Options for biochemical production of 4-hydroxybutyrate and its lactone as a substitute for petrochemical production

Options are discussed for biochemical production of 4-hydroxybutyrate (4-HB) and its lactone, gamma-butyrolactone (GBL), from renewable sources. In the first part of the study, the thermodynamic feasibility of four potential metabolic pathways from glucose to 4-HB are analyzed. The calculations reveal that when the pathways are NAD(+) dependent the intermediate succinate semialdehyde (SSA) accumulates leading to low 4-HB yields at equilibrium. For NADP(+) dependent pathways the calculated yield of 4-HB improves, up to almost 100%. In the second part of this study, continuous removal of 4-HB from the solution is considered to shift SSA conversion into 4-HB so that SSA accumulation is minimized. One option is the enzymatic production of GBL from 4-HB. Candida antarctica Lipase B shows good lactonization rates at pH 4, but unfortunately this conversion cannot be performed in-vivo during 4-HB production because of the neutral intracellular pH.     

Resolution of N-(2-ethyl-6-methylphenyl) alanine catalyzed by Lipase B from Candida antarctica

A biotransformation process has been developed for the production of (S)-N-(2-ethyl-6-methylphenyl) alanine by enantioselective hydrolysis of racemic methyl ester in the presence of Candida antarctica lipase B (CAL-B). However, the enantioselectivity of CAL-B towards the resolution is not high enough to obtain enantiomerically pure product. In order to improve the enantioselectivity of the enzyme, the effects of surfactants on CAL-B-catalyzed hydrolysis were tested. The results suggest that surfactants can influence the microenvironment of the enzyme, and the addition of Tween-80, in particular, to the reaction medium markedly enhanced the selectivity of CAL-B towards the substrate used, with the enantiomeric ratio (E-value) increasing from 11.3 to 60.1.     

酵母脂肪酶分子进化及催化合成(S)-1,4-苯并二口恶烷*

Objective: 1,4-Benzodioxane is an important chiral intermediate for antihypertensive (Proroxan and Doxazosin), antidepressant (MCK-242) and other drugs, and it displays a broad spectrum of applications in the pharmaceutical field. Currently, in spite of high-yield advantage of chemical synthesis, there are some problems of environmental pollution and low production safety. Using lipase to catalyze synthesis of 1,4-benzodioxane provides a new pathway of green synthesis of 1,4-benzodioxane. However, natural enzymes face the dilemma of poor enantioselectivity. Therefore, molecular evolution was performed on Candida antarctica lipase B, and a technical route for the catalytic synthesis of 1,4-benzodioxane was established. Methods: Firstly, the key amino acid residues involved in substrate binding and conversion in the active center of Candida antarctica lipase B were analyzed, and saturation mutagenesis libraries on the interaction sites were constructed. Improved mutants with high efficiency and high enantioselectivity were then obtained using HPLC detection. Furthermore, catalytic synthesis conditions of mutant D223N/A225K were systematically optimized. Results: The results indicated that the mutants mainly derived from the pairwise site D223/A225 (such as D223N/A225K and D223G/A225W) were biased towards the synthesis of (S)-isoforms, while most of the mutants derived from the pairwise site E188/I189 (such as E188D/I189M) showed a bias for the synthesis of (R)-isoforms. Compared with WT, the ee_s value of the best mutant D223N/A225K to synthesize (S)-1,4-benzodioxane was increased from 11.9% to 29.3%. After systematic optimization of the reaction conditions, an ee_s value of (93.9±0.16)% and a conversion rate of (47.5±2.33)% were achieved using mutant D223N/A225K to catalyze kinetic resolution of methyl (R,S)-2,3-dihydro-1,4-benzodioxin-2-carboxylate in n-butanol/phosphate buffered saline (20∶80, V/V) biphasic solvent at 37℃ for 50 min. Conclusion: An efficient kinetic resolution of methyl (R,S)-2,3-dihydro-1,4-benzodioxin-2-carboxylate was successfully achieved by molecular evolution and optimization of conditions, which provides a new example for the creation of new enzymes by protein engineering technology, and also provides a theoretical and technical foundation for the efficient synthesis of (S)-1,4-benzodioxane molecules by enzymatic methods.     

Resolution of N-(2-ethyl-6-methylphenyl) alanine catalyzed by Lipase B from Candida antarctica

A biotransformation process has been developed for the production of (S)-N-(2-ethyl-6-methylphenyl) alanine by enantioselective hydrolysis of racemic methyl ester in the presence of Candida antarctica lipase B (CAL-B). However, the enantioselectivity of CAL-B towards the resolution is not high enough to obtain enantiomerically pure product. In order to improve the enantioselectivity of the enzyme, the effects of surfactants on CAL-B-catalyzed hydrolysis were tested. The results suggest that surfactants can influence the microenvironment of the enzyme, and the addition of Tween-80, in particular, to the reaction medium markedly enhanced the selectivity of CAL-B towards the substrate used, with the enantiomeric ratio (E-value) increasing from 11.3 to 60.1.     

Hydrolytic resolution of (R,S)-naproxen 2,2,2-trifluoroethyl thioester by Carica papaya lipase in water-saturated organic solvents

For the first time, the Carica papaya lipase (CPL) stored in crude papain is explored as a potential enantioselective biocatalyst for obtaining chiral acids from their racemic thioesters. Hydrolytic resolution of (R,S)-naproxen 2,2,2-trifluoroethyl thioester in water-saturated organic solvents is employed as a model system for studying the effects of temperature and solvents on lipase activity and enantioselectivity. An optimal temperature of 60 degrees C, based on the initial rate of (S)-thioester and a high enantiomeric ratio (i.e., E-value defined as the ratio of initial rates for both substrates) of >100 at 45 degrees C in isooctane, is obtained. Kinetic analysis, considering product inhibition and enzyme deactivation, is also performed, showing agreement between the experimental and best-fit conversions for (S)-thioester. A comparison of the kinetic and thermodynamic behaviors of CPL and Candida rugosa lipase (CRL) in isooctane and cyclohexane indicates that both lipases are very similar in terms of thermodynamic parameters DeltaDeltaH and DeltaDeltaS, initial rate of (S)-substrate, and E-value when (R,S)-naproxen 2,2,2-trifluoroethyl thioester or ester is employed as substrate.     

Efficient preparation of (R)-alpha-monobenzoyl glycerol by lipase catalyzed asymmetric esterification: optimization and operation in packed bed reactor

Optically active (R)-alpha-monobenzoyl glycerol (MBG) was synthesized by Candida antarctica lipase B (CHIRAZYME L-2) catalyzed asymmetric esterification of glycerol with benzoic anhydride in organic solvents. Various conditions, such as the type and composition of the organic solvent, water content of the system, reaction temperature, and concentrations of the substrates were systematically examined and optimized in screw-capped test tubes with respect to both the reaction rate and the enzyme selectivity. 1,4-Dioxane was found to be the best solvent and no additional water was needed for the system. The optimum temperature was around 30 degrees C, while the most suitable substrate concentrations were 100 mM each for glycerol and benzoic anhydride, respectively. However, when excessive anhydride (e.g., 200 mM) was used, the produced MBG could be further transformed into 1,3-dibenzoyl glycerol (DBG) by the same enzyme with a priority to (S)-MBG, resulting in a significant improvement of the product optical purity from ca. 50-70% e.e. Under optimal conditions (100 mM glycerol, 100-200 mM benzoic anhydride, dioxane, 25-30 degrees C), the enzymatic synthesis of (R)-MBG was successfully operated in a packed-bed reactor for about 1 week, with an average productivity of 0.79 g MBG/day/g biocatalyst in the case of continuous operation and 0.94 g MBG/day/g biocatalyst in the case of semicontinuous operation. After refinement and preferential crystallization of the crude product, (R)-MBG could be obtained in an almost optically pure form (>98% e.e.).     

Functional characterization of salt‐tolerant microbial esterase WDEst17 and its use in the generation of optically pure ethyl (R)‐3‐hydroxybutyrate

The two enantiomers of ethyl 3‐hydroxybutyrate are important intermediates for the synthesis of a great variety of valuable chiral drugs. The preparation of chiral drug intermediates through kinetic resolution reactions catalyzed by esterases/lipases has been demonstrated to be an efficient and environmentally friendly method. We previously functionally characterized microbial esterase PHE21 and used PHE21 as a biocatalyst to generate optically pure ethyl (S)‐3‐hydroxybutyrate. Herein, we also functionally characterized one novel salt‐tolerant microbial esterase WDEst17 from the genome of Dactylosporangium aurantiacum subsp. Hamdenensis NRRL 18085. Esterase WDEst17 was further developed as an efficient biocatalyst to generate (R)‐3‐hydroxybutyrate, an important chiral drug intermediate, with the enantiomeric excess being 99% and the conversion rate being 65.05%, respectively, after process optimization. Notably, the enantio‐selectivity of esterase WDEst17 was opposite than that of esterase PHE21. The identification of esterases WDEst17 and PHE21 through genome mining of microorganisms provides useful biocatalysts for the preparation of valuable chiral drug intermediates.     

Effective enzymatic resolution of rac-1-(3-trifluoromethylphenyl)propan-2-ol, precursor of fenfluramine

Lipases from Candida antarctica and from Mucor miehei efficiently catalyze the enantioselective esterification of rac-1-(3-trifluoromethylphenyl)propan-2-ol. The obtained enantioforms are suitable to prepare both enantiomers of fenfluramine.     

Enzymatic resolution of 3-(3-indolyl)butyric acid: a key intermediate for indolmycin synthesis

Both enantiomers of 3-(3-indolyl)butyric acid, a key intermediate of indolmycin, were successfully prepared by lipase-catalysed enantioselective hydrolysis. Of the enzymes examined, Pseudomonas fluorescens lipase (lipase AK) showed the best enantioselectivity and highest reactivity for the hydrolysis of (±)-trifluoroethyl 3-(3-indolyl)butyrate. Under optimal conditions, optical resolution was completed in one enzyme-catalysed step, the S-acid and unreacted R-ester being obtained in high optical purity.     

Immobilized Candida antarctica lipase B on ZnO nanowires/macroporous silica composites for catalyzing chiral resolution of (R,S)-2-octanol

ZnO nanowires were successfully introduced into a macroporous SiO₂ by in situ hydrothermal growth in 3D pores. The obtained composites were characterized by SEM and XRD, and used as supports to immobilize Candida antarctica lipase B (CALB) through adsorption. The high specific surface area (233 m²/g) and strong electrostatic interaction resulted that the average loading amount of the composite supports (196.8 mg/g) was 3–4 times of that of macroporous SiO₂ and approximate to that of a silica-based mesoporous material. Both adsorption capacity and the activity of the CALB immobilized on the composite supports almost kept unchanged as the samples were soaked in buffer solution for 48 h. The chiral resolution of 2-octanol was catalyzed by immobilized CALB. A maximum molar conversion of 49.1% was achieved with 99% enantiomeric excess of (R)-2-octanol acetate under the optimal condition: a reaction using 1.0 mol/L (R,S)-2-octanol, 2.0 mol/L vinyl acetate and 4.0 wt.% water content at 60 °C for 8 h. After fifteen recycles the immobilized lipase could retain 96.9% of relative activity and 93.8% of relative enantioselectivity.     

Biocatalyzed approach for the surface functionalization of poly(L‐lactic acid) films using hydrolytic enzymes

Poly(lactic acid) as a biodegradable thermoplastic polyester has received increasing attention. This renewable polyester has found applications in a wide range of products such as food packaging, textiles and biomedical devices. Its major drawbacks are poor toughness, slow degradation rate and lack of reactive side‐chain groups. An enzymatic process for the grafting of carboxylic acids onto the surface of poly(L‐lactic acid) (PLLA) films was developed using Candida antarctica lipase B as a catalyst. Enzymatic hydrolysis of the PLLA film using Humicola insolens cutinase in order to increase the number of hydroxyl and carboxylic groups on the outer polymer chains for grafting was also assessed and showed a change of water contact angle from 74.6 to 33.1° while the roughness and waviness were an order of magnitude higher in comparison to the blank. Surface functionalization was demonstrated using two different techniques, ¹⁴C‐radiochemical analysis and X‐ray photoelectron spectroscopy (XPS) using ¹⁴C‐butyric acid sodium salt and 4,4,4‐trifluorobutyric acid as model molecules, respectively. XPS analysis showed that 4,4,4‐trifluorobutyric acid was enzymatically coupled based on an increase of the fluor content from 0.19 to 0.40%. The presented ¹⁴C‐radiochemical analyses are consistent with the XPS data indicating the potential of enzymatic functionalization in different reaction conditions.     

Experiment and simulation on kinetic resolution of (R,S)-2-chloromandelic acid by enzymatic transesterification

Optically pure 2-chloromandelic acid (ClMA) is a very important chiral drug intermediate for synthesis of (S)-clopidogrel, belonging to the platelet aggregation inhibitor. Enantioselective resolution of (R,S)-2-chloromandelic acid was carried out in organic solvent through irreversible transesterification catalyzed by lipase AK with vinyl acetate acting as the acyl donor. Effects of various conditions on enantioselectivity and activity of lipase were investigated, including organic solvents, temperature, water content, substrate ratio, enzyme loading, and reaction time. Based on homogeneous reaction and Ping-Pong bi-bi mechanism, a quantitative model was constructed to simulate and optimize the reaction process. Under the optimal conditions, excellent results were obtained with high conversion of (R)-2-ClMA (c _R, ≥98.85%) and large enantiomeric excess of substrate (ee _s, ≥98.15%). There is a good agreement between predicted values and experiment data, which indicates that the established method is a powerful tool for optimization of the enantioselective transesterification process for enantiomers separation.     

Partially purified Carica papaya lipase: a versatile biocatalyst for the hydrolytic resolution of (R,S)-2-arylpropionic thioesters in water-saturated organic solvents

With the hydrolytic resolution of (R,S)-naproxen 2,2,2-trifluoroethyl thioesters in water-saturated isooctane as a model system, improvements of the specific lipase activity and thermal stability were found when a crude Carica papaya lipase (CPL) was partially purified and employed as the biocatalyst. The partially purified Carica papaya lipase (PCPL) was furthermore explored as an effective enantioselective biocatalyst for the hydrolytic resolution of (R,S)-profen thioesters in water-saturated organic solvents. The kinetic analysis in water-saturated isooctane indicated that both acyl donor and acyl acceptor have profound influences on the lipase activity, E-value, and enantioselectivity. Inversion of the enantioselectivity from (S)- to (R)-thioester was found for (R,S)-fenoprofen and (R,S)-ketoprofen thioesters that contained a bulky substituent at the meta-position of 2-phenyl moiety of the acyl part. Kinetic constants for the acylation step were furthermore estimated for elucidating the kinetic data and postulating an active site model. The thermodynamic analysis indicated that the enantiomer discrimination was driven by the difference of activation enthalpy (DeltaDeltaH) and that of activation entropy (DeltaDeltaS), yet the latter was dominated for most of the reacting systems. The postulated active site model was supported from the variation of DeltaDeltaH and DeltaDeltaS with the acyl moiety, in which a good linear enthalpy-entropy compensation relationship was also illustrated. A comparison of the performances between Candida rugosa lipase (CRL) and PCPL indicated that PCPL was superior to CRL in terms of the better thermal stability, similar or better lipase activity for the fast-reacting substrate, time-course-stability, and lower enzyme cost.     

Simultaneous monitoring of the enantiomeric purities of both substrate and product in the enzymatic resolution of (R,S)-2-acetoxy-3-bromopropyl para-toluenesulfonate by HPLC chiral column chromatography

The enantiomeric purities of both substrate, 2-acetoxy-3-bromopropyl para-toluenesulfonate (I), and product, 2-hydroxy-3-bromopropyl p-toluenesulfonate (II) were examined in one analysis. The enzymatic resolution was conducted by Amano lipase AK and the enantiomeric excess as well as the conversion rate were monitored by HPLC analysis utilizing a Chiralcel OD column. After 7 h of reaction, HPLC results indicated that the enantiomeric purities of both substrate (I) and product (II) were greater than 95% and the conversion rate was around 55%. © 1995 Wiley-Liss, Inc.     

Synthesis of enantiomerically pure trans-(1R,2R)- and cis-(1S,2R)-1-amino-2-indanol by lipase and omega-transaminase

Syntheses of trans-(1R,2R) and cis-(1S,2R)-1-amino-2-indanol (AI) were accomplished by a series of enantioselective enzymatic reactions using lipase and transaminase (TA). Lipase catalysed enantioselective hydrolysis of 2-acetoxyindanone was employed to prepare (R)-2-hydroxy indanone (HI). trans-AI (5 mM) (de > 98%) was produced from 20 mM (R)-2- HI using omega-TA and 50 mM (S)-1-aminoindan as an amino donor in water-saturated ethyl acetate. For the production of cis-AI, the diastereomeric (2R)-AI was synthesized from (R)-2-HI using reductive amination, and the kinetic resolution was performed with omega-TA. The enantioselectivity of omega-TA for (2R)-AI was increased to 22.1 in the presence of 5% gamma-cyclodextrin. cis-AI (15.4 mM) (96% de) was obtained from 40 mM (2R)-AI using 30 mM pyruvate and omega-TA (25 mg) in 10 mL of 100 mM phosphate buffer (pH 7.0).     

Enzymatic formation and esterification of (S)-mandelonitrile

The (S)-selective hydroxynitrile lyase from Hevea brasiliensis (HbHNL) catalyzes the trans-cyanohydrin reaction (transcyanation). The equilibrium of this two-step reaction sequence is not favorable unless a large excess of acetone cyanohydrin (1) is used. Therefore, the coupling of this reaction with a follow-up reaction was investigated. It was established that the trans-cyanohydrin reaction could be performed in organic media, making it possible to couple it with a lipase-catalyzed acylation. Candida antarctica lipase B (CAL-B) shows a high selectivity (E=100) for (S)-mandelonitrile (4) and is, therefore, the ideal candidate for this type of multi-step one-pot reaction.     

In silico prediction and validation of the importance of the Entner-Doudoroff pathway in poly(3-hydroxybutyrate) production by metabolically engineered Escherichia coli

The metabolic network of Escherichia coli was constructed and was used to simulate the distribution of metabolic fluxes in wild-type E. coli and recombinant E. coli producing poly(3-hydroxybutyrate) [P(3HB)]. The flux of acetyl-CoA into the tricarboxylic acid (TCA) cycle, which competes with the P(3HB) biosynthesis pathway, decreased significantly during P(3HB) production. It was notable to find from in silico analysis that the Entner-Doudoroff (ED) pathway flux increased significantly under P(3HB)-accumulating conditions. To prove the role of ED pathway on P(3HB) production, a mutant E. coli strain, KEDA, which is defective in the activity of 2-keto-3-deoxy-6-phosphogluconate aldolase (Eda), was examined as a host strain for the production of P(3HB) by transforming it with pJC4, a plasmid containing the Alcaligenes latus P(3HB) biosynthesis operon. The P(3HB) content obtained with KEDA (pJC4) was lower than that obtained with its parent strain KS272 (pJC4). The reduced P(3HB) biosynthetic capacity of KEDA (pJC4) could be restored by the co-expression of the E. coli eda gene, which proves the important role of ED pathway on P(3HB) synthesis in recombinant E. coli as predicted by metabolic flux analysis.     

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.     

Practical access to the proline analogs (S,S,S)- and (R,R,R)-2-methyloctahydroindole-2-carboxylic acids by HPLC enantioseparation

An efficient methodology for the preparation of the α‐tetrasubstituted proline analog (S,S,S)‐2‐methyloctahydroindole‐2‐carboxylic acid, (S,S,S)‐(αMe)Oic, and its enantiomer, (R,R,R)‐(αMe)Oic, has been developed. Starting from easily available substrates and through simple transformations, a racemic precursor has been synthesized in excellent yield and further subjected to HPLC resolution using a cellulose‐derived chiral stationary phase. Specifically, a semipreparative (250 mm × 20 mm ID) Chiralpak® IC column has allowed the efficient resolution of more than 4 g of racemate using a mixture of n‐hexane/tert‐butyl methyl ether/2‐propanol as the eluent. Multigram quantities of the target amino acids have been isolated in enantiomerically pure form and suitably protected for incorporation into peptides. Chirality, 2011. © 2011 Wiley‐Liss, Inc.     

(R,S)-2-chlorophenoxyl pyrazolides as novel substrates for improving lipase-catalyzed hydrolytic resolution

The best reaction condition of Candida antartica lipase B as biocatalyst, 3-(2-pyridyl)pyrazole as leaving azole, and water-saturated methyl t-butyl ether as reaction medium at 45°C were first selected for performing the hydrolytic resolution of (R,S)-2-(4-chlorophenoxyl) azolides (1-4). In comparison with the kinetic resolution of (R,S)-2-phenylpropionyl 3-(2-pyridyl)pyrazolide or (R,S)-α-methoxyphenylacetyl 3-(2-pyridyl)pyrazolide at the same reaction condition, excellent enantioselectivity with more than two order-of-magnitudes higher activity for each enantiomer was obtained. The resolution was then extended to other (R,S)-3-(2-pyridyl)pyrazolides (5-7) containing 2-chloro, 3-chloro, or 2,4-dichloro substituent, giving good (E > 48) to excellent (E > 100) enantioselectivity. The thermodynamic analysis for 1, 2, and 4-7 demonstrates profound effects of the acyl or leaving moiety on varying enthalpic and entropic contributions to the difference of Gibbs free energies. A thorough kinetic analysis further indicates that on the basis of 6, the excellent enantiomeric ratio for 4 and 7 is due to the higher reactivity of (S)-4 and lower reactivity of (R)-7, respectively.