Design of the pH profile for asymmetric bioreduction of ethyl 4-chloro-3-oxobutyrate on the basis of a data-driven method

The goal of this paper was to design the optimal time-varying operating pH profile in the asymmetric reduction of ethyl 4-chloro-3-oxobutyrate by baker's yeast. Ethyl (S)-4-chloro-3-hydroxybutyrate was produced to reach two important quality indices: reaction yield and product optical purity. The method integrated an orthogonal function approximation and an orthogonal array. The technique used a set of orthonormal functions as the basis for representing the possible profile. The optimal profile could be obtained if the orthogonal coefficients were properly adjusted. The orthogonal array was used to design and analyze the effect of each orthogonal coefficient in order to reach the optimal objective (quality) function. The performance based on the proposed strategy was significantly improved by over 10% compared with the traditional fixed pH or uncontrolled pH values during the reaction. The proposed method can be applied to the required dynamic profile in the bioreactor process to effectively improve the product quality, given good design directions and the advantage of the traditional statistical approach.     

Applying slow-release biocatalysis to the asymmetric reduction of ethyl 4-chloroacetoacetate

Amberlite XAD 2 resin enhanced the asymmetric reduction of ethyl 4-chloroacetoacetate (ECA) to S-4-chloro-3-hydroxybutyric acid ethyl ester as catalyzed by Saccharomyces cerevisiae. The absorbed ECA was released slowly to the solution during the reaction so that the substrate inhibition and the spontaneous chemical hydrolysis of ECA were considerably lessened. With 75 g resin l^–1 and ECA at 74 mM, the reaction yield and the product's optical purity increased from 75% to 84% and from 88% to 93%, respectively.     

Mathematical modeling of asymmetric reduction of ethyl 4-chloro acetoacetate by bakers’ yeast

A mathematic model was developed to simulate the asymmetric reduction of ethyl 4-chloro acetoacetate (ECA) by bakers’ yeast. The model of the process considered the kinetics of enzymatic reaction, the effect of substrate inhibition and the spontaneous degradation of the substrate. The reaction kinetics of the ECA degradation was determined empirically. The inhibition by the substrate was analyzed and the apparent kinetic constants of the overall enzymatic reaction, of the S-enzymes and of the R-enzymes, were estimated individually. The system of equations was solved numerically using the Runge–Kutta method. The close correlation between the predicted and experimental results concerning product formation, reaction yield and optical purity of product under various substrate concentrations, implied the reliability of the established model.     

Enantioselective microbial reduction of 3,5-dioxo-6-(benzyloxy) hexanoic acid, ethyl ester

The key chiral intermediate 3,5-dihydroxy-6-(benzyloxy) hexanoic acid, ethyl ester 2a, was made by the stereoselective microbial reduction of 3,5-dioxo-6-(benzyloxy) hexanoic acid, ethyl ester 1. Among various microbial cultures evaluated, cell suspensions of Acinetobacter calcoaceticus SC 13876 reduced 1 to 2a. The reaction yield of 85% and optical purity of 97% was obtained using glycerol-grown cells. The substrate was used at 2 g l⁻¹ and cells were used at 20% (w/v, wet cells) concentrations. The optimum pH for the reduction of 1 to 2a was 5.5 and the optimum temperature was 32°C. Cell extracts of A. calcoaceticus SC 13876 in the presence of NAD⁺, glucose, and glucose dehydrogenase reduced 1 to the corresponding monohydroxy compounds 3 and 4 [3-hydroxy-5-oxo-6-(benzyloxy) hexanoic acid ethyl ester 3, and 5-hydroxy-3-oxo-6-(benzyloxy) hexanoic acid ethyl ester 4]. Both 3 and 4 were further reduced to 2a by cell extracts. Reaction yield of 92% and optical purity of 99% were obtained when the reaction was carried out in a 1-l batch using cell extracts. The substrate was used at 10 g l⁻¹. Product 2a was isolated from the reaction mixture in 72% overall yield. The GC and HPLC area % purity of the isolated product was 99% and the optical purity was 99.5%. The reductase which converted 1 to 2a was purified about 200-fold from cell extracts of A. calcoaceticus SC 13876. The purified enzyme gave a single protein band on SDS-PAGE corresponding to 35,000 daltons.     

固定化细胞有机相催化不对称还原β-羰基酯

将酵母细胞用海藻酸钙包埋后用于有机相催化不对称还原4-氯乙酰乙酸乙酯制备光学活性的4-氯-3-羟基丁酸乙酯,从中筛选得到具有较高立体选择性和还原能力的菌株假丝酵母SW0401,将此菌株的细胞固定化细胞作为研究对象,系统考察了固定化条件、固定化细胞大小、反应溶剂、初始底物浓度、辅助底物、固定化细胞热处理和抑制剂对还原反应的影响。结果表明,上述因素对反应的摩尔转化率和产物（S）-CHBE光学纯度有显著影响。固定化时所用缓冲液的pH值为7．0时和固定化细胞颗粒平均直径为2.5mm较合适,以正己烷为反应介质时反应的摩尔转化率和产物光学纯度最优,初始底物浓度以54.7mmol／L为宜,辅助底物以1-己醇为佳。对固定化细胞的热处理和添加抑制剂烯丙醇均能够明显改善产物的光学纯度,但对提高摩尔转化率有负面影响。     

Mathematical modeling of elution curves for a protein mixture in ion exchange chromatography and for the optimal selection of operational conditions

Elution curves in ionic exchange chromatography (IEC) for a three-protein mixture (alpha-lactoalbumin, ovalbumin, and beta-lactoglobulin), carried out under different flow rates and ionic strength conditions, were simulated using two different mathematical models. These models were the Plate Model and the more fundamentally based Rate Model. Relatively low protein concentrations were used to avoid protein-protein interactions. Simulated elution curves were compared with experimental data not used for parameter identification. Deviation between experimental data and the simulated curves using the Plate Model was less than 0.0189 (absorbance units); a slightly higher deviation [0.0252 (absorbance units)] was obtained when the Rate Model was used. A cost function was built that included the effect of the different production stages, namely fermentation, purification, and concentration. These considered the effect on the performance of IEC; yield, purity, concentration and the time needed to accomplish the separation. Operational conditions in the IEC such as flow rate, ionic strength gradient and the operational time can be selected using this model in order to find the minimum cost for the protein production process depending on the characteristics of the final product desired such as purity and yield. This cost function was successfully used for the selection of the operational conditions as well as the fraction of the product to be collected (peak cutting) in IEC. It can be used for protein products with different characteristics and qualities, such as purity and yield, by choosing the appropriate parameters.     

Integration of enzyme, strain and reaction engineering to overcome limitations of baker's yeast in the asymmetric reduction of alpha-keto esters

We report on the development of a whole-cell biocatalytic system based on the popular host Saccharomyces cerevisiae that shows programmable performance and good atom economy in the reduction of alpha-keto ester substrates. The NADPH-dependent yeast reductase background was suppressed through the combined effects of overexpression of a biosynthetic NADH-active reductase (xylose reductase from Candida tenuis) to the highest possible level and the use of anaerobic reaction conditions in the presence of an ethanol co-substrate where mainly NADH is recycled. The presented multi-level engineering approach leads to significant improvements in product optical purity along with increases in the efficiency of alpha-keto ester reduction and co-substrate yield (molar ratio of formed alpha-hydroxy ester to consumed ethanol). The corresponding alpha-hydroxy esters were obtained in useful yields (>50%) with purities of > or =99.4% enantiomeric excess. The obtained co-substrate yield reached values of greater than 1.0 with acetate as the only by-product formed.     

“大豆分离蛋白”工艺用复合酶制剂的研究

大豆分离蛋白的碱浸提酸沉淀工艺,目前普遍存在蛋白质回收率低,分离蛋白成品得率低,纯度低的问题,利用复合酶制剂的作用可改变这一状况。针对工艺需要,从十余种工业酶制剂中筛选出两种蛋白酶活低、多糖降解酶活高的酶制剂;Ａｎ－７６半纤维酶和果胶酶ｕｌｔｒａｚｙｒｍ＾ＴＭ。对两种酶制剂的酶活组分、主体酶的部分酶学性质,以及酶的用量与蛋白质浸出率的关系等方面进行了大量基础实验。经优化实验确定了复合酶制剂的复配比     

Optimization of Recombinant Cytochrome P450 2C9 Protein Production in Escherichia coli DH5α by Statistically-Based Experimental Design

Cytochrome P450 (CYP) 2C9 is of major importance in drug metabolism. However, the low yield of recombinant CYP2C9 protein in E. coli strains prevents its extensive use in the study of in vitro drug metabolism. In the present study, Taguchi design and desirability function were first used to investigate the effect of medium components (glycerol, δ-ALA, IPTG, ampicillin, chloramphenicol, inoculum density, peptone, thiamine, trace elements, NH₄Cl, and MgSO₄) on recombinant human CYP2C9 production by E. coli DH5α. An L₁₂ (2¹¹) orthogonal array was used to design the experiments to screen out the most influential factors. The CYP concentration and the specific content of CYP were considered as two product quality variables. A desirability function was applied to combine these two qualities as a single objective function. Optimization via central composite design (CCD) was then undertaken to yield the best performance. The confirmation experiments indicated that the expression performance under the optimized conditions was better than those obtained under other conditions. A compromise between conflicting goals, such as achievement of good yield of recombinant CYP2C9 and facility of the following purification, was found by means of the desirability function D. This is the first report that combined Taguchi design and CCD, and performed experiments in a multiresponse framework to optimize the production of human CYP in a recombinant E. coli strain.     

固定化啤酒酵母细胞催化有机硅酮不对称还原

研究了固定化啤酒酵母细胞催化三甲基硅乙酮不对称还原反应,系统探讨了振荡速度、底物浓度、固定化细胞浓度、pH值和反应温度对反应速度、产率和产物光学纯度的影响。结果表明,上述因素对固定化啤酒酵母细胞催化三甲基硅乙酮不对称还原反应均有较显著的影响。振荡速度以150r/min为宜,底物浓度和固定化细胞浓度分别为14mmol/L和0.15g/mL较佳,适宜的pH值为7.3,最佳反应温度为25℃～30℃。在该优化反应条件下,反应最大产率和产物的光学纯度分别高达84.9%和90.2%ee。     

One‐pot synthesis of (R)‐1‐(pyridin‐4‐yl)ethyl acetate using tandem catalyst prepared by co‐immobilization of palladium and lipase on mesoporous foam: Optimization and kinetic modeling

The synthesis of (R )‐1‐(pyridin‐4‐yl)ethyl acetate was achieved over tandem palladium‐lipase catalyst with 100% selectivity using 4‐acetyl pyridine as a reactant. The 2% w /w palladium and lipase catalyst was successfully co‐immobilized in the microenvironment of the mesocellular foam and characterized by various techniques. The palladium metal from catalyst hydrogenated 4‐acetyl pyridine to form 1‐(pyridin‐4‐yl)ethanol. The generated intermediate product then underwent kinetic resolution over lipase and selectively gave (R )‐1‐(pyridin‐4‐ yl)ethyl acetate. The catalytic conditions were then studied for optimal performance of both steps. The reaction conditions were optimized to 50 °C and toluene as a solvent. Both chemical and enzymatic kinetic models of the reaction were developed for a given set of reaction conditions and kinetic parameters were predicted. At optimal conditions, the obtained selectivity of intermediate (1‐(pyridin‐4‐yl)ethanol) was 51.38%. The final product yield of ((R )‐1‐(pyridin‐4‐yl)ethyl acetate) was 48.62%.     

蛋白质体积对其亚细胞定位的影响

为了探索N(2)-L-丙氨酰-L-谷氨酰胺的合成工艺,确定最佳工艺以符合工业化生产的需求,以L-谷氨酰胺为原料,通过氨解反应合成L-丙氨酰-L-谷氨酰胺,并对反应条件及精制工艺进行优化.实验结果显示,当设定氨解反应温度为60℃,反应压力为0.5 MPa,反应时间为5.5h,以及氨水体积与α-D-氯丙酰-L-谷氨酰胺质量之比(V∶m)为1.5∶1时,将所得粗品用75％乙醇溶液进行精制,合成得到的N(2)-L-丙氨酰-L-谷氨酰胺经检测含量为99.65％,总收率约为55.77％.结果表明,经优化后的工艺简便安全、条件温和易控、生产周期短,适合工业化大量生产,且产品纯度、收率较高.     

OPTIMIZATION OF FERMENTATION CONDITIONS AND PURIFICATION OF CORDYCEPIN FROM Cordyceps militaris

The fermentation medium and conditions for the production of cordycepin were optimized in static culture using single-factor experiments, Placket–Burman design, a central composite design, and response surface methodology. Among seven variables including temperature, pH, and the concentrations of glucose, tryptone, yeast extract, KH₂PO₄, and MgSO₄ · 7H₂O, temperature and the concentrations of yeast extract and tryptone were found to be the important factors that significantly affected cordycepin production. The optimized medium consisted of yeast extract 9.00 g/L and tryptone 17.10 g/L, while the optimized culture conditions consisted of seed age 3 days, with an inoculum size of 10% and incubation temperature of 27.1°C. A maximum cordycepin yield of 7.35 g/L was achieved in a 5-L fermenter under the optimized conditions. Next, cordycepin was partially purified and determined. The resulting product showed 90.54% high-performance liquid chromatography (HPLC)–ultraviolet (UV) purity. Therefore, cordycepin was applied to a cell viability assay on SH-SY5Y cells and RM-1 cells. Cordycepin can inhibit the proliferation of RM-1 cells with IC₅₀ of 133 µmol/L, but it has no inhibitory effect on SH-SY5Y cells.

Supplemental materials are available for this article. Go to the publisher's online edition of Preparative Biochemistry and Biotechnology to view the supplemental file.     

Enhancement of asymmetric bioreduction of ethyl 4-chloro acetoacetate by the design of composition of culture medium and reaction conditions

This study investigated the feasibility of improving the stereospecificity of yeast by the adjustment of the culture medium composition and the reaction conditions. The investigation was performed systematically, using an approach that integrates the Taguchi's array method and the steepest ascent method. The reaction yield and the product's ee were the two indexes of reaction performance. A desirability function was applied to combine these two indexes as a single objective function. The removal of peptone and malt extract from the YM medium increased the yeast's stereoselectivity, without reducing the production of biomass. The medium composition and the reaction conditions were then simultaneously optimized. The resulting optimal conditions were 30 g/l glucose for cultivation, 12 g/l yeast extract, a cultivation time of 12 h, 15 g/l glucose for reaction, 150 g/l yeast for reaction, a reaction buffer concentration of 0.2 M and a buffer pH of 8.5. Compared to the one before this study, the product's ee was improved from 82.1 to 92.3%, and the reaction yield was enhanced from 77.3 to 82.3%. Furthermore, the biomass production was increased considerably from 5.94 to 10.14 g DCW/l.     

Enzymatic synthesis of fatty acid ethyl esters by utilizing camellia oil soapstocks and diethyl carbonate

This study was reported on a novel process for fatty acid ethyl esters preparation by transesterification and esterification from renewable low-cost feedstock camellia oil soapstocks and friendly acyl acceptor diethyl carbonate. The main components of product were 83.9% ethyl oleate, 8.9% ethyl palmitate, 4.7% ethyl linoleate and 2.1% ethyl stearate, which could be used as eco-friendly renewable resources or additives of industrial solvent and fossil fuel. The effects of molar ratio of diethyl carbonate to soapstocks oil, lipases, organic solvent, reaction temperature and time were investigated, and process conditions were optimized. The yield was up to 98.4% in solvent-free system with molar ratio of diethyl carbonate to soapstocks oil 3:1 and 5% Novozym 435 (based on the weight of soapstocks oil) at 50 °C and 180 rpm for 24 h. Moreover, there was no obvious loss in the yield after lipases were reused for 10 batches without treatment under optimized conditions.     

High-level production of heterologous proteins using untreated cane molasses and corn steep liquor in Escherichia coli medium

To develop an economical industrial medium, untreated cane molasses (UCM) was tested as a carbon source for fermentation culturing of Escherichia coli. To test the industrial application of this medium, we chose a strain co-expressing a carbonyl reductase (PsCR) and a glucose dehydrogenase (BmGDH). Although corn steep liquor (CSL) could be used as an inexpensive nitrogen source to replace peptone, yeast extract could not be replaced in E. coli media. In a volume of 40 ml per 1-l flask, a cell concentration of optical density (OD₆₀₀) 15.1 and enzyme activities of 6.51 U/ml PsCR and 3.32 U/ml BmGDH were obtained in an optimized medium containing 25.66 g/l yeast extract, 3.88 g/l UCM, and 7.1% (v/v) CSL. When 3.88 g/l UCM was added to the medium at 6 h in a fed-batch process, the E. coli concentration increased to OD₆₀₀ of 24, and expression of both PsCR and BmGDH were twofold higher than that of a batch process. Recombinant cells from batch or fed-batch cultures were assayed for recombinant enzyme activity by testing the reduction of ethyl 4-chloro-3-oxobutanoate to ethyl (S)-4-chloro-3-hydroxybutanoate (CHBE). Compared to cells from batch cultures, fed-batch cultured cells showed higher recombinant enzyme expression, producing 560 mM CHBE in the organic phase with a molar yield of 92% and an optical purity of the (S)-isomer of >99% enantiomeric excess.     

Production of Ethyl Butyrate by Candida rugosa Lipase Immobilized in Polyurethane

The enzymatic production of ethyl butyrate was studied: the lipase of Candida rugosa (E.C. 3.1.1.3.) was immobilized in a polyurethane matrix and subsequently introduced in an organic medium containing the substrates in appropriate concentrations. The large majority of experiments was carried out in n-hexane. Two further solvents were tested, namely n-heptane and n-dodecane. The partition coefficients matrix/solvent were estimated for the various solvent systems. The initial esterification rate, the molar yield ester/acid and the degree of conversion were found to be solvent independent when the reaction media were designed so that similar concentrations were created in the microenvironment. Initial rate experiments indicated that in n-hexane the threshold of inhibitory substrate concentrations lies (i) between 0.40 M and 0.50 M for butyric acid, according to the purity of the enzyme preparation and (ii) at 0.30 M for ethanol. Batch operational stability tests indicate that no enzyme deactivation occurs after 20 consecutive batches.     

Studies of the reductive biotransformation of selected carbonyl compounds by whole cells and extracts of baker's yeast, Saccharomyces carevisiae

The progress of reductive biotransformations of a variety of earbonyl compounds by whole cells of baker's yeast was monitored with time. Biotransformations rates ranged from 0.11 to 112.12 mg product formed per g dry yeast per h. While rapid biotransformations of citronellal and ethyl benzoylformate were observed, complete conversion of substrate to product did not occur. Reductive conversions of ethyl- and methyl-acetoacetate went to completion in 6 and 12 h respectively. Ethyl mandelate was produced stereoselectively, favoring the (R)- stereoisomer and ethyl and methyl-3-hydroxybutyrate were produced with (S)-enantiospecificity. Yeast crude extract and resuspended presence of NAD(P)H. Ethyl benzoylformate and methyl-and ethyl-acetoacetate were preferentially reduced by yeast crude extract as compared to resuspended pellet and, in the case of the former two substrates, the reaction manifested a preference for NADPH over NADH.     

Chemometric optimization of an asymmetric reduction catalyzed by baker's yeast

The stereoselective reduction of ethyl acetoacetate to (+)-(S)-ethyl 3-hydroxybutyrate catalyzed by Saccharomyces cerevisiae was optimized by means of chemometric methods. The quantitative effects of temperature, time of incubation, and concentrations of yeast and substrate on the optical purity and on the percent of reduced substrate were investigated using a factorial design at two levels. This approach gave information about the chemical behavior of the catalyst. The variability of the two responses was expressed by means of their corresponding response surfaces. Use of desirability functions allowed the overall optimization of the process, also taking into account the importance of economic factors. The investigation showed that it is possible to reduce the substrate completely obtaining (+)-(S)-ethyl 3-hydroxybutyrate with percent of enantiomeric excess>98% and, at the same time, to operate in more convenient experimental conditions than those previously reported.     

Asymmetric reduction of ethyl 4-chloro-3-oxobutanoate to ethyl (<Emphasis Type="Italic">R</Emphasis>)-4-chloro-3-hydroxybutanoate with two co-existing,recombinant<Emphasis Type="Italic"> Escherichia coli</Emphasis> strains

Two recombinant strains, E. coli M15 (pQE30-alr0307) and E. coli M15 (pQE30-gdh0310), which were constructed to express, respectively, an NADPH-dependent aldehyde reductase gene and a glucose dehydrogenase gene, were mixed in an appropriate ratio and used for the asymmetric reduction of ethyl 4-chloro-3-oxobutanoate to ethyl (R)-4-chloro-3-hydroxybutanoate. The former strain acted as catalyst and the latter functioned in NADPH regeneration. The biotransformation was completed effectively without any addition of glucose dehydrogenase or NADP⁺/NADPH. An optical purity of 99% (ee) was obtained and the product yield reached 90.5% from 28.5 mM substrate. Revisions requested 27 July 2004/23 September 2004; Revisions received 21 September 2004/29 November 2004