Esterification and hydrolytic activities of Candida rugosa lipase isoform 1 (LIP1) immobilized on celite 545, duolite A7, and sephadex G-25

The esterification and hydrolytic activities of free and immobilized Candida rugosa lipase isoform 1 (LIP1) were investigated. Esterification activity was determined by reacting caprylic acid with glycerol in the presence of molecular sieves (30%, w/w), and the volume of 1.0 M NaOH consumed by the reaction products upon titration was used to calculate esterification activity. Caprylic acid was also reacted with cottonseed oil, and the amount of caprylic acid incorporated after 12 h of reaction was determined. Results indicated that LIP1 had little esterification activity, which was not significantly improved upon immobilization. Hydrolytic activity was determined by incubating tricaprylin emulsion (15%, w/w) with the respective lipases for 60 min, and the reaction products were titrated against 0.5 M NaOH. LIP1 showed hydrolytic activity comparable to Lipozyme RM IM. The hydrolytic activity improved significantly upon immobilization. Immobilization on Celite 545 produced the highest increase in hydrolytic activity.     

Solvent-free enzymatic preparation of feruloylated monoacylglycerols optimized by response surface methodology

The ability of immobilized lipase B from Candida antarctica (Novozym 435) to catalyze the direct esterification of glyceryl ferulate (FG) and oleic acid for feruloylated monoacylglycerols (FMAG) preparation in a solvent-free system was investigated. Enzyme screening and the effect of glycerol on the initial reaction rate of esterification were also investigated. Response surface methodology (RSM) was used to optimize the effects of the reaction temperature (55-65 degrees C), the enzyme load (8-14%; relative to the weight of total substrates), oleic acid/(FG + glycerol) (6:1-9:1; w/w), and the reaction time (1-2 h) on the conversion of FG and yield of FMAG. Validation of the RSM model was verified by the good agreement between the experimental and the predicted values of FG conversion and FMAG yield. The optimum preparation conditions were as follows: temperature, 60 degrees C; enzyme load, 8.2%; substrate ratio, 8.65:1 (oleic acid/(FG + glycerol), w/w); and reaction time, 1.8 h. Under these conditions, the conversion of FG and yield of FMAG are 96.7 +/- 1.0% and 87.6 +/- 1.2%, respectively.     

Enzymatically catalyzed synthesis of low-calorie structured lipid in a solvent-free system: optimization by response surface methodology

A kind of low-calorie structured lipid (LCSL) was obtained by interesterification of tributyrin (TB) and methyl stearate (St-ME), catalyzed by a commercially immobilized 1,3-specific lipase, Lipozyme RM IM from Rhizomucor miehei . The condition optimization of the process was conducted by using response surface methodology (RSM). The optimal conditions for highest conversion of St-ME and lowest content LLL-TAG (SSS and SSP; S, stearic acid; P, palmitic acid) were determined to be a reaction time 6.52 h, a substrate molar ratio (St-ME:TB) of 1.77:1, and an enzyme amount of 10.34% at a reaction temperature of 65 °C; under these conditions, the actually measured conversion of St-ME and content of LLL-TAG were 78.47 and 4.89% respectively, in good agreement with predicted values. The target product under optimal conditions after short-range molecular distillation showed solid fat content (SFC) values similar to those of cocoa butter substitutes (CBS), cocoa butter equivalent (CBE), and cocoa butters (CB), indicating its application for inclusion with other fats as cocoa butter substitutes.     

Water activity effects on geranyl acetate synthesis catalyzed by novozym in supercritical ethane and in supercritical carbon dioxide

The esterification reaction of geraniol with acetic acid catalyzed by Novozym was studied in supercritical ethane (sc-ethane) and in supercritical carbon dioxide (sc-CO(2)). Water activity (a(W)) had a very strong effect on enzyme activity, with reaction rates increasing up to a(W) = 0.25 and then decreasing for higher a(W). Salt hydrate pairs could not prevent changes in a(W) during the course of reaction but were able to control a(W) to some extent and had a beneficial effect on both initial rates of esterification and conversion in sc-ethane. The enzyme was more active in sc-ethane than in sc-CO(2), confirming the deleterious effect of the latter already observed with some enzymes. Temperatures between 40 and 60 degrees C did not have a strong effect on initial rates of esterification, although reaction progress declined considerably in that temperature range. For the mixture of 50 mM acetic acid plus 200 mM geraniol, 100% conversion was achieved at a reaction time of 10 h at 40 degrees C, 100 bar, an a(W) of incubation of 0.25, and a Novozym concentration of 0.55 mg cm(-)(3) in sc-ethane. Conversion was below 50% in sc-CO(2) at otherwise identical conditions. With an equimolar mixture of the two substrates (100 mM), 98% conversion was reached at 10 h of reaction in sc-ethane (73% conversion in sc-CO(2)).     

Factors affecting the resolution of dl-menthol by immobilized lipase-catalyzed esterification in organic solvent.

Among 10 lipases tested, Candida rugosa lipase exhibited the best ability to catalyze the resolution of dl-menthol in organic solvent. The lipase was immobilized on different carriers, and the experiment was carried out with different acyl donors. The high yield and optical purity of the product were achieved in cyclohexane with valeric acid as acyl donor using C. rugosa lipase immobilized on DEAE-Sephadex A-25. The conversion of dl-menthol depended on the water content of immobilized lipase and on the pH of the aqueous solution from which lipase was immobilized. The operational stability of the DEAE-Sephadex A-25 immobilized lipase in catalysis of the esterification reaction showed that >85% activity remained after 34 days of repeated use. The resolution of racemic menthol in organic medium catalyzed by immobilized C. rugosa lipase-catalyzed esterification is very convenient, and it represents a significant improvement in the use of enzyme for the preparative production of optically active menthol. This process is readily applicable to large-scale preparation.     

Novel fatty acid esters of p-coumaryl alcohol in epicuticular wax of apple fruit

Hexane extracts of epicuticular wax from cv. Gala apples were noted to have an unusual, broad absorbance maximum at approximately 258 nm, which led us to isolate and identify the primary UV-absorbing compounds. Column and thin-layer chromatography yielded a fraction that gave a series of paired, 260-nm-absorbing peaks on C(18) HPLC. These were shown to be a family of phenolic fatty acid esters, for which retention times increased with increasing fatty acid chain length, and paired peaks were esters of two related phenolics with the same fatty acid moiety. Alkaline hydrolysis of the esters released two water-soluble phenolics separable by C(18) HPLC. Electrospray ionization mass spectrometry gave a molecular mass of 150 for both, and (1)H NMR plus UV absorbance spectra identified them as E and Z isomers of p-coumaryl alcohol. Alkaline cleavage of the fatty acid esters in the presence of methanol or ethanol resulted in partial derivatization of E-p-coumaryl alcohol to the corresponding gamma-O-methyl or O-ethyl ether. Gradient HMQC NMR of the HPLC-purified stearate ester of E-p-coumaryl alcohol indicated that fatty acid esterification occurs at the gamma-OH rather than at the 4-OH on the phenyl ring. This is the first report of fatty acid esters of monolignols as a natural plant product.     

Solvent-free acid-catalyzed ring-opening of epoxidized oleochemicals using stearates/stearic acid, and its applications

Toxic solvent and strong acid catalysts causing environmental issues have been mainly used for ring-opening of epoxidized oleochemicals. Here, we demonstrated that magnesium stearate (Mg-stearate) was a high efficient catalyst for solvent-free ring-opening of epoxidized methyl oleate, a model compound of midchain epoxide. Mg-stearate resulted in the highest yield (95%) and conversion rate (99%) toward midchain alkoxyesters under the same conditions (160 °C, 12 h) superior to other fatty acid derivatives such as a Lewis acid (lithium and sodium stearate) and Br?nsted acid (stearic acid). Based on this chemical study, we synthesized biogrease and thermoplastic using epoxidized soybean oil (ESO) and Mg-stearate via one-pot, solvent-free, and purification-free process. Mg-stearate played a significant role as a reactant for epoxide ring-opening and as a thickener when excess loading rate was used; viscosity increased from 1800 to 4500 Pa·s at 25 °C when ESO:Mg-stearate increased from 1:1 equiv to 1:2, then behaved like thermoplastics (T(g) = -27 °C, T(m) = 90 °C) with 1:4.     

Absorption and urinary excretion of quercetin,rutin, and alphaG-rutin,a water soluble flavonoid,in rats

Quercetin, rutin, alphaG-rutin (a water soluble flavonoid), and a mixture of rutin and alphaG-rutin were administered to rats by a single gastric intubation, and their absorption and urinary excretion were examined. The plasma and 24 h urinary levels of aglycons (quercetin and tamarixetin/isorhamnetin) were measured by HPLC after deconjugation with beta-glucuronidase/sulfatase treatment. alphaG-rutin was absorbed more rapidly than quercetin or rutin, and the plasma concentrations of quercetin and tamarixetin/isorhamnetin reached the highest peak level 30 min after dosing. Quercetin, rutin, and the mixture of rutin and alphaG-rutin showed the first peak level 8 h, 8 h, and 30 min after dosing, respectively. The area under the concentration-time curve (AUC) for quercetin in rats administered alphaG-rutin was approximately 4.5- and 2-fold higher than those in rats administered quercetin and rutin, respectively, and was almost the same as that in rats administered a mixture of rutin and alphaG-rutin. The highest 24 h urinary excretion was observed in alphaG-rutin-administered rats. These results suggest that alphaG-rutin is absorbed more efficiently than either quercetin or rutin and that a high plasma concentration can be maintained by supplying rutin and alphaG-rutin in combination.     

Steryl and stanyl esters of fatty acids by solvent-free esterification and transesterification in vacuo using lipases from Rhizomucor miehei, Candida antarctica, and Carica papaya.

Sitostanol has been converted in high to near-quantitative extent to the corresponding long-chain acyl esters via esterification with oleic acid or transesterification with methyl oleate or trioleoylglycerol using immobilized lipases from Rhizomucor miehei (Lipozyme IM) and Candida antarctica (lipase B, Novozym 435) as biocatalysts in vacuo (20-40 mbar) at 80 degrees C, whereas the conversion was markedly lower at 60 and 40 degrees C. Corresponding conversions observed with papaya (Carica papaya) latex lipase were generally lower. High conversion rates observed in transesterification of sitostanol with methyl oleate at 80 degrees C using Lipozyme IM were retained even after 10 repeated uses of the biocatalyst. Saturated sterols such as sitostanol and 5alpha-cholestan-3beta-ol were the preferred substrates as compared to Delta(5)-unsaturated cholesterol in transesterification reactions with methyl oleate using Lipozyme IM. Transesterification of cholesterol with dimethyl 1,8-octanedioate using Lipozyme IM in vacuo yielded methylcholesteryl 1,8-octanedioate (75%) and dicholesteryl 1,8-octanedioate (5%). However, transesterification of cholesterol with diethyl carbonate and that of oleyl alcohol with ethylcholesteryl carbonate, both catalyzed by Lipozyme IM, gave ethylcholesteryl carbonate and oleylcholesteryl carbonate, respectively, in low yield (20%). Moreover, cholesterol was transesterified with ethyl dihydrocinnamate using Lipozyme IM to give cholesteryl dihydrocinnamate in moderate yield (56%), whereas the corresponding reaction of lanosterol gave lanosteryl oleate in low yield (14%).     

Vegetable oil stability at elevated temperatures in the presence of ferric stearate and ferrous octanoate

The thermoxidative stability of partially hydrogenated soybean oil (PHSBO) was examined after addition of ferric stearate and ferrous octanoate, and then heating the samples at 120, 160, 180, and 200 degrees C. In a second experiment, the effect of iron concentration (ferric stearate) on PHSBO stability was examined at 180 degrees C, and at concentrations of approximately 0.5 and 1.2 mg of added iron/kg PHSBO. Oil samples were heated continuously for 72 h and sampled every 12 h. The acid value, p-anisidine value, color, dielectric constant and the triacylglycerol polymer content of oil samples were compared to oil samples containing no added iron. Generally, the value of each oxidative index increased with (1) an increase in temperature, (2) an increase in heating time, and/or (3) an increase in iron. The results demonstrate that low concentrations of iron will substantially increase the rate of oxidation for vegetable oil samples heated to temperatures of 120 degrees C to 200 degrees C.     

Enzymatic production of galactooligosaccharides by beta-galactosidase from Bifidobacterium longum BCRC 15708

The production of galactooligosaccharides (GOSs) by transgalactosylation using beta-galactosidase from Bifidobacterium longum BCRC 15708 was studied. Other than lactose, galactose, and glucose, two types of GOSs, tri- and tetrasaccharides, were formed after beta-galactosidase action on 40% lactose. Trisaccharides were the major type of GOS formed. Generally, an increase of the initial lactose concentration in the reaction mixture resulted in a higher GOS production. A maximum yield of 32.5% (w/w) GOSs could be achieved from 40% lactose solution at 45 degrees C, pH 6.8, when the lactose conversion was 59.4%. The corresponding productivity of GOSs was 13.0 g/(L.h). Transgalactosylation activity of beta-galactosidase from a test organism showed a relatively lower sensitivity toward glucose and galactose than that from other organisms. The addition of 5% or 10% glucose or galactose to the reaction mixture did not significantly (p>0.05) reduce the transgalactosylation reaction of beta-galactosidase.     

碳基固体酸催化高酸值生物柴油原料降酸效果

以淀粉和对甲苯磺酸合成的碳基固体酸为催化剂,油酸模拟高酸值生物柴油原料进行酯化降酸的试验研究,考察醇油比、催化剂用量、反应温度、反应时间及重复利用性等因素对转化率的影响。通过单因素与正交试验确定最佳工艺条件：醇油质量比为1︰4,催化剂用量为油酸质量的6.5%,反应时间6 h,反应温度85℃,在此条件下转化率可达80.21%,重复使用6次,转化率仍保持在70%以上。碳基固体酸催化剂对高酸值原料酯化降酸有很好的催化活性,易于分离且具有良好的稳定性。     

Lipase-catalyzed modification of rice bran oil to incorporate capric acid

Capric acid (C10:0) was incorporated into rice bran oil with an immobilized lipase from Rhizomucor miehei as the biocatalyst. Effects of incubation time, substrate mole ratio, enzyme load, and water addition on mole percent incorporation of C10:0 were studied. Transesterification was performed in an organic solvent, hexane, and under solvent-free condition. Pancreatic lipase-catalyzed sn-2 positional analysis and tocopherol analysis were performed before and after enzymatic modification. Products were analyzed by gas-liquid chromatography (GLC) for fatty acid composition. After 24 h of incubation in hexane, there was an average of 26.5 +/- 1.8 mol % incorporation of C10:0 into rice bran oil. The solvent-free reaction produced an average of 24.5 +/- 3.7 mol % capric acid. In general, as the enzyme load, substrate mole ratio, and incubation time increased, the mole percent of capric acid incorporation also increased. Time course reaction indicated C10:0 incorporation increased up to 27.0 mol % at 72 h, for the reaction in hexane, and up to 29.6 mol % at 12 h, for the solvent-free reaction. The highest C10:0 incorporations (53.1 and 43.2 mol %) for the mole ratio experiment occurred at a mole ratio of 1:8 for solvent and solvent-free reactions, respectively. The highest C10:0 incorporation (27.9 mol %) for the reaction in hexane occurred at 10% enzyme load, and the highest incorporation (34.4 mol %) for the solvent-free reaction occurred at 20% enzyme load. Incorporation of C10:0 into rice bran oil declined with the addition of increasing amounts of water after reaching 30.3 mol % at 2% water addition in hexane, and in the solvent-free reaction after reaching 35.9 mol %.     

Optimization of lipase-catalyzed synthesis of octyl hydroxyphenylpropionate by response surface methodology

The ability of immobilized lipase Candida antarctica (Novozyme 435) to catalyze the direct esterification of hydroxyphenylpropionic acid and octanol in a solvent-free system was investigated in this study. Response surface methodology (RSM) and five-level-four-factor central composite rotatable design (CCRD) were employed to evaluate the effects of synthesis parameters, such as reaction time, temperature, enzyme amount, and pH memory, on percentage molar conversion of phenolic acid esters. Reaction time, temperature, and enzyme amount were the most important variables. On the basis of canonical analysis and ridge max analysis, the optimum synthesis conditions with 95.9% molar conversion were reaction time of 58.2 h, temperature of 52.9 degrees C, enzyme amount of 37.8% (w/w), and pH memory of pH 7.     

生物油酯化-加氢提质制备醇酯类燃料

作为清洁可再生的化石燃料取代燃料,生物油的酸性及不稳定性是阻碍其规模化应用的主要障碍之一。该文基于生物油高酮、醛及酸类含量,研究了生物油轻质组分分步酯化加氢(SHE,separated esterification and hydrogenation)、一步酯化加氢(OEH,one step esterification-hydrogenation)及一步酯化加氢后二次加氢(OEH plus,one step esterification-hydrogenation plus second hydrogenation process)的提质过程,考察了钼改性雷尼镍催化剂(Mo-RN,Mo-Raney Ni)及Ru/C催化剂催化生物油制备醇类燃料的重复使用性能,并研究了酯化-加氢反应过程及反应路径。结果表明,生物油经不同酯化-加氢方法处理后,饱和醇酯含量均显著提高,生物油品质得到改善。其中以OEH plus提质处理后的生物油产物中,饱和醇、酯含量最高,分别达74.21%和9.96%。此外,提质后的生物油p H值及酸量下降最为显著,生物油的p H值由反应前的3.67提高到5.88,酸量由111.52 mg/g降至11.75 mg/g。Mo-RN及Ru/C催化剂在酯化-加氢路径下的重复使用性能良好,催化活性均无明显降低。试验证明利用酯化-加氢提质生物油为生物油精制制备含氧燃料提供有效途径。     

Production of ascorbyl palmitate by surfactant-coated lipase in organic media

The surface of a lipase from Burkholderia cepacia was coated with a nonionic surfactant, propylene glycol monostearate, and was used as a biocatalyst in the production of ascorbic acid in tert-butyl alcohol. The influence of various factors such as the type of surfactant, the pH of the buffer used for coating, the amount of surfactant in the coating, the organic solvent, and the temperature and molar ratio of the substrates used in the reaction on the conversion of ascorbyl palmitate were studied. After 24 h of reaction at 50 degrees C, a conversion of 47% was obtained using an ascorbic acid to palmitic acid molar ratio of 1:6. The native lipase showed only 6% conversion.     

Carbon and nitrogen degradation on molecular scale of grass-derived pyrogenic organic material during 28 months of incubation in soil

The present study focuses on the microbial recalcitrance of pyrogenic organic material (PyOM) on a molecular scale. We performed microcosm incubation experiments using ¹³C- and ¹⁵N-enriched grass-derived PyOM mixed with a sub soil material taken from a Haplic Cambisol. Solid-state ¹³C and ¹⁵N NMR studies were conducted to elucidate the humification processes at different stages of PyOM degradation. The chemical structure of the remaining PyOM after incubation was clearly different from the initial pyrogenic material. The proportion of O-containing functional groups was increased, whereas that of aryl C and of N-containing heterocyclic structures had decreased, probably due to mineralisation and conversion to other C and N groups. After 20 months of incubation the aryl C loss reached up to 40% of the initial amount and up to 29% of the remaining PyOM C was assigned to carboxyl/carbonyl C and O-aryl C. These reactions alter the chemical and physical properties of the char residue and make it more available for further microbial attack but also for adsorption processes. Our study presents direct evidence for the degradation of N-heterocyclic domains in charred plant remains adding new aspects to the understanding of the N cycling in fire-affected ecosystems.     

Microbial community changes during humification of 14C-labelled maize straw in heat-treated and native Orthic Luvisol

The microbial communities in agricultural soils are responsible for nutrient cycling and thus for maintaining soil fertility. However, there is still a considerable lack of knowledge on anthropogenic impacts on soils, their microflora, and the associated nutrient cycles. In this microcosm study, microorganisms involved in the conversion of crop residues were investigated by means of classical microbiological and molecular methods such as denaturing gradient gel electrophoresis (DGGE) of PCR (polymerase chain reaction) amplified 16S rRNA genes. ¹⁴C‐labelled maize straw was humified by the naturally occurring microflora in native and in ashed soils, from which organic carbon was removed by heating at 600°C. The humic acids synthesized in the microcosms served as indicators of the humification process and were analysed by ¹³C‐NMR spectroscopy. Ashed, autoclaved and native soil exhibited similar microbial and physicochemical dynamics after inoculation with a soil suspension. Bacterial counts and DGGE analyses showed that in the first few weeks a small number of rapidly growing r‐strategists were principally responsible for the conversion of maize straw. As the incubation continued, the bacterial diversity increased as well as the fungal biomass. ¹³C‐NMR spectroscopy of 26‐week old soil extracts revealed that structures typical of humic substances also evolved from the plant material.     

NaHSO4催化椰子油皂脚油酯化降酸工艺优化

以椰子油皂脚油为原料的生物柴油酯化效率与催化剂和结合反应装置的操作方法有关。以硫酸氢钠为催化剂结合设计的反应装置,对高酸值椰子油皂脚油进行预酯化反应研究。通过单因子试验探讨适用于反应装置的反应条件,并讨论不同的操作方式对反应速率和反应进程的影响。结果表明:最佳条件为:反应温度105℃,甲醇通入流速为0.825 mL/min,催化剂用量为5.0%,反应2 h下转化率>95%。催化剂重复使用9次后转化率78.15%;改变操作方法,0.1 MPa条件下反应,采用通入甲醇1.32 mL/min反应30 min,后常压条件下通入甲醇量0.825 mL/min,反应30 min,椰子油皂脚油酸值由106变为1.2 mg/g,转化率98.9%,并可缩短酯化时间1 h,油脂成品满足酯交换工序要求。精制的生物柴油成品所测试的技术指标符合德国现行生物柴油标准(DIN V 51606)。     

In vitro biogeneration of pure thiosulfinates and propanethial-S-oxide

A model reaction system was developed for generating pure thiosulfinates and propanethial-S-oxide (PTSO) using an isolated alliinase (EC 4.4.1.4) and isolated or synthetic alk(en)yl-L-cysteine sulfoxides (ACSO). Reaction yields ranged from 30 to 60% after 3 h at 21-23 degrees C, and organosulfur reaction products were extracted into CHCl3 to yield product preparations of controlled composition. A pure thiosulfinate or PTSO was derived from a single ACSO, and a preparation containing a mixture of four thiosulfinate species was derived from reaction mixtures employing binary ACSO substrate systems. Identities of homologous thiosulfinates and PTSO were confirmed by 1H NMR. This approach has the potential to be used as a preparative tool for yielding pure thiosulfinates and PTSO to facilitate the study of chemical and biological properties of this group of compounds or as a means to study the dynamics of organosulfur chemistry in preparations from Allium spp.