex tubules, isolated by collagenase treatment. Half-maximal concentrations of both substrates were determined with regard to uptake rates and product formations. Maximal uptake rates were 643 and 329 mumol/h per g of protein for dihydroxyacetone and glycerol respectively. Glucose and lactate were found as major metabolic products. Glycerol kinase, the enzyme catalysing the first step in renal glycerol and dihydroxyacetone metabolism, was measured radiochemically as described by Newsholme, Robinson & Taylor [(1967) Biochim, Biophys. Acta 132, 338-346] and adapted for studies of the localization of this enzyme along the different structures of rabbit nephron. The results show that glycerol kinase is located exclusively in the proximal segments, i.e. the proximal convoluted tubules and the pars recta, but is negligible in the other structures studied. The activities were close to the maximal dihydroxyacetone uptake rates measured in tubule suspensions.     

Lipase-catalyzed synthesis of glycerol carbonate from renewable glycerol and dimethyl carbonate through transesterification

Glycerol carbonate is a key multifunctional compound employed as solvent, additive, monomer, and chemical intermediate. Enzymatic synthesis of glycerol carbonate from renewable starting materials (glycerol and dimethyl carbonate) was successfully achieved by immobilized lipase from Candida antarctica (CALB, Novozym 435). Addition of molecular sieves as scavenger for the removal of methanol, which was generated from dimethyl carbonate during the reaction, accelerated a reaction rate. After the optimization, the equimolar use of glycerol and dimethyl carbonate in the Novozym 435-catalyzed reaction yielded a glycerol carbonate with almost quantitative yield. The resulting glycerol carbonate from 60 °C reaction has shown the low enantiomeric excess (13% ee) as configuration of (R)-enantiomer.     

X-Ray structure of glycerol kinase complexed with an ATP analog implies a novel mechanism for the ATP-dependent glycerol phosphorylation by glycerol kinase.

Glycerol kinase (GK) catalyzes the Mg-ATP-dependent phosphorylation of glycerol which yields glycerol 3-phosphate. The 2.8 A new crystal structure of GK complexed with an ATP analog revealed an unexpected position of the gamma-phosphoryl group, which was 7.2 A distant from the 3-hydroxyl group of glycerol, 5.5 A away from the 3-phosphate of the product (glycerol 3-phosphate) and is stabilized by a beta-hairpin structure. Based on the presented crystal structure and the previously determined structures of GK product complexes, we propose a 3-D model of a nucleophilic in-line transfer mechanism for the ATP-dependent phosphorylation of glycerol by GK.     

Proportional activities of glycerol kinase and glycerol 3-phosphate dehydrogenase in rat hepatomas.

The activities of glycerol 3-phosphate dehydrogenase (EC 1.1.1.8), glycerol kinase (EC 2.7.1.30), lactate dehydrogenase (EC 1.1.1.27), "malic' enzyme (L-malate-NADP+ oxidoreductase; EC 1.1.1.40) and the beta-oxoacyl-(acyl-carrier protein) reductase component of the fatty acid synthetase complex were measured in nine hepatoma lines (8 in rats, 1 in mouse) and in the livers of host animals. With the single exception of Morris hepatoma 16, which had unusually high glycerol 3-phosphate dehydrogenase activity, the activities of glycerol 3-phosphate dehydrogenase and glycerol kinase were highly correlated in normal livers and hepatomas (r = 0.97; P less than 0.01). The activities of these two enzymes were not strongly correlated with the activities of any of the other three enzymes. The primary function of hepatic glycerol 3-phosphate dehydrogenase appears to be in gluconeogenesis from glycerol.     

Optimization of glycerol feeding for clavulanic acid production by Streptomyces clavuligerus with glycerol feeding

Glycerol at 10–20 g l^–1 increased clavulanic acid production by Streptomyces clavuligerus in shake-flask culture. The biosynthesis of clavulanic acid continued for longer by feeding glycerol and production increased to 250 mg l^–1 compared with 115 mg l^–1 without feeding. In fermenter batch culture, degradation of clavulanic acid began after 72 h. With glycerol feeding in fed-batch culture, clavulanic acid production was not only increased further to about 280 mg l^–1 but also remained stable up to 130 h.     

Biosynthesis of glycerol carbonate from glycerol by lipase in dimethyl carbonate as the solvent

Glycerol carbonate was synthesized from renewable glycerol and dimethyl carbonate using lipase in solvent-free reaction system in which excess dimethyl carbonate played as the reaction medium. A variety of lipases have been tested for their abilities to catalyze transesterification reaction, and Candida antartica lipase B and Novozyme 435 exhibited higher catalytic activities. The silica-coated glycerol with a 1:1 ratio was supplied to prevent two-phase formation between hydrophobic dimethyl carbonate and hydrophilic glycerol. Glycerol carbonate was successfully synthesized with more than 90% conversion from dimethyl carbonate and glycerol with a molar ratio of 10 using Novozyme 435-catalyzed transesterification at 70 °C. The Novozyme 435 [5% (w/w) and 20% (w/w)] and silica gel were more than four times recycled with good stability in a repeated batch operation for the solvent-free synthesis of glycerol carbonate.     

Pathways of glyceride glycerol synthesis

Isolated rat liver parenchymal cells were incubated for various periods with [U-(14)C,2-(3)H]glycerol and the radioisotopic yields in the major products were determined, as well as the (3)H/(14)C ratios in glyceride glycerol and intracellular glycerol phosphate. Under the conditions used (0.1mm-glycerol+10mm-l-lactate or 10mm-glycerol as substrates), only small differences were found between these (3)H/(14)C ratios. The results suggest a minor role for a pathway of glyceride glycerol synthesis involving reduction of acylated dihydroxyacetone phosphate, under these experimental conditions.     

Activation of aspartase by glycerol

Aspartase [EC 4.3.1.1] of Escherichia coli, which exhibits a sigmoidicity in the substrate saturation profile at alkaline pH, was markedly activated by 10–20% glycerol at low substrate concentrations and pH 8.5. In contrast, no activation, but an inhibition was observed at pH 7.0 throughout the substrate concentrations tested. The activation profile of the enzyme as a function of glycerol concentration was considerably influenced by L-aspartate concentration. Neither alteration of the cooperative nature of the enzyme nor subunit dissociation was associated with the activation. Besides glycerol, ethylene glycol, propylene glycol, dimethylsulfoxide, and dioxane also activated the enzyme.