非水体系中脂肪酶催化合成乳酸乙基糖苷酯的工艺研究

在非水体系中 ,通过固定化脂肪酶催化合成一种新型α 羟基酸衍生物 乳酸糖苷酯。考察了常压下有机溶剂、酰基供体、不同种固定化酶、乙基糖苷的浓度、酶量和反应温度对反应的影响。研究表明在无溶剂体系中以乳酸丁酯作为酰基供体可有效地合成乳酸糖苷酯 ,固定化酶Novozym435和来源于Candida sp .菌株的细胞固定化酶 ,化学修饰的干酶粉均是合适的催化剂。最佳反应条件为 :酶浓度 75g L ,乙基葡萄糖苷的浓度为 0.4mol L ,温度为 70℃ ,转速 200r min ,反应 50h ,转化率可达 71%。在真空度为 0.09MPa的压力下 ,反应温度 65℃ ,酶浓度 75g L ,乙基葡萄糖苷 0.35mol L时 ,反应初速率可达到 607(mmol·L^-1·h^-1 ) ,40h后转化率可达到 90%。反应产物经过萃取法和硅胶柱层析方法分离 ,纯度达到 95 % (W/W)。     

Enzymatic synthesis of unsaturated fatty acid glucoside esters for dermo-cosmetic applications.

Unsaturated fatty acid alpha-butylglucoside esters were prepared by enzymatic esterification of alpha-butylglucoside in nonaqueous media. Conditions were firstly optimized using oleic acid as acyl group. Synthesis was possible in several solvents but the presence of water co-product in the medium limited the reaction to a thermodynamic equilibrium corresponding to a maximal conversion yield of 62%. In pure molten substrates, the removal of water under reduced pressure enabled yields superior to 95% to be obtained. Product profiles depended on enzyme origin : whatever the support, immobilized lipase B from Candida antarctica proved to be far more regioselective for the primary hydroxyl group of glucose than immobilized lipase from Rhizomucor miehei. Results obtained could be easily transposed to the acylation of alpha-butylglucoside with a commercial mixture of unsaturated fatty acids containing more than 60% of linoleic acid. The biocatalyst could be recycled more than ten times without any significant activity loss.     

Low-cost propionate salt as road deicer: evaluation of cheese whey and other media constituents

Propionate and acetate salts are environmentally friendly, effective road deicer substitutes for widely used sodium chloride. A low-cost medium, using raw cheese whey and hydrolyzed whey permeate/whey permeate powder as substrates, and corn-steep liquor as a nutrient supplement, was studied for lactic acid production, replacing synthetic lactose and other high-cost nutrients. A non-sterile stage-I fermentation process for improved lactate productivity using an inexpensive commercial medium was performed at a 20-L fermenter level. A lactate yield of 0.98 g/g lactose and a productivity of 1.1 g/L/h was obtained with complete lactose utilization. When synthetic lactate and glucose were used as substrates in propionate and acetate fermentation, a total acid yield of 0.55 g/g glucose and lactate consumed and a batch productivity of 0.22 g/L/h was obtained. A stage-II fermentation process to produce propionate and acetate salts from cheese whey-derived lactate (stage-I fermentation broth) resulted in 1.6%( w/v) propionate after a total of 161 h (stages I and II).     

Lysophosphatidylcholine synthesis with Candida antarctica lipase B (Novozym 435)

Immobilized lipase from Candida antarctica lipase B (Novozym 435) was effective in the synthesis of lysophosphatidylcholine (LPC). The transesterification of L-alpha-glycerophosphorylcholine (GPC) and vinyl laurate was carried out in a solvent free system or in the presence of 50% (v/v) t95%) were easily achieved. The lipase was selective for the sn10 times). High purity products could be produced by a decrease of the reaction temperature to induce precipitation of the product. The temperature needed depended on the fatty acid chain length. Thus, only lysophosphatidylcholine was produced with palmitic acid vinyl ester at 45 degrees C, whereas for the vinyl esters of lauric acid, capric acid, and caprylic acid, a lower reaction temperature (25 degrees C) was necessary to obtain solely the lysophospholipid products.     

Kinetics of lipase-catalysed methyl gallate production in the presence of deep eutectic solvent

Production of methyl gallate (MG), which is an important phenolic acid ester for pharmaceutical industry, was carried out by Novozym 435-catalysed transesterification of propyl gallate (PG) with methanol in a deep eutectic solvent. Reaction parameters governing substrate molar ratio, enzyme concentration, temperature and agitation rate were investigated batch-wise in choline chloride:glycerol-water binary mixture. The results were evaluated in terms of conversion of PG, yield of MG and hydrolysis of PG to gallic acid. 10% (w/w) of water was found to be favourable in the reaction medium for low hydrolysis percent. The highest conversion (17.4%) and yield (60.4%) but the lowest hydrolysis (2%) after 120?h of transesterification were found at PG/methanol molar ratio of 1:6, enzyme concentration of 40?g/L, 50?°C and 200?rpm. A kinetic model based on the Ping-Pong Bi–Bi mechanism for transesterification of PG was proposed with the assumption that there were no internal and external mass transfer resistances.     

Thiol-functionalization of acrylic ester monomers catalyzed by immobilized Humicola insolens cutinase

Immobilized cutinase HiC from the ascomycete Humicola insolens was applied as a novel biocatalyst for the synthesis of functionalized acryclic esters by transesterification. As a model reaction, transesterification of methyl acrylate with 6-mercapto-1-hexanol at a high molar ratio in a solvent free system was chosen. Besides two minor Michael-addition by-products, 6-mercaptohexyl acrylic ester was identified as the main product with the thiol as the functional end group. Reaction conditions were optimized regarding the influence of water (0-1.72 M), temperature (22-50 °C), product inhibition and addition of the radical inhibitor butylated hydroxytoluol (BHT; 0.14-0.71 M) on conversion and by-product formation. Highest conversion of 6-mercapto-1-hexanol to 6-mercaptohexyl acrylic ester (95.4 ± 0.3%) was achieved after 6h at 40 °C in the presence of 0.025% (w/w) water without formation of by-products in a solvent free system. Applying methyl methacrylate, transesterification with 6-mercapto-1-hexanol was significantly lower (43.6 ± 0.1%) compared to transesterification of methyl acrylate with 6-mercapto-1-hexanol.     

Esterification and transesterification reactions catalysed by addition of fermented solids to organic reaction media

We report for the first time both the production of the lipase of Burkholderia cepacia in solid-state fermentation and the biocatalysis of esterification and transesterification reactions through the direct addition of the lyophilised fermented solids to organic reaction media. B. cepacia produced a lipolytic activity equivalent to 108 U of pNPP-hydrolysing activity per gram of dry solids after 72 h growth on corn bran with 5% (v/w) commercial corn oil as the inducer. The fermented solid material was lyophilised and added directly to the reaction medium in esterification and transesterification reactions. A factorial design was used to study the effects on esterification of temperature, alcohol-to-acid molar ratio and amount of lipolytic activity added. All three variables affected the ester yield significantly, with the amount of enzyme being most important. A 94% ester yield was obtained at 18 h at 37 °C, with an alcohol-to-acid molar ratio of 5:1 and 60 U of added lipolytic activity. For the transesterification reaction, a factorial design was undertaken with the variables being the alcohol-to-acid molar ratio and the added lipolytic activity. Ester yields of over 95% were obtained after 120 h. Our results suggest that biocatalysis using direct addition of fermented solids to organic reaction medium should be further explored.     

High yield chromatographic purification of enzymatically produced tyrosine fructosyl ester

The synthesis of L-tyrosine fructosyl ester, from fructose and L-tyrosine methyl ester, was carried out by a transesterification reaction catalyzed by α-chymotrypsin in water without any organic cosolvent. The yield was optimized by regulating the pH?of the reaction medium and a maximum yield of 63% was obtained. A two-step process of tyrosine fructosyl ester purification was proposed using adsorbent resin and activated carbon. Different elutions and supports easily applicable to industrial process have been studied. Solutions of tyrosine fructosyl ester with a 96% purity and 70% recovery yield were obtained by chromatography on a column of acrylic polyester resins with a solution of 0.5?M NaCl as eluant.     

Direct preparation of biodiesel from rapeseed oil leached by two-phase solvent extraction

A new method which coupled the two-phase solvent extraction (TSE) with the synthesis of biodiesel was studied. Investigations were carried out on transesterification of methanol with oil-hexane solution coming from TSE process in the presence of sodium hydroxide as the catalyst. Biodiesel (fatty acid methyl esters) were the products of transesterification. The influential factors of transesterification, such as reaction time, catalyst concentration, mole ratio of methanol to oil and reaction temperature were optimized. The results showed that the optimal reaction parameters were sodium hydroxide concentration 1.1% by weight of rapeseed oil, mole ratio of methanol to oil 9:1, reaction time 120 min, and reaction temperature 55-60 degrees C. Under these conditions, the TG conversion would rise up to 98.2%. Based on the new method, biodiesel production process could be simplified and the biodiesel cost could be reduced.     

Synthesis of phosphatidylcholine with defined fatty acid in the sn-1 position by lipase-catalyzed esterification and transesterification reaction

The incorporation of caproic acid in the sn-1 position of phosphatidylcholine (PC) catalyzed by lipase from Rhizopus oryzae was investigated in a water activity-controlled organic medium. The reaction was carried out either as esterification or transesterification. A comparison between these two reaction modes was made with regard to product yield, product purity, reaction time, and byproduct formation as a consequence of acyl migration. The yield in the esterification and transesterification reaction was the same under identical conditions. The highest yield (78%) was obtained at a water activity (a(w)) of 0.11 and a caproic acid concentration of 0.8 M. The reaction time was shorter in the esterification reaction than in the transesterification reaction. The difference in reaction time was especially pronounced at low water activities and high fatty acid concentrations. The loss in yield due to acyl migration and consequent enzymatic side reactions was around 16% under a wide range of conditions. The incorporation of a fatty acid in the sn-1 position of PC proved to be thermodynamically much more favorable than the incorporation of a fatty acid in the sn-2 position.     

Low-cost effective culture medium optimization for <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid production by <Emphasis Type="Italic">Lactobacillus coryniformis</Emphasis> subsp. <Emphasis Type="Italic">torquens</Emphasis> under oxygen-deprived condition

Lactic acid is considered a commodity and its production is boosted by the synthesis of polylactic acid. d-lactic acid (DLA) isomer offers greater flexibility and biodegradability and it can only be obtained in its pure form through fermentation. The lactate dehydrogenase is stereospecific for homofermentative production of DLA isomer in the metabolic pathway of Lactobacillus coryniformis subsp. torquens, with optical purity of ≥?99.9% under oxygen-deprived condition. A simple culture medium that increases DLA production and reduces fermentation costs is fundamental for industrial applicability. A central composite rotatable design was used to evaluate significant components influencing the DLA production. Concentrations were adjusted using the Design-Expert 7.0 optimization tool with a desirability coefficient of 0.693 and the best concentrations of each component were determined. Finally, an assay in the bioreactor with the modified culture medium resulted in a product yield of 0.95 g/g, volumetric productivity of 0.85 g/L.h and 95% of efficiency.     

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.     

Synthesis of L-tyrosine glyceryl ester catalyzed by α-chymotrypsin in water-miscible organic solvents: A possible sun-tan accelerator product

Summary The synthesis of L-tyrosine glyceryl ester, from glycerol and L-tyrosine methyl ester, was carried out by a transesterification reaction catalyzed by -chymotrypsin. Values of 60 % (v/v) for glycerol and 200 mM for L-tyrosine methyl ester were optimal for the transesterification reaction. Additionally to glycerol, several other water miscible cosolvents (acetonitrile, N,N'-dimetyl formamide and tetrahydrofurane) were tested in the reaction media, but their presence did not give an enhancement on the transesterification activity with respect to the glycerol/water medium. However, increasing the hydrophobicity of the cosolvent resulted in a reduction of the enzyme activity, the water:glycerol mixture being the best reaction media.     

Simultaneous saccharification and L-(+)-lactic acid fermentation of protease-treated wheat bran using mixed culture of lactobacilli

Protease-treated wheat bran (20% w/v) of particle size less than 300 μm containing 65% (w/w) starch was used for the simultaneous saccharification and l-(+)-lactic acid fermentation by the mixed cultures of Lactobacillus casei and Lactobacillus delbrueckii. Maximum lactate yield after various process optimizations was 123 gl⁻¹ with a productivity of 2.3 gl⁻¹ h⁻¹ corresponding to a conversion of 0.95 g lactic acid per gram starch after 54 h at 37°C. By using protease-treated wheat bran around tenfold decrease in supplementation of the costly medium component, like yeast extract, was achieved together with a considerable increase in the production level.     

Synthesis of trimethylolpropane esters of oleic acid by Lipoprime 50T

The ability of the commercial lipolytic enzyme Lipoprime 50T to catalyze the biotechnologically important synthesis of the biodegradable and environmentally acceptable trimethylolpropane (2-ethyl-2-(hydroxymethyl)-1,3-propanediol) ester of oleic acid was investigated. Simple and accurate thin-layer chromatography and computer analysis methods were used that enable one to follow changes of all reaction mixture components simultaneously. The processes of transesterification and esterification were compared. The effects of the molar ratio of the substrates, reaction temperature, time, and medium on the composition of the reaction mixture were analyzed. Esterification was determined to be more preferable than transesterification in both studied solvents. Under the optimal conditions identified (15% w/w water, temperature 60°C, trimethylolpropane to oleic acid molar ratio 1:3.5, and reaction time 72 h), the highest trimethylolpropane trioleate yield of around 62% and trimethylolpropane mono-, di-, and trioleate overall yield of about 83% were obtained. Although the yields are not high enough for industrial application, the process shows the potential to be optimized for higher yields in the near future as the conversions were obtained at ambient pressure, whereas many other processes described in the literature are conducted under vacuum at a specific pressure.     

Production of biodiesel and lactic acid from rapeseed oil using sodium silicate as catalyst

Biodiesel and lactic acid from rapeseed oil was produced using sodium silicate as catalyst. The transesterification in the presence of the catalyst proceeded with a maximum yield of 99.6% under optimized conditions [3% (w/w) sodium silicate, methanol/oil molar ratio 9/1, reaction time 60 min, reaction temperature 60 °C, and stirring rate 250 rpm]. After six consecutive transesterification reactions, the catalyst was collected and used for catalysis of the conversion of glycerol to lactic acid. A maximum yield of 80.5% was achieved when the reaction was carried out at a temperature of 300 °C for 90 min. Thus, sodium silicate is an effective catalyst for transesterification and lactic acid production from the biodiesel by-product, glycerol.     

L(+)-Lactic acid from sweet sorghum by submerged and solid-state fermentations

Sweet sorghum was used as the raw material for the lactate production by a strain of Lactobacillus paracasei. The submerged conversion of sugar juice obtained from sweet sorghum by extraction could be accomplished with the same efficiency as observed in a control experiment with MRS-glucose medium (final lactate concentration of 88–106 g/l, lactate yield of 91–95%, duration of the fermentation of 24–32 h). Finely ground stalks of sorghum served as the substrate in the solid-state fermentation. The lactate accumulation in the solid medium and the lactate yield were optimized up to values comparable with the results from the submerged fermentation (final lactate concentration of 90 g/kg, lactate yield of 91–95%). However, the duration of the fermentation amounted to 120–200 h in the solid-state process. The data from a series of experiments performed at variable values of temperatures between 30°C and 36°C and initial sugar concentrations between 60 g/kg and 115 g/kg, and degrees of moisture between 78% and 82% was the basis of a polynomial multidimensional regression. As a result, simple three-dimensional model functions were obtained for the maximum productivity of lactate formation, the lactate yield and the time required for a 90% conversion.     

Effect of immobilization support, water activity, and enzyme ionization state on cutinase activity and enantioselectivity in organic media

We studied the reaction between vinyl butyrate and 2-phenyl-1-propanol in acetonitrile catalyzed by Fusarium solani pisi cutinase immobilized on zeolites NaA and NaY and on Accurel PA-6. The choice of 2-phenyl-1-propanol was based on modeling studies that suggested moderate cutinase enantioselectivity towards this substrate. With all the supports, initial rates of transesterification were higher at a water activity (a(w)) of 0.2 than at a(w) = 0.7, and the reverse was true for initial rates of hydrolysis. By providing acid-base control in the medium through the use of solid-state buffers that control the parameter pH-pNa, which we monitored using an organo-soluble chromoionophoric indicator, we were able, in some cases, to completely eliminate dissolved butyric acid. However, none of the buffers used were able to improve the rates of transesterification relative to the blanks (no added buffer) when the enzyme was immobilized at an optimum pH of 8.5. When the enzyme was immobilized at pH 5 and exhibited only marginal activity, however, even a relatively acidic buffer with a pK(a) of 4.3 was able to restore catalytic activity to about 20% of that displayed for a pH of immobilization of 8.5, at otherwise identical conditions. As a(w) was increased from 0.2 to 0.7, rates of transesterification first increased slightly and then decreased. Rates of hydrolysis showed a steady increase in that a(w) range, and so did total initial reaction rates. The presence or absence of the buffers did not impact on the competition between transesterification and hydrolysis, regardless of whether the butyric acid formed remained as such in the reaction medium or was eliminated from the microenvironment of the enzyme through conversion into an insoluble salt. Cutinase enantioselectivity towards 2-phenyl-1-propanol was indeed low and was not affected by differences in immobilization support, enzyme protonation state, or a(w).     

Synthesis of biodiesel in column fixed-bed bioreactor using the fermented solid produced by Burkholderia cepacia LTEB11

We produced a lipase from Burkholderia cepacia in solid-state fermentation and used it to catalyze the synthesis of biodiesel in a fixed-bed reactor. In the solid-state fermentation step, a 50:50 (by mass) mixture of sugarcane bagasse and sunflower seed meal gave 234 units of pNPP-hydrolyzing activity per gram of dry solids at 96 h. This fermented solid was lyophilized and delipidated, packed into a column and used to catalyze the synthesis of biodiesel through the ethanolysis of soybean oil in a medium free of co-solvents, with the reaction mixture being continuously circulated through the column. The best conversion was 95% after 46 h, which was obtained at 50 °C, with an alcohol:oil molar ratio of 3:1, alcohol addition in two steps and the addition of 1% of (m/m) water to the reaction medium. This system has potential to decrease the costs of enzyme-catalyzed transesterification reactions.     

Enzymatic Preparation of Mono- and Di-Stearin by Glycerolysis of Ethyl Stearate and Direct Esterification of Glycerol in the Presence of a Lipase from Candida Antarctica (Novozym 435)

Mixtures of 1(3)-monostearin and distearin were prepared by direct esterification of glycerol with stearic acid or transesterification using ethyl stearate as acyl donor in the presence of Candida antarctica lipase (Novozym 435) using a variety of solvents of differing polarity. In all cases, the transesterification resulted in higher product yields. In n-heptane as reaction medium the addition of water (3%) was essential for high product yields, with mono- and distearin being produced in almost equal amounts. Using more polar solvents as reaction media, such as acetonitrile or acetone, again the highest yields were obtained in the transesterification mode; employing these solvents the reactions were much more selective towards the formation of monostearin.