Using a water-immiscible ionic liquid to improve asymmetric reduction of 4-(trimethylsilyl)-3-butyn-2-one catalyzed by immobilized Candida parapsilosis CCTCC M203011 cells

Background  

Whole cells are usually employed for biocatalytic reduction reactions to ensure efficient coenzyme regeneration and to avoid problems with enzyme purification and stability. The efficiency of whole cell-catalyzed bioreduction is frequently restricted by pronounced toxicity of substrate and/or product to the microbial cells and in many instances the use of two-phase reaction systems can solve such problems. Therefore, we developed new, biphasic reaction systems with biocompatible water-immiscible ionic liquids (ILs) as alternatives to conventional organic solvents, in order to improve the asymmetric reduction of 4-(trimethylsilyl)-3-butyn-2-one (TMSB) to (S)-4-(trimethylsilyl)-3-butyn-2-ol {(S)-TMSBOL}, a key intermediate for synthesis of 5-lipoxygenase inhibitors, using immobilized Candida parapsilosis CCTCC M203011 cells as the biocatalyst.     

固定化细胞生物催化制备(S)-苯基乙二醇的研究

采用海藻酸钙固定化细胞方法能有效提高近平滑假丝酵母催化(RS)-苯基乙二醇(S-PED)去消旋化反应的稳定性,在优化反应条件下,固定化细胞催化批次由游离细胞的2次提高至6次,产物可保持高光学纯度(>95%)、高产率(>85%)。与化学合成液晶掺杂剂S-1011的理化特性对比结果表明,生物催化法制备获得的(S)-PED能够替代化学法制备的相同产品。     

Purification, characterization, gene cloning, and expression of a novel alcohol dehydrogenase with anti-prelog stereospecificity from Candida parapsilosis

An alcohol dehydrogenase from Candida parapsilosis CCTCC M203011 was characterized along with its biochemical activity and structural gene. The amino acid sequence shows similarity to those of the short-chain dehydrogenase/reductases but no overall identity to known proteins. This enzyme with unusual stereospecificity catalyzes an anti-Prelog reduction of 2-hydroxyacetophenone to (S)-1-phenyl-1,2-ethanediol.     

Efficient asymmetric reduction of 4-(trimethylsilyl)-3-butyn-2-one by Candida parapsilosis cells in an ionic liquid-containing system

Hydrophilic ionic liquids (ILs) were employed as green solvents to construct an IL-containing co-solvent system for improving the asymmetric reduction of 4-(trimethylsilyl)-3-butyn-2-one by immobilized Candida parapsilosis cells. Among 14 hydrophilic ILs examined, 1-(2'-hydroxyl)ethyl-3-methylimidazolium nitrate (C(2)OHMIM·NO(3)) was considered as the most suitable IL for the bioreduction with the fastest initial reaction rate, the highest yield and the highest product e.e., which may be due to the good biocompatibility with the cells. For a better understanding of the bioreduction performed in the C(2)OHMIM·NO(3)-containing co-solvent system, the effects of several crucial variables were systematically investigated. The optimal C(2)OHMIM·NO(3) content, substrate concentration, buffer pH, co-substrate concentration and temperature were 10% (v/v), 3.0 mmol/L, 5.0, 98.1 mmol/L and 30°C, respectively. Under the optimal conditions, the initial reaction rate, the maximum yield and the product e.e. were 17.3 μmol/h g(cell), 95.2% and >99.9%, respectively, which are much better than the corresponding results previously reported. Moreover, the immobilized cells remained more than 83% of their initial activity even after being used repeatedly for 10 batches in the C(2)OHMIM·NO(3)-containing system, exhibiting excellent operational stability.     

Efficient kinetic resolution of (R,S)-1-trimethylsilylethanol via lipase-mediated enantioselective acylation in ionic liquids

Efficient enantioselective acylation of (R,S)-1-trimethylsilylethanol {(R,S)-1-TMSE} with vinyl acetate catalyzed by immobilized lipase from Candida antarctica B (i.e., Novozym 435) was successfully conducted in ionic liquids (ILs). A remarkable enhancement in the initial rate and the enantioselectivity of the acylation was observed by using ILs as the reaction media when compared to the organic solvents tested. Also, the activity, enantioselectivity, and thermostability of Novozym 435 increased with increasing hydrophobicity of ILs. Of the six ILs examined, the IL C4MIm.PF6 gave the fastest initial rate and the highest enantioselectivity, and was consequently chosen as the favorable medium for the reaction. The optimal molar ratio of vinyl acetate to (R,S)-1-TMSE, water activity, and reaction temperature range were 4:1, 0.75, and 40 -50 degrees C, respectively, under which the initial rate and the enantioselectivity (E value) were 27.6 mM/h and 149, respectively. After a reaction time of 6 h, the ee of the remaining (S)-1-TMSE reached 97.1% at the substrate conversion of 50.7%. Additionally, Novozym 435 was effectively recycled and reused in C4MIm.PF6 for five consecutive runs without substantial lose in activity and enantioselectivity. The preparative scale kinetic resolution of (R,S)-1-TMSE in C4MIm.PF6 is shown to be very promising and useful for the industrial production of enantiopure (S)-1-TMSE.     

Highly enantioselective reduction of 4-(trimethylsilyl)-3-butyn-2-one to enantiopure (R)-4-(trimethylsilyl)-3-butyn-2-ol using a novel strain Acetobacter sp. CCTCC M209061

The biocatalytic reduction of 4-(trimethylsilyl)-3-butyn-2-one to enantiopure (R)-4-(trimethylsilyl)-3-butyn-2-ol was successfully conducted with high enantioselectivity using immobilized whole cells of a novel strain Acetobacter sp. CCTCC M209061, newly isolated from kefir. Compared with other microorganisms that were investigated, Acetobacter sp. CCTCC M209061 was shown to be more effective for the bioreduction reaction, and afforded much higher yield and product enantiomeric excess (e.e.). The optimal buffer pH, co-substrate concentration, reaction temperature, substrate concentration and shaking rate were 5.0, 130.6 mM, 30 °C, 6.0 mM and 180 r/min, respectively. Under the optimized conditions, the maximum yield and the product e.e. were 71% and >99%, respectively, which are much higher than those reported previously. Additionally, the established biocatalytic system proved to be efficient for the bioreduction of acetyltrimethylsilane to (R)-1-trimethylsilylethanol with excellent yield and product e.e. The immobilized cells manifested a good operational stability under the above reaction conditions since they retained 70% of their catalytic activity after ten cycles of use.     

Use of an ionic liquid to improve asymmetric reduction of 4′-methoxyacetophenone catalyzed by immobilized Rhodotorula sp. AS2.2241 cells

Rhodotorula sp. AS2.2241, a newly isolated strain, was used as biocatalyst for asymmetric reduction of 4′-methoxyacetophenone (MOAP) to enantiopure (S)-1-(4-methoxyphenyl)ethanol {(S)-MOPE}. Despite the improved efficiency of the reaction with immobilized cells compared to free cells, the inhibition of the reaction by substrate and product in monophasic aqueous system proved to be big problem. For high efficient biotransformation, several water-immiscible ionic liquids (ILs) were employed as green solvents to construct ionic liquid-involving biphasic systems. Of the six ILs tested, C₄MIM·PF₆ exhibited the best biocompatibility with the cells, and consequently the biocatalytic reduction proceeded with the fastest initial reaction rate and the highest maximum substrate conversion in the C₄MIM·PF₆-based biphasic system. To better understand the bioreduction conducted in the C₄MIM·PF₆-based biphasic system, various variables that influenced the performance of the reaction were examined. The optimal buffer pH, reaction temperature, volume ratio of buffer to C₄MIM·PF₆ and substrate concentration were 7.5, 25 °C, 4/1 and 40 mM, respectively. Under the optimal conditions, the initial reaction rate, maximum substrate conversion and product e.e. were 1.6 μmol/h, 95.5% and >99%, respectively. Additionally, the cells still remained above 90% of their original activity in the C₄MIM·PF₆-based biphasic system, which was much higher than that in the monophasic buffer system (about 25% of their original activity), after being repeatedly used for 8 batches (50 h per batch), indicating that C₄MIM·PF₆ markedly enhanced the operational stability of the cells.     

Purification, Characterization, Gene Cloning, and Expression of a Novel Alcohol Dehydrogenase with Anti-Prelog Stereospecificity from Candida parapsilosis

An alcohol dehydrogenase from Candida parapsilosis CCTCC M203011 was characterized along with its biochemical activity and structural gene. The amino acid sequence shows similarity to those of the short-chain dehydrogenase/reductases but no overall identity to known proteins. This enzyme with unusual stereospecificity catalyzes an anti-Prelog reduction of 2-hydroxyacetophenone to (S)-1-phenyl-1,2-ethanediol.     

重组大肠杆菌表达氧化还原酶不对称还原2-羟基苯乙酮的研究

将来源于7种微生物的13个氧化还原酶基因分别与表达载体pET21c连接后,转化入Escherichia coli BL21(DE3)中,得到13株重组菌.重组菌在IPTG诱导下进行表达,并对2-羟基苯乙酮进行不对称催化还原.研究发现,在17℃下诱导表达的重组酶比活明显高于30℃和37℃下诱导表达的比活.另外,来源于Ca...     

Enhancement of substrate concentration in microbial stereoinversion through one-pot oxidation and reduction by aqueous two-phase system

An extractive biocatalytic method of aqueous two-phase system was employed for stereoinversing (R)-1-phenyl-1,2-ethanediol into (S)-1-phenyl-1,2-ethanediol by Candida parapsilosis CCTCC M203011. It was observed that substrate and product inhibitions in microbial stereoinversion through one-pot oxidation and reduction were removed efficiently by extractive biocatalysis in aqueous two-phase system with PEG 4000/phosphate potassium system, and that the substrate concentration was enhanced from 15 to 30 g/L with product optical purity of 99.02% e.e. and yield of 90% after 60 h. Simultaneously, it was observed that change in cell morphology impedes the further enhancement of substrate concentration in this system but can be reversibly changed after stereoinversion or cultivation in systems without PEG.     

Efficient enantioselective reduction of 4′-methoxyacetophenone with immobilized Rhodotorula sp. AS2.2241 cells in a hydrophilic ionic liquid-containing co-solvent system

The biocatalytic enantioselective reduction of 4′-methoxyacetophenone to (S)-1-(4-methoxyphenyl)ethanol was successfully conducted in a hydrophilic IL-containing co-solvent system using immobilized Rhodotorula sp. AS2.2241 cells. Of all the tested ILs, the best results were observed with the novel IL 1-(2′-hydroxy)ethyl-3-methylimidazolium nitrate (C₂OHMIM·NO₃), which showed a good biocompatibility with the cells and increased the cell membrane permeability moderately, thus improving the efficiency of the bioreduction. To better understand the bioreduction, several crucial influential variables were also examined. The optimal C₂OHMIM·NO₃ content, buffer pH, reaction temperature and substrate concentration were 5.0% (v/v), 8.5, 25 °C and 12 mM, respectively. Under the optimized conditions, the initial reaction rate, the maximum yield and the product e.e. were 9.8 μmol/h g_cell, 98.3% and >99%, respectively, which are much better than the results previously reported. The established biocatalytic system has proven to be highly effective for the reduction of other aryl ketones. Also, the cells exhibited excellent operational stability in the presence of C₂OHMIM·NO₃. Moreover, the ILs can accumulate within the cells, suggesting that ILs are likely to interact with the related enzymes within the cells.     

Efficient synthesis of enantiopure (S)-4-(trimethylsilyl)-3-butyn-2-ol via asymmetric reduction of 4-(trimethylsilyl)-3-butyn-2-one with immobilized Candida parapsilosis CCTCC M203011 cells

In this paper, we show the substrate 4-(trimethylsilyl)-3-butyn-2-one is unstable, and can be easily cleaved into a carbonyl alkyne and trimethylhydroxysilane in aqueous buffer with pH above 6.0. However, this problem could be effectively solved by lowering the buffer pH. Meanwhile, the efficient synthesis of enantiopure (S)-4-(trimethylsilyl)-3-butyn-2-ol, a key intermediate for preparing a 5-lipoxygenase inhibitor, has been successfully conducted through the asymmetric reduction of 4-(trimethylsilyl)-3-butyn-2-one with immobilized Candida parapsilosis CCTCC M203011 cells. For optimization of the reaction, various influential variables, such as buffer pH, co-substrate concentration, reaction temperature and substrate concentration, were systematically examined. All the factors mentioned above had effect on the reaction to some extent. The optimal buffer pH, co-substrate concentration, reaction temperature and substrate concentration were 5.0, 65.3 mM, 30 °C and 3.0 mM, respectively, under which the maximum yield and product e.e. were as high as 81.3% and >99.9% after a reaction time of 1 h, which are much higher than the corresponding values previously reported.     

Cloning and expression of the gene encoding (R)-specific carbonyl reductase from Candida parapsilosis CCTCC M203011

The gene which encodes (R)-specific carbonyl reductase (rCR) from Candida parapsilosis CCTCC M203011 was cloned, sequenced and compared with genes from the GenBank. The results indicated that rCR gene was 1011 bp, encoding a protein of 336 amino acids with a molecular weight of 35.9 kDa, and its nucleotide sequence showed 99% similarity to those of other members of the alcohol dehydrogenase superfamily. The rCR gene could express in recombinant strain Escherichia coli JM 109, and the expression plasmid could produce (R)-1-pheny-1,2-ethanediol (100% e.e., 80.14% yield) fromβ-hydroxyacetophenone without any additive to regenerate NAD+ from NADH.     

含离子液体体系中固定化细胞转化甘草酸生成单葡萄糖醛酸基甘草次酸

研究疏水性离子液体1-丁基-3-甲基咪唑六氟磷酸盐([BMIM]PF6)与醋酸-醋酸钠缓冲液两相体系中,固定化产紫青霉Penicillium purpurogenum Li-3细胞转化甘草酸(GL)生成单葡萄糖醛酸基甘草次酸(GAMG)的反应,并与缓冲液单相体系作为对照.确定了在[BMIM]PF6/缓冲液两相体系中,最适离子液体加入比例、缓冲液pH、反应温度、底物浓度分别为10%、5.8、35℃和6.0mmol/L,在此条件下反应58h,甘草酸转化率为87.03%,比缓冲液单相体系提高了15.02%.离子液体循环使用8次后,回收利用率维持在85.28%.主产物GAMG和副产物甘草次酸(GA)在两相体系中得到有效分离,为后续产物分离带来便利.     

Whole-cell biocatalysis: Evaluation of new hydrophobic ionic liquids for efficient asymmetric reduction of prochiral ketones

A biphasic process design is often applied in whole-cell biocatalysis if substrate and product have low water solubility, are unstable in water or toxic for the biocatalyst. Some water immiscible ionic liquids (ILs) with adequate distribution coefficients have already been applied successfully as second liquid phase, which acts as a substrate reservoir and in situ extractant for the product. In this work, 12 new ILs were evaluated with respect to their applicability in biphasic asymmetric reductions of prochiral ketones in comparison to 9 already published ILs. The ILs under study are composed of seven different cations and three different anions. Recombinant Escherichia coli was used as whole-cell biocatalyst overexpressing the genes of a Lactobacillus brevis alcohol dehydrogenase (LB-ADH) and a Candida boidinii formate dehydrogenase (CB-FDH) for cofactor regeneration. Best results were achieved if ionic liquids with [PF6]- and [NTF]-anions were applied, whereas [FAP]-ILs showed minor qualification, e.g., the use of [HMPL][NTF] as second liquid phase for asymmetric synthesis of (R)-2-octanol resulted in a space–time-yield of 180 g L⁻¹ d⁻¹, a chemical yield of 95% and an enantiomeric excess of 99.7% in a simple batch process.     

一种新型(S)-羰基还原酶的克隆及其功能表达

【目的】从近平滑假丝酵母(Candida parapsilosis CCTCC M203011)基因组中钓取新型(S)-羰基还原酶基因(scrⅡ),对其生物转化手性醇的功能进行了验证。【方法】采用PCR的方法,从C.parapsilosis基因组中扩增出一段可能的羰基还原酶基因scrⅡ。以构建的重组菌Escherichia coli BL21/pET28a-scrⅡ为生物催化剂,2-羟基苯乙酮为底物进行催化反应,经HPLC分析,计算终产物的光学纯度和产率,确定了转化反应的最适温度和pH值。【结果】scrⅡ基因全长为840bp,编码279个氨基酸,与已报道的(S)-羰基还原酶基因scr的一致性为85%。氨基酸序列分析表明SCRⅡ具有典型短链醇脱氢酶的功能域:辅酶结合区域Thr40-Gly41-(X)3-Gly45-X-Gly47和催化三联体结构Ser172-(X)n-Tyr187-(X)3-Lys191。在30℃,0.1mmol/LIPTG的诱导下,(S)-羰基还原酶(SCRⅡ)在E.coli中过量表达。以10%(w/v)的重组菌为催化剂,高浓度(6g/L)2-羟基苯乙酮为底物,在最适反应温度35℃和pH5.5的条件下,转化产物(S)-苯基乙二醇的光学纯度高达99.1%e.e.,产率为89.6%。与(S)-羰基还原酶SCR相比较,底物浓度提高了一倍,产物的光学纯度和产率分别提高了10%和28%。【结论】采用分子克隆技术分离出新型羰基还原酶SCRⅡ的编码基因,该酶的发现为手性醇的高效制备奠定了坚实的研究基础。     

Enzymatic production of 2-amino-2,3-dimethylbutyramide by cyanide-resistant nitrile hydratase

A novel enzymatic route for the synthesis of 2-amino-2,3-dimethylbutyramide (ADBA), important intermediate of highly potent and broad-spectrum imidazolinone herbicides, from 2-amino-2,3-dimethylbutyronitrile (ADBN) was developed. Strain Rhodococcus boritolerans CCTCC M 208108 harboring nitrile hydratase (NHase) towards ADBN was screened through a sophisticated colorimetric screening method and was found to be resistant to cyanide (5 mM). Resting cells of R. boritolerans CCTCC M 208108 also proved to be tolerant against high product concentration (40 g l⁻¹) and alkaline pH (pH 9.3). A preparative scale process for continuous production of ADBA in both aqueous and biphasic systems was developed and some key parameters of the biocatalytic process were optimized. Inhibition of NHase by cyanide dissociated from ADBN was successfully overcome by temperature control (at 10°C). The product concentration, yield and catalyst productivity were further improved to 50 g l⁻¹, 91% and 6.3 g product/g catalyst using a 30/70 (v/v) n-hexane/water biphasic system. Furthermore, cells of R. boritolerans CCTCC M 208108 could be reused for at lease twice by stopping the continuous reaction before cyanide concentration rose to 2 mM, with the catalyst productivity increasing to 12.3 g product/g catalyst. These results demonstrated that enzymatic synthesis of ADBA using whole cells of R. boritolerans CCTCC M 208108 showed potential for industrial application.     

Enantioselective reduction of aryl ketones using immobilized cells of Candida viswanathii

Enantioselective reduction of 1-acetonapthone to S(−)-1-(1-naphthyl) ethanol, a key intermediate for the synthesis of HMG Co-A reductase inhibitor, was successfully carried out using immobilized cells of a newly isolated carbonyl reductase producing yeast strain Candida viswanathii MTCC 5158. Calcium alginate (1.5%, w/v) gave the best immobilization efficiency. Among different organic solvents and ionic liquids tried as reaction media, isopropanol gave the best enantioselectivity with moderate conversion. The immobilized cells (100 mg/ml in 50 mM Tris buffer pH 9) showed best results at a substrate concentration of 0.2 mg/ml at 30 °C. After twelve cycles of reaction, no significant decrease in bioreduction efficiency of the immobilized cells was observed as compared to the free cells.     

Enzymatic enantioselective transcyanation of silicon-containing aliphatic ketone with (S)-hydroxynitrile lyase from Manihot esculenta

(S)-Hydroxynitrile lyase from Manihot esculenta (MeHNL) was shown for the first time to be able to catalyze the enantioselective transcyanation of acetyltrimethylsilane (ATMS) with acetone cyanohydrin to form (S)-2-trimethylsilyl-2-hydroxyl-propionitrile in an aqueous/organic biphasic system. To better understand the reaction, various influential variables were examined. The most suitable organic phase, optimal buffer pH, aqueous phase content, shaking rate, temperature, concentration of ATMS, acetone cyanohydrin and crude enzyme were diisopropyl ether (DIPE), 5.4, 13% (v/v), 190 rpm, 40°C, 10 mM, 20 mM, and 35 U/ml, respectively, under which the initial reaction rate, substrate conversion and product enantiomeric excess (e.e.) were 19.5 mM/h, 99.0% and 93.5%, respectively. A comparative study demonstrated that silicon atoms in the substrate had a great effect on the reaction, and that ATMS was a much better substrate for MeHNL than its carbon analogue 3,3-dimethyl-2-butanone (DMBO) with respect to the initial reaction rate, substrate conversion and product e.e. MeHNL has greater affinity towards ATMS than its carbon analogue as indicated by the much lower K_m. The activation energy of MeHNL-catalyzed transcyanation of ATMS was also markedly lower than that of DMBO. The silicon effect on the reaction was rationalized on the basis of the special characteristics of silicon atoms and the catalytic mechanism of MeHNL.     

Steady-state kinetics of the oxidation of (S)-1-phenyl-1,2-ethanediol catalyzed by alcohol dehydrogenase from Candida parapsilosis CCTCC M203011

Kinetic studies of oxidation reaction of (S)-1-phenyl-1,2-ethanediol (PED) catalyzed by a NAD⁺-dependent alcohol dehydrogenase from Candida parapsilosis CCTCC M203011 obtained from China Center for Type Culture Collection (CPADH) were observed for getting insight into the deracemization redox reaction. The data of initial velocity experiments in the absence of product, product (β-hydroxy-hypnone) inhibition experiments and dead-end (pyrazole) inhibition experiments strongly suggest that the reaction follows Theorell-Chance BiBi mechanism in which the coenzymes bind to the free form of the enzyme firstly. The kinetic parameters of this model were estimated by using non-linear regression analysis software.