Studies on the lipozyme-catalyzed synthesis of butyl laurate

The effects of temperature, speed of agitation, enzyme concentration, etc., on butyl laurate synthessis using Mucor miehei lipase (Lipozymetrade mark) have been studied. Although the soluble enzyme was quite thermcstable in aqeous solution, it deactivated rapidly at and above 40 degrees C in the presence of butanol. This enzyme immobilized on an anion-exchange resin (Lipozymetrade mark) showed enhanced stability (as compared to the soluble form) to denaturation by butanol under the same conditions. The denaturation of M. miehei lipase was found to be a function of the butanol concentration in the aqueous phase, and rapid denaturation takes place at the concentration corresponding to its saturation at that temperature. (c) 1995 John Wiley & Sons, Inc.     

The role of retinol in the initiation of sporangiophores of Phycomyces blakesleeanus

The initiation of sporangiophores of Phycomyces was analyzed under oxygen-limiting conditions. Mutants lacking -carotene have a higher oxygen threshold than the wild type depending on the residual amount of -carotene. The supersensitivity to low oxygen tension is specific for sporangiophore initiation and can be suppressed by addition of either retinal, retinol or retinol acetate to the medium. It is suggested that retinol is a natural regulator of differentiation in Phycomyces.     

Effects of chilling on tomato fruit texture

The effects of chilling on tomato ( Lycopersicon esculentum Mill cv. Caruso) texture were investigated using fruit stored at 22°C (nonchilled) or 5°C (chilled) for 28 days. or at 5°C for 15 days before transfer to 22°C to facilitate ripening during and additional 13 days (prechilled). Prechilled fruit exhibited symptoms of slight chilling injury, i.e. development of mealiness, accelerated softening relative to that of nonchilled fruit and nonuniform surface colour development. The firmness of all fruit decreased during ripening and chilled storage when measured by flat plate compression and puncture, especially during the early stages of ripening of nonchilled and prechilled fruit. The compression firmness of pericarp tissue similarly decreased during ripening of nonchilled and prechilled fruit, but was maintained during chilling. Total moisture content (ca 94%) of tissue, uronide content (32-35% w/w) and extracted β-galactosidase activity did not differ significantly ( P > 0.05) among fruit during ripening and chilled storage. The degree of uronide methyl esterification in ethanol-insoluble solids prepared from pericarp tissue (EIS) was relatively low for all fruit. i.e. <40%. EIS from which greater levels of pectinesterase were extracted (i.e. nonchilled>chilled>prechilled) exhibited decreased levels of uronide methyl esterification. Markedly elevated levels of β-glucosidase activity were extracted from prechilled EIS. Total polygalacturonase activity (mainly as PGI) and autolysis of enzyme-extracted EIS were inversely correlated ( P ≤ 0.05) only with the loss of nonchilled fruit and tissue firmness and prechilled fruit firmness. Results suggest a possible role for β-glucosidase in textural changes of prechilled fruit and tissue (e.g. loss of firmness, development of mealiness) and also implicate loss of skin strength in the softening of whole fruit during chilling.     

High resolution NMR for studying lipid hydrolysis and esterification in cod (Gadus morhua) gonads

High resolution NMR was applied to study biochemical changes of lipids in cod (Gadus morhua) gonads during 7 days storage at 4 degrees C. Changes were observed in the (13)C and (1)H resonances of cholesterol which were due to esterification of fatty acids at the hydroxyl position in roe and milt. Furthermore, the (13)C NMR spectra showed that the lipolytic changes in milt and roe where different. New resonances appeared during storage, due to formation of specific free fatty acids, with the corresponding changes in resonances of the esterified carbonyls and glycerols. The highly unsaturated n-3 fatty acids were hydrolysed from the sn-1 and sn-2 position of both phosphatidylcholine and phosphatidylethanolamine in milt. The lipolytical changes in roe were less prominent compared to the changes in milt, however significant levels of sn-1-lysophospholipids was detected both in roe and milt. The current data demonstrate that high resolution NMR may be a suitable method to non-destructively study hydrolysis and esterification reactions occurring in heterogeneous marine lipids in a one step procedure.     

Effect of acyl donor chain length and sugar/acyl donor molar ratio on enzymatic synthesis of fatty acid fructose esters

Lipase-catalyzed synthesis of fatty acid sugar esters through direct esterification was performed in 2-methyl 2-butanol as solvent. Fructose and saturated fatty acids were used as substrates and the reaction was catalyzed by immobilized Candida antarctica lipase. The effect of the initial fructose/acyl donor molar ratio and the carbon-chain length of the acyl donor as well as their reciprocal interactions on the reaction performance were investigated. For this purpose, an experimental design taking into account variations of the molar ratio (from 1:1 to 1:5) and the carbon-chain length of the fatty acid (from C8 to C18) was employed. Statistical analysis of the data indicated that the two factors as well as their interactions had significant effects on the sugar esters synthesis. The obtained results showed that whatever the molar ratio used, the highest concentration (73 g l⁻¹), fructose and fatty acid conversion yields (100% and 80%, respectively) and initial reaction rate (40 g l⁻¹ h⁻¹) were reached when using the C18 fatty acid as acyl donor. Low molar ratios gave the best fatty acid conversion yields and initial reaction rates, whereas the best total sugar ester concentrations and fructose conversion yields were obtained for high molar ratios.     

Relationship of photocarotenogenesis to other behavioural and regulatory responses inPhycomyces

The effect of light on carotene accumulation was studied by analyzing the -carotene content of 4--old mycelia continuously exposed to illumination of different intensities. The wild-type, mutants defective in phototropism, mutants defective in carotene regulation, and newpic mutants specifically defective for photocarotenogenesis were examined. The results indicate that photocarotenogenesis depends on a single sensory pathway which shares its earlier steps (governed by genesmadA andmadB) with the sensory pathway for phototropism. It shares its later steps (probably governed by genescarA andpicB) with one of the pathways for carotene regulation, and includes at least one specific step (governed by genepicA) not known to be involved in other responses.     

Enzymatic esterification between n-alcohol homologs and n-caprylic acid in non-aqueous medium under microwave irradiation

The enzymatic esterification between n-alcohol homologs and n-caprylic acid catalyzed by lipozyme RM IM (LRI) in microwave field was investigated. Some interesting findings were obtained. The optimum reaction temperature slightly shifted from that in enzymatic esterification by conventional heating. n-Alcohol homologs used in this experiment showed substrate specificity in terms of the odd and even carbon numbers. THF expressed abnormal solvent effect. Whereas in the contrastive enzymatic esterification by conventional heating, the above mentioned substrate specificity and solvent effect were not observed. All the above phenomena could be explained by both thermal and non-thermal effect of microwave on enzyme and substrates. Further investigation revealed that microwave irradiation reduced the apparent activation energy of the enzymatic reaction according to Arrhenius equation, which is considered as one of the causes increasing initial reaction rate.     

Stability of individual carotenoids, retinol and tocopherols in human plasma during exposure to light and after extraction

We have modified gradient HPLC procedures for simultaneous quantification of retinol, γ-tocopherol, α-tocopherol, lutein/zeaxanthin, β-cryptoxanthin, trans-lycopene, cis-lycopene, α-carotene and β-carotene in 200-μl aliquots of human plasma. The photosensitivity of these analytes in plasma exposed to fluorescent lighting for up to 72 h was investigated and most were stable under these conditions. The stability of these analytes held in darkness at −20°C, 4°C or room temperature for up to 48 h after extraction from plasma was also investigated. Variability in measurement of most analytes was greater at room temperature than at 4°C or −20°C. There were statistically significant variations in the measured concentrations of some analytes in samples kept cold. However, the magnitude of these variations was small and of little biological significance, particularly over the first 24 h.     

Alcohol Induced Reversal of Enantioselectivity in a Lipase Catalyzed Resolution of 2-Chloropropionic Acid

Alcohol induced reversal of enantioselectivity in the esterification of 2-chloropropionic acid using lipase from Candida cylindracea has been investigated. It was found that an alcohol having substituents both at the α- and the β-carbon preferentially esterified the S-acid, while a straight chain alcohol preferentially esterified the R-acid. Intermediate enantioselectivities were obtained with alcohols having substituents either at the α- or the β-carbon, but still in favor of the R-acid. An acyl binding domain composed of three subsites is proposed for this lipase; one for the hydrocarbon chain, a second for a methyl substituent and a third for an electronegative substituent.