Chemo-enzymatic synthesis of unnatural amino acids

A general chemo-enzymatic process has been developed to prepare enantiomerically pure L- and D-amino acids in high yield by deracemisation of racemic starting materials. The method has been developed from initial academic studies to be a robust, scalable industrial process. Unnatural amino acids, in high optical purity, are a rapidly growing class of intermediates required for pharmaceuticals, agrochemicals and other fine chemical applications. However, no single method has proven sufficiently adaptable to prepare these compounds generally at large scale. Our approach uses an enantioselective oxidase biocatalyst and a non-selective chemical reducing agent to effect the stereoinversion of one enantiomer and can result in an enantiomeric excess of > 99 % from a starting racemate, and product yields over 90 %. The current approach compares very favourably to resolution methods which have a maximum single pass yield of 50 %. Efficient methods have been developed to adapt the biocatalyst used in this process towards new target compounds and to optimise key factors which improve the process efficiency and offer competitive economics at scale.

Kinetics of enzymatic synthesis of geranyl butyrate by transesterification in various supercritical fluids

The transesterification of methyl butyrate, ethyl butyrate and butyl butyrate to geranyl butyrate was investigated in supercritical carbon dioxide. The effect of chain length of the butyrate on the rate of transesterification was investigated. The initial rates followed the trend: ethyl butyrate < butyl butyrate < methyl butyrate. The transesterification of butyl butyrate to geranyl butyrate in various supercritical fluids such as ethylene, methane, ethane was also examined. The initial rate of transesterification of butyl butyrate in different supercritical fluids followed the order: ScCO2 < ScC2H6 < ScC2H4 < ScCH4. The highest initial rate was obtained in supercritical methane and the reasons for this observation were proposed. The Ping-Pong Bi-Bi model with inhibition by both acid and alcohol was used to model the experimental data and determine the kinetics of the reaction. (C) 2010 Elsevier B.V. All rights reserved.

Enzymatic synthesis of flavors in supercritical carbon dioxide

Commercially important flavor esters of isoamyl alcohol, catalyzed by crude hog pancreas lipase (HPL), were synthesized under solvent-free conditions and in supercritical carbon dioxide. The esters synthesized were isoamyl acetate, isoamyl propionate, isoamyl butyrate, and isoamyl octanoate. Very low yields (3-4%) of isoamyl acetate were obtained, but high yields for the other three esters were obtained under both supercritical and solvent-free conditions. The yields of esters of the even-carbon acids, isoamyl acetate, butyrate, and octanoate, increased with increasing chain length, whereas the yield of isoamyl propionate was higher than that of isoamyl butyrate. The optimum temperature of the reaction was higher under supercritical conditions (45 degreesC) than under solvent-free conditions (35-40 degreesC). The effects of other parameters such as alcohol concentration, water concentration, and enzyme loading were investigated. An increase in the water concentration decreased the conversion significantly in supercritical carbon dioxide but not under solvent-free conditions. The optimum ratio of alcohol to acid was dependent on the extent of inhibition by the acid. Although providing a higher apparent yield by being run in a highly concentrated medium, the overall conversion under solvent-free conditions was lower than that under supercritical conditions for similar enzyme concentrations, indicating that the synthesis of esters in supercritical carbon dioxide might be a viable option.

Enzymatic synthesis of isopropyl myristate using immobilized lipase from Bacillus cereus MTCC 8372

A purified alkaline thermo-tolerant bacterial lipase from Bacillus cereus MTCC 8372 was immobilized on a Poly (MAc- co -DMA- cl -MBAm) hydrogel. The hydrogel showed approximately 94% binding capacity for lipase. The immobilized lipase (2.36 IU) was used to achieve esterification of myristic acid and isopropanol in n -heptane at 65 °C under continuous shaking. The myristic acid and isopropanol when used at a concentration of 100 mM each in n -heptane resulted in formation of isopropyl myristate (66.0 ± 0.3 mM) in 15 h. The reaction temperature below or higher than 65°C markedly reduced the formation of isopropyl myristate. Addition of a molecular sieve (3 Å × 1.5 mm) to the reaction mixture drastically reduced the ester formation. The hydrogel bound lipase when repetitively used to perform esterification under optimized conditions resulted in 38.0 ± 0.2 mM isopropyl myristate after the 3 ^rd cycle of esterification.

Enzymatic synthesis of isopropyl acetate catalysed by immobilized bacillus cereus lipase in organic medium

Selective production of fragrance fatty acid ester from isopropanol and acetic acid has been achieved using silica-immobilizedlipase of Bacillus cereus MTCC 8372. A purified thermoalkalophilic extracellular lipase was immobilized by adsorption onto the silica. The effects of various parameters like molar ratio of substrates (isopropanol and acetic acid; 25 to 100 mM), concentration ofbiocatalyst (25–125 mg/mL), reaction time, reaction temperature, organic solvents,molecular sieves, and initial water activity werestudied for optimal ester synthesis. Under optimized conditions, 66.0mM of isopropyl acetate was produced when isopropanol and acetic acid were used at 100mM: 75mM in 9h at 55◦C in n-heptane under continuous shaking (160 rpm) using bound lipase(25mg). Addition of molecular sieves (3 °A ×1.5mm) resulted in a marked increase in ester synthesis (73.0mM). Ester synthesiswas enhanced by water activity associated with pre-equilibrated saturated salt solution of LiCl. The immobilized lipase retained more than 50% of its activity after the 6th cycle of reuse.

Microwave-assisted enzymatic synthesis of beef tallow biodiesel

Optimal conditions for the microwave-assisted enzymatic synthesis of biodiesel have been developed by a full 2(2) factorial design leading to a set of seven runs with different combinations of molar ratio and temperature. The main goal was to reduce the reaction time preliminarily established by a process of conventional heating. Reactions yielding biodiesel, in which beef tallow and ethanol used as raw materials were catalyzed by lipase from Burkholderia cepacia immobilized on silica-PVA and microwave irradiations within the range of 8-15 W were performed to reach the reaction temperature. Under optimized conditions (1:6 molar ratio of beef tallow to ethanol molar ratio at 50A degrees C) almost total conversion of the fatty acid presented in the original beef tallow was converted into ethyl esters in a reaction that required 8 h, i.e., a productivity of about 92 mg ethyl esters g(-1) h(-1). This represents an increase of sixfold for the process carried out under conventional heating. In general, the process promises low energy demand and higher biodiesel productivity. The microwave assistance speeds up the enzyme catalyzed reactions, decreases the destructive effects on the enzyme of the operational conditions such as, higher temperature, stability, and specificity to its substrate, and allows the entire reaction medium to be heated uniformly.

Floral Scent Production in Clarkia breweri1 : III. Enzymatic Synthesis and Emission of Benzenoid Esters

The fragrance of Clarkia breweri (Onagraceae), a California annual plant, includes three benzenoid esters: benzylacetate, benzylbenzoate, and methylsalicylate. Here we report that petal tissue was responsible for the benzylacetate and methylsalicylate emission, whereas the pistil was the main source of benzylbenzoate. The activities of two novel enzymes, acetyl-coenzyme A:benzylalcohol acetyltransferase (BEAT), which catalyzes the acetyl esterification of benzylalcohol, and S-adenosyl-l-methionine:salicylic acid carboxyl methyltransferase, which catalyzes the methyl esterification of salicylic acid, were also highest in petal tissue and absent in leaves. In addition, the activity of both enzymes in the various floral organs was developmentally and differentially regulated. S-Adenosyl-l-methionine:salicylic acid carboxyl methyltransferase activity in petals peaked in mature buds and declined during the next few days after anthesis, and it showed a strong, positive correlation with the emission of methylsalicylate. The levels of BEAT activity and benzylacetate emission in petals also increased in parallel as the buds matured and the flowers opened, but as emission began to decline on the 2nd d after anthesis, BEAT activity continued to increase and remained high until the end of the lifespan of the flower.

Synthesis of biodiesel in supercritical alcohols and supercritical carbon dioxide

Biodiesel was synthesized in supercritical fluids by two routes: non-catalytically in supercritical alcohols and by enzyme catalysis in supercritical carbon dioxide. Two oils, sesame oil and mustard oil, and two alcohols, methanol and ethanol, were used for the synthesis. Complete conversion was observed for synthesis in supercritical alcohols whereas only a maximum of 70% conversion was observed for the enzymatic synthesis in supercritical carbon dioxide. For the synthesis in supercritical alcohols, the activation energies and pseudo-first order rate constants were determined. For the reactions in supercritical carbon dioxide, a mechanism based on ping pong bi-bi was proposed and the kinetic parameters were determined. (C) 2009 Elsevier Ltd. All rights reserved.

Enzymatic Polymerization of High Molecular Weight DNA

The use of DNA as a polymeric building material transcends its function in biology and is exciting in bionanotechnology for applications ranging from biosensing, to diagnostics, and to targeted drug delivery. These applications are enabled by DNA’s unique structural and chemical properties, embodied as a directional polyanion that exhibits molecular recognition capabilities. Hence, the efficient and precise synthesis of high molecular weight DNA materials has become key to advance DNA bionanotechnology. Current synthesis methods largely rely on either solid phase chemical synthesis or template-dependent polymerase amplification. The inherent step-by-step fashion of solid phase synthesis limits the length of the resulting DNA to typically less than 150 nucleotides. In contrast, polymerase based enzymatic synthesis methods (e.g., polymerase chain reaction) are not limited by product length, but require a DNA template to guide the synthesis. Furthermore, advanced DNA bionanotechnology requires tailorable structural and self-assembly properties. Current synthesis methods, however, often involve multiple conjugating reactions and extensive purification steps.

The research described in this dissertation aims to develop a facile method to synthesize high molecular weight, single stranded DNA (or polynucleotide) with versatile functionalities. We exploit the ability of a template-independent DNA polymerase−terminal deoxynucleotidyl transferase (TdT) to catalyze the polymerization of 2’-deoxyribonucleoside 5’-triphosphates (dNTP, monomer) from the 3’-hydroxyl group of an oligodeoxyribonucleotide (initiator). We termed this enzymatic synthesis method: TdT catalyzed enzymatic polymerization, or TcEP.

Specifically, this dissertation is structured to address three specific research aims. With the objective to generate high molecular weight polynucleotides, Specific Aim 1 studies the reaction kinetics of TcEP by investigating the polymerization of 2’-deoxythymidine 5’-triphosphates (monomer) from the 3’-hydroxyl group of oligodeoxyribothymidine (initiator) using in situ 1H NMR and fluorescent gel electrophoresis. We found that TcEP kinetics follows the “living” chain-growth polycondensation mechanism, and like in “living” polymerizations, the molecular weight of the final product is determined by the starting molar ratio of monomer to initiator. The distribution of the molecular weight is crucially influenced by the molar ratio of initiator to TdT. We developed a reaction kinetics model that allows us to quantitatively describe the reaction and predict the molecular weight of the reaction products.

Specific Aim 2 further explores TcEP’s ability to transcend homo-polynucleotide synthesis by varying the choices of initiators and monomers. We investigated the effects of initiator length and sequence on TcEP, and found that the minimum length of an effective initiator should be 10 nucleotides and that the formation of secondary structures close to the 3’-hydroxyl group can impede the polymerization reaction. We also demonstrated TcEP’s capacity to incorporate a wide range of unnatural dNTPs into the growing chain, such as, hydrophobic fluorescent dNTP and fluoro modified dNTP. By harnessing the encoded nucleotide sequence of an initiator and the chemical diversity of monomers, TcEP enables us to introduce molecular recognition capabilities and chemical functionalities on the 5’-terminus and 3’-terminus, respectively.

Building on TcEP’s synthesis capacities, in Specific Aim 3 we invented a two-step strategy to synthesize diblock amphiphilic polynucleotides, in which the first, hydrophilic block serves as a macro-initiator for the growth of the second block, comprised of natural and/or unnatural nucleotides. By tuning the hydrophilic length, we synthesized the amphiphilic diblock polynucleotides that can self-assemble into micellar structures ranging from star-like to crew-cut morphologies. The observed self-assembly behaviors agree with predictions from dissipative particle dynamics simulations as well as scaling law for polyelectrolyte block copolymers.

In summary, we developed an enzymatic synthesis method (i.e., TcEP) that enables the facile synthesis of high molecular weight polynucleotides with low polydispersity. Although we can control the nucleotide sequence only to a limited extent, TcEP offers a method to integrate an oligodeoxyribonucleotide with specific sequence at the 5’-terminus and to incorporate functional groups along the growing chains simultaneously. Additionally, we used TcEP to synthesize amphiphilic polynucleotides that display self-assemble ability. We anticipate that our facile synthesis method will not only advance molecular biology, but also invigorate materials science and bionanotechnology.

Produção de biodiesel a partir da transesterificação de óleo de soja ácido por via enzimática

Neste trabalho, foi estudada a transesterificação enzimática do óleo de soja com álcool catalisada por lipase comercial imobilizada. Inicialmente foram investigados os efeitos da temperatura, do tipo de enzima (Novozym 435, Lipozyme RM-IM e Lipozyme TL-IM) e do tipo de álcool (etanol ou butanol) na síntese de biodiesel utilizando óleo de soja refinado. A melhor temperatura observada para as reações empregando Lipozyme TL IM e Lipozyme RM IM foi de 50C, enquanto que para a Novozym 435, a temperatura ótima foi de 70C. O maior teor em biodiesel (~60%) foi obtido na etanólise do óleo de soja a 70C utilizando Novozym 435 a 5% m/m. Também foi avaliada a síntese de biodiesel por via enzimática a partir da etanólise de óleos ácidos com índice de acidez (IA) (de 8,5, 54,4 e 93,7). A conversão do ácido graxo livre foi superior a 90% nas reações conduzidas com Novozym 435 nas reações com os três óleos ácidos testados. O teor em biodiesel foi próximo a 50% na etanólise do óleo de soja com índice de acidez de 8,5 empregando Lipozyme TL IM, porém para índices de acidez maiores este rendimento diminuiu. Para as reações conduzidas com Lipozyme RM-IM, o teor em biodiesel manteve-se em torno de 30% para todos os óleos ácidos investigados. A etanólise do óleo de soja refinado empregando reator de leito fixo em modo contínuo, a 50C, foi investigada variando a velocidade espacial dos reagentes (0,255 e 0,508 h-1), o tipo de lipase (Novozym 435, Lipozyme RM-IM e Lipozyme TL-IM) e a possibilidade de reuso do biocatalisador após lavagem com butanol. Na reação com Novozym 435, o teor em biodiesel foi de aproximadamente 50% e se manteve estável durante 4,5 h para as duas velocidades espaciais testadas. A lipase Lipozyme TL-IM apresentou teor em biodiesel maior do que o obtido nas reações com os outros biocatalisadores (~80%), porém não apresentou a mesma estabilidade. Foi observada uma queda significativa na produção de biodiesel nas reações empregando o reuso do biocatalisador

真菌单宁酶发酵五倍子和有机溶剂中酶法合成没食子酸丙酯的研究

本文报告了丝状真菌单宁酶发酵五倍子及有机溶剂中酶法合成没食子酸丙酯的研究。利用单宁和/或五倍子诱导丝状真菌产生单宁 酶的原理，借助二级发酵程序，对从天然源得到的75株菌进行了生物转化实验研究。选择出既能水解单宁或五倍子成没食子酸，又 能把没食子酸和丙醇合成没食子酸丙酯，而且生物催化活性都较高的1株菌，这株菌经初步鉴定为黑曲霉(Aspergillus niger No.17)。随后对它开展了产酶条件和参数优化实验，得出了最佳培养条件。立足于参数优化实验方案的基础上，经由液体培养发酵 制备单宁酶制剂，并把该酶通过化学手段共价结合到一种新型载体—聚乙烯醇和戊二醛反应生成的缩醛上，制备得到固定化单宁酶 。这种固定化生物催化剂在两种有机介质体系中都具有逆向催化合成没食子酸丙酯的能力。最后建立起来一条有效可行的微生物酶 法制备没食子酸的技术途径，没食子酸产率达到70%。对这种物质进行元素 分析：含C,49.45%;含H,3.63%。它的熔点为237℃～243 ℃，三种溶剂系统的TLC均只给出一个斑点。这些数据都与标准品一致。有机溶剂中酶法合成没食子酸丙酯的技术途径已经建立。 水溶性单宁酶在潜溶剂体系中也能催化上述酯化反应，反应混合物中的PG浓度为16.4mmol/L，制备薄层被用于分离反应混合物所含 的PG，这种产物被红外、质谱及三种溶剂系统的TLC等方法鉴定，确证为目标产物。在这一学位论文的实验研究过程中，还包括一 些生化分析方法的建立和应用，这些方法用于鉴定底物和产物及测定它们的浓度，其内容主要包括TLC定性/半定量分析、元素分析 、质谱、红外等手段的综合运用。本工作为开发我国特有的天然产物资源—五倍子的生物化工加工技术及非水相生物催化技术的开 发，提供了有用的基础数据资料，具有应用基础研究工作的重要性。In this thesis, the studies on the fermentation of Chinese gallotannin by filamentous fungi with tannase activity and enzymatic synthesis of propyl gallate(PG) in organic solvents were described through these biocatalysts. Based on the principles of induction enzyme, the tannase produced from filamentous fungi by adding tannic acid(TA) and/or Chinese gallotannin into media was investigated, and the screening experiments of bioconversion were done with 75 strains by means of a two-stage fermentation procedure. These strains were isolated with the enrichment culture technique from natural sources. Hence we selected one strain (Aspergillus niger No.17) that can not only catalyze the hydrolyses of TA and/or Chinese gallotannin into gallic acid(GA) in the liquid cultures, but also be used to synthesize PG from propanol and GA in the non-aqueous media. At the same time both of its biocatalytical activities were higher. This strain was calssified to be Aspergillus niger by the primary identification. Then optimum conditions for production of the tannase and its parameters were examined. In this way, one set of optimum culture conditions was selected. Making use of the optimum proposal, the tanase was prepared through a liquid fermentation procedure. The enzyme was convalently coupled to a new type of carrier which was made chemically from polyvinyl alcohol(PVA)and glutaraldehyde. The immobilized enzymes were able to synthesize PG reversely in two organic media. Finally, an effective enzymatic technique for production of GA was developed. The yield of GA products was up to 70%。Element analysis for this substance: calce: C, 49.42%; H, 3.56%; found: C, 49.45%, H, 3.63%. Its melting point was 237℃～ 243℃ and TLCs on three solvent systems gave only one spot respectively. These data were identical with theauthentic GA. The enzymatic synthesis of PG in organic solvents was extablished with reverse route of tannase catalytical hydrolysis. Aqueous enzyme perparation also catalyzed above esterification in a buffer system. The PG concentration in the reaction mixture was 16.4mmol/L. The reparative-scale TLC was used to isolate PG from the reaction mixture. This product separated was identified by IR, MS and TLC on three solvent systems. In this study of thesis, some biochemical analytical mehtods were developed and used to identify substrates and products, and to determinate their concentration. These methods, including TLC qualitative/half quantitative analysis, element analysis, MS, IR and so on, were useful, available and performable. This work provided basic data and information for developing the biochemical engineering and bio-processing of Chinese gallotannin-a special natural resource in China and the non-aqueous phase biocatalysis. Thus, this study possesses importance in the applied and basic research work.