Biodiesel production via enzymatic catalysis

A review of current work in biodiesel production via enzymatic catalysis has been done. The parameters of the process as determined by laboratories are represented and analyzed. The main factors affecting interesterification are considered. The major types of oils and alcohols used in biodiesel synthesis are listed. The means of lipase enzyme immobilization, including exposure on the cell surface, are discussed.     

洋葱伯克霍尔德菌脂肪酶：一种应用前景广阔的生物催化剂

洋葱伯克霍尔德菌脂肪酶对有机溶剂（醇）、热、氧化剂、表面活性剂、去污剂、蛋白酶等具有良好的抗性,在有机合成、对映体拆分、非水相催化等领域应用十分广泛。综述了洋葱伯克霍尔德菌脂肪酶的发酵生产、分离纯化、基因克隆与表达、固定化与生物印迹、蛋白质结构解析及应用研究等,并展望了其未来发展方向,以期为该工业酶的研发与广泛应用提供参考。     

Chemoenzymatic Preparation of Optically Active Long-Chain 3-Hydroxyalkanoates

The synthesis of optically active 3-hydroxyalkanoates of varying chain length (C_lo, C₁₄ and C₁₈) was investigated using chemoenzymatic methods. While bakers' yeast mediated reduction of 3-oxoalkanoates gave (R)-3-hydroxyalkanoates (e.e. >80%, yield —10%), both enantiomers were obtained by enzymatic resolution of racemic methyl 3-butanoyloxyalkanoates using Geotrichum candidum lipase with moderate to good optical purity (e.e. 32–92%, yield 40–60% for both enantiomers). Lipase-catalysed interesterification, however, was found to proceed with similar enantioselectivity but with slow rate of conversion.     

pH-imprinted lipase catalyzed synthesis of dextran fatty acid ester

The application of enzymatic catalysis for the synthesis of polysaccharide-based surfactants was investigated. The polysaccharide dextran, a neutral bacterial polysaccharide consisting of -1,6 linked glucose units, was chemically modified by the attachment of hydrophobic groups through a transesterification reaction with a vinyl decanoate. A screening of commercially available lipases and protease for the synthesis of amphiphilic polysaccharides in DMSO suggested that lipase AY from Candida rugosa modified dextran T-40 with vinyl decanoate at the highest conversion. A pH-adjustment in a phosphate buffer at pH 7.5 prior to use is crucial to make this enzyme active in DMSO. The effect of enzyme concentration and mole ratio of fatty ester to dextran T-40 on the conversion and the rate of reaction were studied. Finally, investigation of the kinetics and regioselectivity of lipase AY-catalyzed modification offer a possibility to regulate the position and the extent of hydrophobic group attached to dextran. These two properties are fundamental for controlling the physico-chemical properties of the final polymeric surfactants.     

Microbial lipases: at the interface of aqueous and non-aqueous media. A review

In recent times, biotechnological applications of microbial lipases in synthesis of many organic molecules have rapidly increased in non-aqueous media. Microbial lipases are the 'working horses' in biocatalysis and have been extensively studied when their exceptionally high stability in non-aqueous media has been discovered. Stability of lipases in organic solvents makes them commercially feasibile in the enzymatic esterification reactions. Their stability is affected by temperature, reaction medium, water concentration and by the biocatalyst's preparation. An optimization process for ester synthesis from pilot scale to industrial scale in the reaction medium is discussed. The water released during the esterification process can be controlled over a wide range and has a profound effect on the activity of the lipases. Approaches to lipase catalysis like protein engineering, directed evolution and metagenome approach were studied. This review reports the recent development in the field ofnon-aqueous microbial lipase catalysis and factors controlling the esterification/transesterification processes in organic media.     

Dynamic kinetic resolutions and asymmetric transformations by enzymes coupled with metal catalysis

The combination of enzyme and metal catalysis is described as a useful method for the synthesis of optically active compounds. A key feature of this new methodology is the use of metal catalysts for the in situ racemization of enzymatically unreactive enantiomers in the enzymatic resolution of racemic substrates. So far, two combinations - lipase-ruthenium and lipase-palladium - have been developed for the efficient dynamic kinetic resolution of alcohols and amines. The use of these combinations has also been extended to catalysis of the asymmetric transformation of ketones, their enol acetates, and ketoximes. In most cases, enzyme-metal combination catalysis has provided good yields and high optical purities.     

Immobilized Candida antarctica lipase B: Hydration, stripping off and application in ring opening polyester synthesis

This work reviews the stripping off, role of water molecules in activity, and flexibility of immobilized Candida antarctica lipase B (CALB). Employment of CALB in ring opening polyester synthesis emphasizing on a polylactide is discussed in detail. Execution of enzymes in place of inorganic catalysts is the most green alternative for sustainable and environment friendly synthesis of products on an industrial scale. Robust immobilization and consequently performance of enzyme is the essential objective of enzyme application in industry. Water bound to the surface of an enzyme (contact class of water molecules) is inevitable for enzyme performance; it controls enzyme dynamics via flexibility changes and has intensive influence on enzyme activity. The value of pH during immobilization of CALB plays a critical role in fixing the active conformation of an enzyme. Comprehensive selection of support and protocol can develop a robust immobilized enzyme thus enhancing its performance. Organic solvents with a log P value higher than four are more suitable for enzymatic catalysis as these solvents tend to strip away very little of the enzyme surface bound water molecules. Alternatively ionic liquid can work as a more promising reaction media. Covalent immobilization is an exclusively reliable technique to circumvent the leaching of enzymes and to enhance stability. Activated polystyrene nanoparticles can prove to be a practical and economical support for chemical immobilization of CALB. In order to reduce the E-factor for the synthesis of biodegradable polymers; enzymatic ring opening polyester synthesis (eROPS) of cyclic monomers is a more sensible route for polyester synthesis. Synergies obtained from ionic liquids and immobilized enzyme can be much effective eROPS.     

Stereoselectivity of Pseudomonas cepacia lipase toward secondary alcohols: a quantitative model

The lipase from Pseudomonas cepacia represents a widely applied catalyst for highly enantioselective resolution of chiral secondary alcohols. While its stereopreference is determined predominantly by the substrate structure, stereoselectivity depends on atomic details of interactions between substrate and lipase. Thirty secondary alcohols with published E values using P. cepacia lipase in hydrolysis or esterification reactions were selected, and models of their octanoic acid esters were docked to the open conformation of P. cepacia lipase. The two enantiomers of 27 substrates bound preferentially in either of two binding modes: the fast-reacting enantiomer in a productive mode and the slow-reacting enantiomer in a nonproductive mode. Nonproductive mode of fast-reacting enantiomers was prohibited by repulsive interactions. For the slow-reacting enantiomers in the productive binding mode, the substrate pushes the active site histidine away from its proper orientation, and the distance d(H(N epsilon) - O(alc)) between the histidine side chain and the alcohol oxygen increases, d(H(N epsilon) - O(alc)) was correlated to experimentally observed enantioselectivity: in substrates for which P. cepacia lipase has high enantioselectivity (E > 100), d(H(N epsilon) - O(alc)) is >2.2 A for slow-reacting enantiomers, thus preventing efficient catalysis of this enantiomer. In substrates of low enantioselectivity (E < 20), the distance d(H(N epsilon) - O(alc)) is less than 2.0 A, and slow- and fast-reacting enantiomers are catalyzed at similar rates. For substrates of medium enantioselectivity (20 < E < 100), d(H(N epsilon) - O(alc)) is around 2.1 A. This simple model can be applied to predict enantioselectivity of P. cepacia lipase toward a broad range of secondary alcohols.     

R-stereopreference analysis of lipase Novozym 435 in kinetic resolution of flurbiprofen

Immobilized lipase from Candida antarctica (Novozym 435) was employed in the kinetic resolution of racemic flurbiprofen by enantioselective esterification with methanol. It was found that the lipase has the R-stereopreference and the reaction matches Bi Bi Ping Pong mechanism with dead-end inhibition of methanol. Furthermore, the R-stereopreference was analyzed in details from the aspects of enzymatic kinetic mechanism and reaction activation energy of both enantiomers. The R-enantiomer shows lower activation energy and higher maximum reaction rate than the S-enantiomer, which implies the R-stereopreference of the lipase and makes the kinetic resolution of flurbiprofen via enzymatic reaction feasible.     

Following the lipase catalyzed enantioselective hydrolysis of amino acid esters with capillary electrophoresis using contactless conductivity detection

The progress of the enzymatic hydrolysis of racemic mixtures of the enantiomers of the methyl esters of serine and threonine was monitored. This was possible in a reaction vessel of 1.5 mL by direct sampling of volumes in the nanoliter‐range directly into an electrophoresis capillary. Contactless conductivity detection was used for quantification as the analytes are not accessible by UV‐detection in capillary electrophoresis. Porcine pancreatic lipase and wheat germ lipase both showed a preference for the L‐enantiomers of both amino acid esters. The selectivity of the porcine lipase between the two L‐esters of the two amino acids was also studied and it was found that the production of L ‐threonine had priority over L ‐serine. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Lipase/esterase-catalyzed ring-opening polymerization: A green polyester synthesis technique

In the last decade, there has been increased interest in lipase/esterase-catalyzed ring-opening polymerization as an alternative to metal-based catalytic processes. This review focuses on three components in the reaction system, namely biocatalysts, reaction medium and monomers. Novel lipases or esterases are described with particular emphasis on, those derived from thermophiles, immobilized enzymes and recombinant whole-cell biocatalysts. Green solvents in enzymatic ring-opening polymerization, including water, ionic liquids, supercritical carbon dioxide and hydrofluorocarbon solvents, are also discussed. Enzymatic ring-opening polymerization is reviewed with regard to the variety of polymers obtainable, such as polyesters, polycarbonates, polyphosphates and polythioesters. Among these, enzymatic synthesis of polyesters has been most widely investigated, and is discussed for lactones with small to large ring sizes. Finally, the mechanism of enzymatic ring-opening polymerization is described, which is generally accepted as a monomer-activated mechanism. Overall, the review demonstrates that lipase/esterase-catalyzed synthesis of polymers via ring-opening polymerization provides an effective platform for conducting “green polymer chemistry”.     

Enantioselective synthesis of (S)-ibuprofen ester prodrug in cyclohexane by Candida rugosa lipase immobilized on Accurel MP1000

An enantioselective esterification process was developed for the synthesis of 2-N-morpholinoethyl (S)-ibuprofen ester prodrug from racemic ibuprofen by using Candida rugosa lipase immobilized on Accurel MP1000 in cyclohexane. Compared with the performance of Lipase MY, the immobilized lipase possesses a higher enzyme activity and thermal stability, but with a slightly suppressed enantioselectivity. A kinetic model was proposed and confirmed from experiments, for the simulation of time-course conversions of both enantiomers at various combinations of substrate concentrations in a batch reactor. Preliminary results of employing the proposed model and the immobilized lipase in a continuous packed-bed reactor were also reported and discussed.     

Enzymatic reactions in biotechnology (48th Bach lecture)]

The paper is the 48th Bach Lecture presented under the same title. It covers the biochemical mechanisms of the biogenesis of microbial biosynthetic products, role of acetyl-CoA, function of the succinate-glycine cycle, reactions of the hexose-monophosphate pathway of carbon metabolism. The reversible action of hydrolases in enzymatic catalysis and degradation of xenobiotics are discussed. The data on redox reactions are pooled. Such modern biotechnological processes as epoxidation, synthesis of acrylamide and some monomers involved in chemical syntheses of polymers, synthesis of oligosaccharide and fluorine-containing amino acids are considered. Promising commercial applications of biocatalysis are discussed.     

Regioselective enzymatic acylation of multi-hydroxyl compounds in organic synthesis

With current developments in enzyme-catalyzed reactions and techniques available for rational redesign of natural biocatalysts, the enzymatic biosynthesis can become one of the most valuable synthetic methods. Enzymatic regioselective catalysis in organic media has played a key role in pursuing asymmetric synthesis for active chiral compounds. Here, we shortly describe some historical issues of the rapidly growing area, enzymatic catalysis in synthetic organic chemistry and then review researches that have been carried out in the regioselective enzymatic catalysis for the past two decades. An application of this technology to the modification of some potential target drug compound will be also presented.     

Novel route for the resolution of both enantiomers of dropropizine by using oxime esters and supported lipases of Pseudomonas cepacia

Resolution of (R)- and (S)-dropropizine which is an antitussive and central sedative therapeutic agent in high optical and chemical yields was achieved by lipases of Pseudomonas cepacia supported on ceramic particles (lipase PS-C) and on diatomite (lipase PS-D) with oxime esters in organic solvents. The influence of several factors (lipase source, structural variations in oxime esters, the amount of lipase and its recyclability) on the enantioselectivity have been investigated. Different properties were used to describe the solvents, namely the hydrophobicity (quantified by log P) and the dielectic constant (epsilon). This enzymatic acylation using oxime esters was significant as only (S)-dropropizine and (R)-dropropizine monoacetate was obtained. (R)-Dropropizine monoacetate was chemically hydrolyzed to obtain (R)-dropropizine. The highest enantioselectivity was observed when O-acetyl benzophenone oxime was used. This enzymatic resolution provides a versatile method for getting the pure enantiomers of dropropizine by effectively optimizing the various reaction parameters.     

脂肪酶活性部位上的盖与界面活性

脂肪酶是一种非常重要的水解酶,在工业催化、医药和科学研究等领域中有广泛应用. 大部分脂肪酶的活性部位上方有一段被称为“盖”的α-螺旋,这种盖赋予脂肪酶在水/油界面上有特殊的催化活性,界面活性.而在单一水相或油相中却表现出低活性或无活性.界面活性与盖的组成、大小、构象及其存在环境等密切相关,探明盖与脂肪酶界面活性的关系对于脂肪酶的开发和利用是非常关键的.因此,长期以来人们对盖在脂肪酶催化作用中所扮演的角色进行了孜孜不倦的探索.本文从盖的构象、移动、组成和删除等方面综述了其对脂肪酶催化作用的影响,期望对人们认识脂肪酶盖与其催化作用之间的关系有一定的帮助.     

Lipase-catalyzed ring-opening polymerization of lactones to polyesters and its mechanistic aspects.

Lipase catalysis induced a ring-opening polymerization of lactones with different ring-sizes. Small-size (four-membered) and medium-size lactones (six- and seven-membered) as well as macrolides (12-, 13-, 16-, and 17-membered) were subjected to lipase-catalyzed polymerization. The polymerization behaviors depended primarily on the lipase origin and the monomer structure. The macrolides showing much lower anionic polymerizability were enzymatically polymerized faster than epsilon-caprolactone. The granular immobilized lipase derived from Candida antartica showed extremely efficient catalysis in the polymerization of epsilon-caprolactone. Single-step terminal functionalization of the polyester was achieved by initiator and terminator methods. The enzymatic polymerizability of lactones was quantitatively evaluated by Michaelis-Menten kinetics.     

Whole-cell biocatalysts for biodiesel fuel production

Biodiesel fuel (BDF), which refers to fatty acid alkyl esters, has attracted considerable attention as an environmentally friendly alternative fuel for diesel engines. Alkali catalysis is widely applied for the commercial production of BDF. However, enzymatic transesterification offers considerable advantages, including reducing process operations in biodiesel fuel production and an easy separation of the glycerol byproduct. The high cost of the lipase enzyme is the main obstacle for a commercially feasible enzymatic production of biodiesel fuels. To reduce enzyme associated process costs, the immobilization of fungal mycelium within biomass support particles (BSPs) as well as expression of the lipase enzyme on the surface of yeast cells has been developed to generate whole-cell biocatalysts for industrial applications.     

Lipase B from Candida antarctica catalyses enantioselective transesterification of 2-(4-methoxybenzyl)-1-cyclohexanols and 2-(4-methoxybenzyl)-1-cyclopentanols

The 2-(4-methoxybenzyl)-1-cyclohexanols and 2-(4-methoxybenzyl)-1-cyclopentanols are the basic structure of a series of juvenile hormone analogs which act as insect growth regulators. Their enantioselective transesterification with the lipase B from Candida antarctica produced pure enantiomers of R-cyclohexyl and R-cyclopentyl acetates (i.e. ee_p > 99%). Differences observed in the resolution of the four racemic compounds are in accordance with model structure of secondary alcohols suitable for catalysis.     

The synthesis of amphipathic prodrugs of 1,2-diol drugs with saccharide conjugates by high regioselective enzymatic protocol

A facile, high regioselective enzymatic synthesis approach for the preparation of amphipathic prodrugs with saccharides of mephenesin and chlorphenesin was developed. Firstly, transesterification of two drugs with divinyl dicarboxylates with different carbon chain length was performed under the catalysis of Candida antarctica lipase acrylic resin and Lipozyme in anhydrous acetone at 50 degrees C, respectively. A series of lipophilic derivatives with vinyl groups of mephenesin and chlorphenesin were prepared. The influences of different organic solvents, enzyme sources, reaction time, and the acylation reagents on the synthesis of vinyl esters were investigated. And then, protease-catalyzed high regioselective acylation of D-glucose and D-mannose with vinyl esters of mephenesin and chlorphenesin gave drug-saccharide derivatives in good yields. The studies of lipophilicity and hydrolysis in vitro of prodrugs verified that drug-saccharide derivatives had amphipathic properties, and both lipophilic and amphipathic drug derivatives had obvious controlled release characteristics.