酶法合成生物柴油工业化研究进展

介绍了北京化工大学近年来酶法合成生物柴油工业化研究的结果。主要内容包括以下几个方面:高产脂肪酶菌株的选育、脂肪酶发酵工艺优化及放大、脂肪酶固定化方法、酶反应器放大、生物柴油分离精制及副产物甘油综合利用。该脂肪酶假丝酵母Candida sp.99-125在5 m3罐发酵活力不低于8 000 IU/mL,然后将该脂肪酶吸附固定在织物膜上并进行表面改性,用于搅拌罐式反应器生产每吨甲酯的需酶量仅为4.2 kg,产品经分离精制调质后,其各项指标完全符合德国生物柴油生产标准。副产物甘油可用于1,3-丙二醇发酵,30 L发酵罐中1,3-丙二醇的产量可达到76.1 g/L。     

Acylation of sucrose with vinyl esters using immobilized hydrolases: demonstration that chemical catalysis may interfere with enzymatic catalysis

The acylation of sucrose with vinyl laurate in dimethylsulfoxide was catalyzed by Celite (28% conversion in 24 h at 40 °C, 150 mg catalyst ml^–1), Eupergit C (11% conversion in 24 h at 60 °C, 150 mg catalyst/ml), and even the simple Na₂HPO₄ (17% conversion in 24 h at 40 °C, 20 mg catalyst ml^–1). These chemical acylations must therefore be taken into account in acylations of hydroxyl-containing compounds with enol esters in polar solvents using immobilized enzymes.     

A simple enzymatic procedure for the synthesis of a hydroxylated polyester from glycerol and adipic acid

Lipase B from Candida antarctica catalyzed regioselectively the polyesterification of glycerol and adipic acid. UV-MALDI-TOF-MS analysis of the polymers shows low molecular weight polyesters (1314-1716) with very narrow polydispersities (1.0–1.2).     

Regioselective enzymatic acylation of ribavirin to give potential multifunctional derivatives

Lipase-catalyzed synthesis of potential multifunctional ribavirin derivatives was performed in acetone. Divinyl dicarboxylates with different chain lengths (C₄, C₆, C₉, C₁₀) were used as acyl donors and the reactions were catalyzed by lipase immobilized on acrylic resin from Candida antarctica (CAL-B). Ribavirin was regioselectivly acylated at the primary hydroxyl groups and the corresponding vinyl esters (C₄, C₆, C₉, C₁₀) were prepared in respective yields of 48%, 65%, 54%, 55%.     

Assay for rapid analysis of the tri-peptidase activity of LTA4 hydrolase

Leukotriene A4 hydrolase is a bifunctional zinc metalloenzyme with an epoxide hydrolase activity as well as an arginyl tri-peptidase activity. Detailed enzymological and mechanistic investigations of the latter activity have been hampered by the lack of a rapid and convenient enzyme assay. Here we have developed a new method allowing direct spectrophotometric assessment of the tri-peptide cleaving activity of leukotriene A4 hydrolase, as well as other peptidases. The method utilizes two competing substrates, one chromogenic reference substrate together with the tri-peptide substrate of interest, and relies on computer-assisted analysis of progress curves. The chromogenic reference substrate serves to disclose the "invisible" tri-peptide substrate for kinetic analysis. The method is fast and simple and will allow detailed kinetic studies and screening for natural peptide substrates of leukotriene A4 hydrolase as well as other members of the M1 family of aminopeptidases.     

Solvent-free enzymatic synthesis of alitame precursor using eutectic substrate mixtures

N-benzyloxycarbonyl-L-aspartic acid ethyl ester-D-alanine amide, a derivative of alitame, was synthesized from a eutectic mixture of the substrates N-benzyloxycarbonyl-L-aspartic acid diethyl ester and D-alanine amide using alpha-chymotrypsin. The hydrophilic solvents DMSO and MEA were found to be the best adjuvants for formation of a eutectic substrate mixture. A low eutectic temperature of 27 degrees C was obtained for the substrate mixture containing 9% DMSO, 18% MEA, and 12% water. Under these conditions a conversion yield of 70.3% (mol/mol) was obtained at 37 degrees C. The optimum molar ratio of the acyl acceptor D-alanine amide and the acyl donor N-benzyloxycarbonyl-L-aspartic acid diethyl ester was 1:1.     

酶反应速率方程的普适形式

酶反应速率方程的普适形式是应用于相互关联的大规模代谢途径动力学建模的重要方法.把酶反应速率方程写成Michaelis-Menten-King-Altman方程形式可以使得动力学参数(或函数)容易与数据库中的实验数据相接轨,并可以处理任意数量的底物和产物,有利于大规模的计算.普适形式可以同时描述正、负反应方向,并能精确地用于准稳态条件.展示了在三类生物体系中广泛存在的酶反应机制中普适方程的严格推导过程,并讨论了普适方程的特点,针对不可逆反应酶反应产生的产物抑制效应可以自然消除,总结了在普适速率方程中体现调节剂的作用和协同作用.     

Development of a recovery and recycle process for a pseudomonas lipase used for large-scale enzymatic synthesis

Enzymes are potential catalysts for a wide range of large-scale chemical synthesis steps, particularly when the creation of a specific chiral center is desired. The efficient recycling of the enzyme catalyst and the removal of carryover impurities were crucial factors in the improvement of a stereoselective ester hydrolysis step used in the synthesis of a selective leukotriene antagonist. In this enzymatic reaction step, the substrate and product were both largely insoluble, while the enzyme was soluble in the aqueous reaction mixture. Microfiltration and ultrafiltration of the slurry reaction mother liquor indicated near 100% enzyme protein recovery, while activity recovery was about 70% to 80%. These activity losses might be accounted for by enzyme degradation (1 to 2 mg/L . h) during the 40-hour reaction period. Dissolved impurities, principally a diacid byproduct, in the enzyme recycling stream were reduced 60% to 70% by either lowering the solution pH to 4.0 or raising the solution ionic strength to 1 M. (c) 1993 John Wiley & Sons, Inc.     

Metabolic control analysis of an enzymatic biofuel cell

Metabolic control analysis (MCA) is an analytical technique that aims to quantify the distribution of control that enzymes exhibit over the steady‐state fluxes through a metabolic network. In an enzymatic biofuel cell, the flux of interest is the electrical current generated by the system. Regardless of transport limitations and other constraints, kinetic limitations can become potential bottlenecks in the operation of a biofuel cell. We have used an indirect approach to MCA to investigate a common osmium‐mediated glucose oxidase/laccase enzymatic biofuel cell. The results of the analysis show that the control of the electron flux strongly depends on the total mediator concentrations and the extent of polarization of the individual electrodes. The effect of varying oxygen concentrations is also examined, as oxygen is required for the cathode, but it participates in a non‐productive reaction at the anode. Under normal operating conditions the electrodes will be highly polarized and will both contain high mediator concentrations. This configuration will result in a dominant FCC at the anode, and the conditions that are needed for balanced flux control between the anode and cathode are explored. As increasingly complex bioelectrocatalytic systems and architectures are envisioned, MCA will be a valuable framework to facilitate their design and subsequent operation. Biotechnol. Bioeng. 2009;102: 1624–1635. © 2008 Wiley Periodicals, Inc.     

Combinatorial array-based enzymatic polyester synthesis.

A combinatorial strategy for biocatalytic polymer synthesis is demonstrated. A library of polymers was synthesized in 96 deep-well plates using AA-BB polycondensations of acyl donors and acceptors. The library was based on four straight-chain diesters as acyl donors (C(3)-C(10)) with aliphatic/aromatic diols as well as more diverse structures including carbohydrates, nucleic acids, and a natural steroid diol used as acyl acceptors. The lipase from Candida antarctica was active in acetonitrile and was capable of catalyzing the polycondensation of the aforementioned monomers to polymers with M(w)'s reaching as high as 20,000 Da, including the preparation of novel sugar-containing polyesters. The combinatorial approach to biocatalytic polymer synthesis described herein serves as a foundation for polymeric materials discovery by demonstrating that polymer arrays can be produced from structurally complex monomers.     

Apparent cooperativity and apparent hyperbolic behavior of enzyme mixtures acting on the same substrate

It is well known that a negative cooperative behavior displayed by a monomeric enzyme may be associated with the simultaneous presence of two enzymes acting on the same substrate. In this paper, emphasis is given to the effect exerted by a rapid equilibrium between the enzyme forms in leading to a hyperbolic behavior, thus masking the presence of multiple enzyme forms.     

Enantiopure building blocks for chiral drugs from racemic mixtures of secondary alcohols by combination of lipase catalysis and Mitsunobu esterification.

The racemic alcohols 3-chloro-1-(2-thienyl)-1-propanol, 3-chloro-1-phenylpropanol, and 1-chloro-3-(3,4-difluorophenoxy)-2-propanol were converted into a mixture of one enantiomer as butanoate and the other as alcohol by lipase catalysis. Subsequent Mitsunobu esterification without separation proceeded with inversion of the unreacted alcohols to give high yield and ee of the three enantiopure butanoates. The butanoates of opposite configuration were produced in a similar manner, but starting with lipase-catalyzed hydrolysis of the racemic butanoates.     

Molecular binding scaffolds increase local substrate concentration enhancing the enzymatic hydrolysis of VX nerve agent

Kinetic enhancement of organophosphate hydrolysis is a long-standing challenge in catalysis. For prophylactic treatment against organophosphate exposure, enzymatic hydrolysis needs to occur at high rates in the presence of low substrate concentrations and enzymatic activity should persist over days and weeks. Here, the conjugation of small DNA scaffolds was used to introduce substrate binding sites with micromolar affinity to VX, paraoxon, and methyl-parathion in close proximity to the enzyme phosphotriesterase (PTE). The result was a decrease in K_M and increase in the rate at low substrate concentrations. An optimized system for paraoxon hydrolysis decreased K_M by 11-fold, with a corresponding increase in second-order rate constant. The initial rates of VX and methyl-parathion hydrolysis were also increased by 3.1- and 6.7-fold, respectively. The designed scaffolds not only increased the local substrate concentration, but they also resulted in increased stability and PTE-DNA particle size tuning between 25 and ~150 nm. The scaffold engineering approach taken here is focused on altering the local chemical and physical microenvironment around the enzyme and is therefore compatible with active site engineering via combinatorial and computational approaches.     

Evaluation of the factors affecting avicel reactivity using multi-stage enzymatic hydrolysis

Multi-stage and single-stage enzymatic hydrolysis of cellulose (Avicel PH-101) were conducted to investigate individual factors that affect the rate-reducing kinetics of enzymatic hydrolysis. Understanding factors affecting enzymatic hydrolysis of Avicel will help improve hydrolysis of various biomasses. Product inhibition, enzyme deactivation, and the changes of substrate are potential factors that can affect the hydrolysis efficiency of Avicel. Multi-stage enzymatic hydrolysis resulted in 36.9% and 25.4% higher carbohydrate conversion as compared to a single-stage enzymatic hydrolysis with an enzyme loading of 5 and 20 FPU/g in a 96 h reaction. However, a decline in carbohydrate conversion of 1.6% and 2.6% was observed through each stage with 5 and 20 FPU/g, respectively. This indicated that the substrate became more recalcitrant as hydrolysis progressed. The decreased reactivity was not due to crystallinity because no significant change in crystallinity was detected by X-ray diffraction. Product inhibition was significant at low enzyme loading, while it was marginal at high enzyme loading. Therefore, product inhibition can only partially explain this decreased conversion. Another important factor, enzyme deactivation, contributed to 20.3% and 25.4% decrease in the total carbohydrate conversion of 96 h hydrolysis with 5 and 20 FPU/g, respectively. This work shows that an important reason for the decreased Avicel digestibility is the effect of enzyme blockage, which refers to the enzymes that irreversibly adsorb on accessible sites of substrate. About 45.3% and 63.2% of the total decreased conversion at the end of the 8th stage with 5 and 20 FPU/g, respectively, was due to the presence of irreversibly adsorbed enzymes. This blockage of active sites by enzymes has been speculated by other researchers, but this article shows further evidence of this effect.     

The effect of organic solvents on the equilibrium position of enzymatic acylglycerol synthesis

The effect of organic solvents on the equilibrium position of lipase-catalyzed esterification of glycerol and decanoic acid has been investigated. The reaction is carried out in an aqueous-organic two-phase system. In polar solvents, high mole fractions of monoacylglycerol and low mole fractions of triacylglycerol and measured, while in nonpolar solvents, the measured differences in the mole fractions of monodi-, and triacylglycerols are less. There is a good correlation between the ester mole fractions at equilibrium and the log P of the solvent (partition coefficient in n-octanolwater), however, only if the group of tertiary alcohols is excluded. In the plot of the easter mole fractions as a function of the logarithm of hte solubility of water in the organic solvent, the tertiary alcohols can be included; however, in this case other deviations appear.For the prediction of the effect of organic solvents on the ester mole fractions at reaction equilibrium in nondilute reaction systems with a water activity below 1, the program TREP (Two-phase Reaction Equilibrium Prediction) is developed, which is based on the UNIFAC group contribution method. With this model the equilibrium data are essentially predicted from basic thermodynamic data. The required equilibrium constants are estimated from experiments without an organic solvent in the reaction medium. The mole fractions calculated by TREP show the same trends as the experimentally measured mole fractions; however, some variation is observed in the absolute values. These deviations may be due to inaccuracies in the UNIFAC group contribution method. TREP is found to be a correct method to predict within some limits the ester mole fractions at equilibrium for all mixtures of solvents, substrates, and products. The production of monoester can be enhanced in reaction system with a sufficient high concentration of a polar solvent. In experiments with a triglymeto-decanoic acid ratio of 5, almost no di-and triesters can be detected at equilibrium. (c) 1993 John Wiley & Sons, Inc.     

Continuous reaction-separation process for enzymatic esterification in supercritical carbon dioxide

The study of enzymatic esterification by an immobilized lipase in supercritical carbon dioxide (SCCO(2)) and in n-hexane, described in our previous works, was extended to continuous operation in a tubular fixed bed. The modeling of the reaction vessel operation was achieved through the use of the simple plug flow model coupled with the appropriate kinetic equation. Comparison with experiments proved to be satisfactory. The study of the postreactional separation, an important feature when using SCCO(2), was undertaken experimentally and good selectivities and product recovery were obtained. (c) 1994 John Wiley & Sons, Inc.     

Total synthesis of (+)-macrosphelides A,C, E,F, and G based on enzymatic function

The total synthesis of (+)-macrosphelide A (1) (18.5% overall yield in 11 steps), (+)-macrosphelide C (2) (25% overall yield in 9 steps), (+)-macrosphelide E (3) (23.9% overall yield in 11 steps), (+)-macrosphelide F (4) (20% overall yield in 9 steps), and (+)-macrosphelide G (5) (22% overall yield in 9 steps) was achieved from a chemoenzymatic reaction product (4R,5S)-4-benzyloxy-5-hydroxy-2(E)-hexenoate 10.