Enzymatic synthesis of hydroxycinnamic acid glycerol esters using type A feruloyl esterase from Aspergillus niger

We found that hydroxycinnamic acid (HA) glycerol esters such as 1-sinapoyl glycerol and 1-p-coumaroyl glycerol can be synthesized through a direct esterification reaction using a type A feruloyl esterase from Aspergillus niger. The water solubilities of HA glycerol esters were higher than those of the original chemicals. HA glycerol esters absorbed ultraviolet light and scavenged 1,1-diphenyl-2-picrylhydrazyl radicals.     

Heterologous expression, characterization and site-directed mutagenesis of cutinase CUTAB1 from Alternaria brassicicola

The cutinase CUTAB1 was cloned from a cutin induced culture of Alternaria brassicicola and heterologously expressed in Pichia pastoris under the control of the methanol-inducible AOX1 promoter. From a 400-ml culture, 36 mg of purified recombinant enzyme were obtained. Biochemical characterization revealed highest catalytic activity of the enzyme at 40°C and pH 7-9 using p-nitrophenyl palmitate (p-NPP) as substrate. Among several fatty acid methyl and ethyl esters, glycerol esters and p-nitrophenyl esters tested, CUTAB1 showed highest activity towards tributyrin (3,302 ± 160 U mg⁻¹) and the activity decreased with increase in chain length of the investigated esters. Lowest activity was found for p-NPP. Replacing Leu80, Leu181 and Ile183, respectively, by the smaller alanine in the hydrophobic binding loop of CUTAB1, drastically reduced the overall activity of the enzyme. On the other hand, mutation A84F located in the small helical flap of CUTAB1 significantly increased the activity of the enzyme towards longer chain substrates like p-NPP.     

Lysis of Bacillus subtilis Cells by Glycerol and Sucrose Esters of Fatty Acids

The lytic action of glycerol and sucrose esters of fatty acids with different carbon chain lengths on the exponentially growing cells of Bacillus subtilis 168 was investigated. Of each series of esters, glycerol dodecanoate and sucrose hexadecanoate were the most active. Lysis at 1 h after the addition of 0.1 mM glycerol dodecanoate or 20 μg of sucrose hexadecanoate per ml was 81 or 79%, respectively, as evaluated by the reduction in optical density. During this treatment a great loss of viability occurred that preceded lysis. The results that were obtained suggest that autolysis is induced by these esters. The esters caused morphological changes in the cells, but a seeming adaptation of the cells to esters was seen.     

Studies on a novel carbon source and cosolvent for lipase production by<Emphasis Type="Italic"> Candida rugosa</Emphasis>

Oleic acid esters were shown to be the best carbon source for both cell growth and lipase production by Candida rugosa. Use of a cosolvent, dodecane, in fermentations improved the solubility of solid substrates and increased oxygen solubility. This resulted in the highest lipase activity in batch fermentation with glycerol trioleate and dodecane. Lipase activity reached 77.1 units ml^–1.     

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%.     

Stereoselective Reduction of α- and β-Keto Esters with Aerobic Thermophiles,Bacillus Strains

The first example of stereoselective reduction with aerobic thermophiles is reported. Various α- and β-keto esters were reduced stereoselectively to the corresponding alcohols by the aerobic thermophiles, Bacillus strains. In particular, the reduction of ethyl 3-methyl-2-oxobutanoate with B. stearothermophilus DSM 297 gave the corresponding (R)-alcohol with high yield in excellent enan-tioselectively (> 99% e.e.). The conversions of keto esters to the corresponding hydroxy esters with Bacillus strains were increased by introduction of glycerol in the reaction mixture as an additive.     

Enzymatic synthesis and antitumor evaluation of mono- and diesters of 3-O-β-D-galactopyranosyl-sn-glycerol

Lipase-catalyzed synthesis of mono- and diesters of 3-O-β-D-galactopyranosyl-sn- glycerol (β-GG) with caproic acid was performed in acetone. The simultaneous production of 1(6’)-monoesters and 1,6’-diesters of β-GG was achieved in this reaction. In order to improve the yield of β-GG esters, four process parameters, enzyme concentration (15?～?25?mg/mL), and substrate molar ratio (caproic acid: β-GG=?1.60?～?2.00?mmol: 0.10?mmol), reaction temperature (40?～?60?°C), and reaction time (8?～?12?h), were optimized via response surface methodology (RSM) employing a three-level-four-variable central composite design. Results showed that enzyme concentration had the most significant (p?β-GG esters. The optimal reaction conditions in acetone were given as follows: Novozyme435 concentration 18.65?mg/mL, molar rate of caproic acid to β-GG 19.46:1, reaction temperature 48?°C, and reaction time 9.83?h. The yield of β-GG esters reached 88.08% under above optimized conditions, which was very close to the predicted value 87.95%. The molar ratio of monoester to diesters was 0.39:0.61. β-GG esters with other fatty acyl chains were synthesized based on the optimized conditions. In vitro antitumor activity indicated that the antitumor activity of β-GG esters was dependent on the nature of fatty acids, such as the length of acyl chain, the degree of saturation, as well as the number of acyl chain.     

Lipase catalyse glycerolysis for kinetic resolution of racemates

Candida antarctica lipase B catalyzed kinetic resolution of representative secondary alcohols, esters, and amine was successfully performed using triacetin or glycerol as solvents and acyl donor/acceptor. High conversions and enantioselectivities were achieved and the product was easily separated by simple extraction with diethyl ether.     

Fatty acid composition of cholesterol esters in brains of patients with Schilder's disease, G M1 -gangliosidosis and Tay-Sachs disease, and its possible relationship to the -position fatty acids of lecithin

Abstract— Cholesterol esters were isolated from cerebral cortex and white matter of patients with Schilder's disease, G_M1-gangliosidosis and Tay-Sachs disease, and the fatty acid composition was determined by gas-liquid chromatography. The fatty acid composition was similar among the three pathological conditions, but it was entirely different from that reported for cholesterol esters of normal brain. Lecithin and ethanolamine phospholipids were isolated from the same brain specimens, treated with snake venom phospholipase A, and the fatty acids at the a’and β-positions of the glycerol moiety were determined separately. The fatty acid composition of cholesterol esters was similar to that of the β-position fatty acids of lecithin of white matter in all samples, and was quite different from those of the a'-position of lecithin, or of the a’or β-position of ethanolamine phospholipids. The results indicate that the source of fatty acids for cholesterol esterification in nonspecific sudanophilic demyelination is different from that in normal brain, and that the most likely source is the β-linked fatty acids of lecithin. There are two possible enzymic mechanisms; activation of phospholipase A and subsequent esterification of the liberated β-position fatty acids to cholesterol, or direct transacylation by lecithin-cholesterol acyl transferase.     

Green enzymatic production of glyceryl monoundecylenate using immobilized Candida antarctica lipase B

Enzymatic synthesis of glyceryl monoundecylenate (GMU) was performed using indigenously immobilized Candida anatarctica lipase B preparation (named as PyCal) using glycerol and undecylenic acid as substrates. The effect of molar ratio, enzyme load, reaction time, and organic solvent on the reaction conversion was determined. Both batch and continuous processes for GMU synthesis with shortened reaction time were developed. Under optimized batch reaction conditions such as 1:5 molar ratio of undecylenic acid and glycerol, 2?h of reaction time at 30% substrate concentration in tert-butyl alcohol, conversion of 82% in the absence of molecular sieve, and conversion of 93% in the presence of molecular sieve were achieved. Packed bed reactor studies resulted in high conversion of 86% in 10-min residence time. Characterization of formed GMU was performed by FTIR, MS/MS. Enzymatic process resulted in GMU as a predominant product in high yield and shorter reaction time periods with GMU content of 92% and DAG content of 8%. Optimized GMU synthesis in the present study can be used as a useful reference for industrial synthesis of fatty acid esters of glycerol by the enzymatic route.     

Metabolism of long-chain alcohols in cell suspension cultures of soya and rape

Heterotrophic cell suspension cultures of soya (Glycine max) and photomixotrophic cell suspension cultures of rape (Brassica napus) were incubated with cis-9-[1-¹⁴C]octadecenol for 3–48 h. It was found that under aerobic conditions large proportions of the alcohol are oxidized to oleic acid, which is incorporated predominantly into phospholipids, whereas up to 30% of the substrate is esterified to wax esters. This is true for both the heterotrophic and the photomixotrophic cell suspension cultures, but the metabolic rates are much higher in the latter. Under anaerobic conditions only small proportions of the radioactively labeled alcohol are oxidized to oleic acid, whereas a major portion of the alcohol is esterified to wax esters both in heterotrophic and photomixotrophic cultures. Incubations of homogenates of photomixotrophic rape cells with labeled cis-9-octadecenol showed that pH 6 is optimum for the formation of wax esters. This monounsaturated alcohol is preferred as a substrate over saturated longchain alcohols, whereas short-chain alcohols, cholesterol, and glycerol are not acylated. Incubations of an enzyme concentrate from a homogenate of rape cells with unlabeled cis-9-octadecenol and [1-¹⁴C]oleic acid, or [1-¹⁴C]stearoyl-CoA, or di[1-¹⁴C]palmitoyl-sn-glycero-3-phosphocholine showed that acylation of the longchain alcohol proceeds predominantly through acyl-CoA. Direct esterification of the alcohol with fatty acid as well as acyl transfer from diacylglycerophosphocholine could be demonstrated to occur to a much smaller extent.     

Eu3+ Sequestration by Biogenic Nano-Hydroxyapatite Synthesized at Neutral and Alkaline pH

Biogenic hydroxyapatite (bio-HA) has the potential for radionuclide capture and remediation of metal-contaminated environments. Biosynthesis of bio-HA was achieved via the phosphatase activity of a Serratia sp. supplemented with various concentrations of CaCl₂ and glycerol 2-phosphate (G2P) provided at pH 7.0 or 8.6. Presence of hydroxyapatite (HA) was confirmed in the samples by X-ray powder diffraction analysis. When provided with limiting (1 mM) G2P and excess (5 mM) Ca²⁺ at pH 8.6, monohydrocalcite was found. This, and bio-HA with less (1 mM) Ca²⁺ accumulated Eu(III) to ～31% and 20% of the biomineral mass, respectively, as compared to 50% of the mineral mass accumulated by commercial HA. Optimally, with bio-HA made at initial pH 7.0 from 2 mM Ca²⁺ and 5 mM G2P, Eu(III) accumulated to ～74% of the weight of bio-HA, which was equal to the mass of the HA mineral component of the biomaterial. The implications with respect to potential bio-HA-barrier development in situ or as a remediation strategy are discussed.     

Screening Botryosphaeria species for lipases: Production of lipase by Botryosphaeria ribis EC-01 grown on soybean oil and other carbon sources

Nine isolates of Botryosphaeria spp. were screened for lipases when cultivated on eight different plant seed oils and glycerol, and all produced lipases. Botryosphaeria ribis EC-01 produced highest lipase titres on soybean oil and glycerol, while eight isolates of Botryosphaeria rhodina produced significantly lower enzyme titres. B. ribis EC-01 produced lipase when grown on different fatty acids, surfactants, carbohydrates and triacylglycerols, with highest enzyme titres produced on Triton X-100-emulsified stearic (316.7 U/mL), palmitic (283.5 U/mL) and oleic (247.4 U/mg) acids, and soybean oil (105.6 U/mL), as well as castor oil (191.2 U/mg); an enhancement of 9-fold over soybean oil-grown cultures. Glycerol was also a good substrate for lipase production. The crude lipase extract was optimally active at pH 8.0 and 55 °C, stable between 30 and 55 °C and pH 1–10, and tolerant to 50% (v/v) glycerol, methanol and ethanol. The crude lipase showed affinity for substrates of short, average and long-chain fatty acids (different esters of p-nitrophenol and triacylglycerols). Zymograms developed with 4-methylumbelliferyl-butyrate showed two bands of lipolytic activity at 45 and 15 kDa. This is the first report on the production of lipases by B. ribis grown on these different carbon sources.     

The gld1 + gene encoding glycerol dehydrogenase is required for glycerol metabolism in Schizosaccharomyces pombe

The budding yeast Saccharomyces cerevisiae is able to utilize glycerol as the sole carbon source via two pathways (glycerol 3-phosphate pathway and dihydroxyacetone [DHA] pathway). In contrast, the fission yeast Schizosaccharomyces pombe does not grow on media containing glycerol as the sole carbon source. However, in the presence of other carbon sources such as galactose and ethanol, S. pombe could assimilate glycerol and glycerol was preferentially utilized over ethanol and galactose. No equivalent of S. cerevisiae Gcy1/glycerol dehydrogenase has been identified in S. pombe. However, we identified a gene in S. pombe, SPAC13F5.03c (gld1 ⁺), that is homologous to bacterial glycerol dehydrogenase. Deletion of gld1 caused a reduction in glycerol dehydrogenase activity and prevented glycerol assimilation. The gld1Δ cells grew on 50 mM DHA as the sole carbon source, indicating that the glycerol dehydrogenase encoded by gld1 ⁺ is essential for glycerol assimilation in S. pombe. Strains of S. pombe deleted for dak1 ⁺ and dak2 ⁺ encoding DHA kinases could not grow on glycerol and showed sensitivity to a higher concentration of DHA. The dak1Δ strain showed a more severe reduction of growth on glycerol and DHA than the dak2Δ strain because the expression of dak1 ⁺ mRNA was higher than that of dak2 ⁺. In wild-type S. pombe, expression of the gld1 ⁺, dak1 ⁺, and dak2 ⁺ genes was repressed at a high concentration of glucose and was derepressed during glucose starvation. We found that gld1 ⁺ was regulated by glucose repression and that it was derepressed in scr1Δ and tup12Δ strains.     

Chitinolytic Activity of Cold Tolerant Antagonistic Species of <Emphasis Type="Italic">Streptomyces</Emphasis> Isolated from Glacial Sites of Indian Himalaya

Seventy-eight isolates of actinomycetes were isolated from the soil samples collected from alpine zones of Pindari glacier region in Indian Himalaya. Following a plate based rapid screening using two test fungi, five efficient isolates (nos. HA1, HA2, HA6, HA40, and HA142) were selected for further characterization with special reference to their antagonistic properties. Based on phenotypic and genotypic characters, the isolates were identified up to species level. All the isolates belonged to the genus Streptomyces. The isolate nos. HA1 and HA2 were S. sampsonii and HA6, HA40 and HA142 were S. griseobrunneus, S. aurantiacus, and S. griseoluteus, respectively. The isolates showed strong antifungal properties against phytopathogenic test fungi in plate assays. All the isolates hydrolyzed glycol–chitin as a substrate in denaturing conditions showing variable amount of different isoforms.     

Conversion of Soybean Oil to Biodiesel Fuel Using Lipozyme TL IM in a Solvent-free Medium

The conversion of soybean oil to biodiesel fuel was investigated in the presence of a lipase from Thermomyces lanuginosus (commercially called Lipozyme TL IM) in a solvent-free medium. The lipase was inactivated when more than 1.5 molar equivalent of methanol was added to the oil mixture. To fully convert the oil to its corresponding methyl esters, the reaction was performed successfully by a three-step addition of 1 molar equivalent of methanol and under the optimized conditions (40°C, 150 rpm, 10% enzyme quantity based on oil weight), the maximum methyl ester (ME) yield was 98% after 12 h reaction. By-product glycerol had a negative effect on enzymatic activity and iso-propanol was found to be effective for glycerol removal, in the presence of which lipase expressed relatively high activity and more than 94% of the ME yield was maintained after being used repeatedly for 15 batches.     

Sequence-directed DNA Curvature in Activator-binding Sequence in the Escherichia coli kdpABC Promoter

Microorganisms that accumulated the eicosapentaenoic acid (EPA)-enriched triacylglycerol (TG), were screened for using yeast-malt medium containing 1% free EPA. The best strain was identified as Mucor hiemalis HA-30. The optimum culture conditions for the accumulation of EPA-enriched TG were : 3% soluble starch, 0.5% polypeptone, 0.3% yeast extract, 0.5% free EPA, and pH 6.0 at 25°C. After the cultivation, 1.77 mg/ml of the TG with EPA purity of 79% was obtained. The EPA content in TG increased in conjunction with the EPA content of the supplemented free fatty acids or ethyl esters. Free EPA were more efficiently incorporated than the ethyl esters. Trieicosapentaenoyl glycerol (EPA, EPA, EPA) accounted for 73% of total TGs.     

THE GLYCEROL PERMEABILITY OF THE PLASMALEMMA OF THE HALOTOLERANT GREEN ALGA DUNALIELLA PARVA (VOLVOCALES)1

The glycerol permeability of the plasmalemma of the green alga Dunaliella parva Lerche was investigated by efflux studies with labelled glycerol, by enzymatic determination of glycerol leakage, and the determination of the reflection coefficient from osmotically induced volume changes (zero flow method). All results indicate that the plasmalemma of D. parva does not exhibit a special low permeability towards glycerol as would be expected from a glycerol accumulating alga. Rather, significant amounts of glycerol diffuse continuously into the medium following the glycerol concentration gradient between the cells and the medium. Efflux rates vary between 0.1 and 2 μmoles glycerol·mg^?1 chlorophyll·h^?1 depending on the external NaCl concentration. After one day up to 25% of the total glycerol of the algal suspension was found in the medium. Within 10 days this value can increase to 60%, depending on the growth constant of the culture. The reflection coefficient σ was determined to be 0.87, the permeability coefficient 2800 × 10^?11 m·sec^?1. To maintain a proper endogenous glycerol level corresponding to the external osmotic pressure, glycerol efflux in D. parva has to be balanced by a continuous synthesis of glycerol. D. parva follows the strategy of “glycerol efflux tolerance” instead of “glycerol efflux avoidance”. The alga has to pay the energetic costs of this strategy of tolerance.     

Deletion of the <Emphasis Type="Italic">CgTPI</Emphasis> Gene Encoding Triose Phosphate Isomerase of <Emphasis Type="Italic">Candida glycerinogenes</Emphasis> Inhibits the Biosynthesis of Glycerol

The yeast Candida glycerinogenes produces a high yield of glycerol only in response to a medium-osmotic stress, but little is known about the relationship between osmoadaptation and glycerol metabolism. The CgTPI gene encoding triose phosphate isomerase of C. glycerinogenes was cloned and sequenced, and its functionality was confirmed by complementation of Saccharomyces cerevisiae tpi1 Δ. The roles of CgTpip in the glycerol biosynthesis and the osmoadaptation were investigated. Unlike S. cerevisiae tpi1 Δ and Klyuveromyces lactis tpi1 Δ, the mutant lacking CgTPI significantly decreased the rate of glucose consumption and the glycerol yield. Furthermore, the mutants decreased osmotolerance to glucose and NaCl. The results suggest that CgTPI might be crucial for a high yield of glycerol by C. glycerinogenes. The inhibition of glycerol biosynthesis might be related to the reduced ability of osmoadaptation to high external osmolarity. To our knowledge, this is the first report that inactivation of a yeast TPI gene inhibits the biosynthesis of glycerol.     

Heterologous Expression of Glycerol 3-Phosphate Dehydrogenase Gene [DhGPD1] from the Osmotolerant Yeast Debaryomyces hansenii in Saccharomyces cerevisiae

The role for the gene encoding glycerol 3-phosphate dehydrogenase (DhGPD1) from the osmotolerant yeast Debaryomyces hansenii, in glycerol production and halotolerance, was studied through its heterologous expression in a Saccharomyces cerevisiae strain deficient in glycerol synthesis (gpd1Δ). The expression of the DhGPD1 gene in the gpd1Δ background restored glycerol production and halotolerance to wild type levels, corroborating its role in the salt-induced production of glycerol. Although the gene was functional in S. cerevisiae, its heterologous expression was not efficient, suggesting that the regulatory mechanism may not be shared by these two yeasts.