Structure investigation of narrow banded spherulites in polyhydroxyalkanoates by microbeam X-ray diffraction with synchrotron radiation

Narrow banded structures of spherulites of poly[(R)-3-hydroxybutyrate] (P(3HB)) and its copolymers, with different second monomer unit of 4-hydroxybutyrate, 3-hydroxyhexanoate or 6-hydroxyhexanoate, were investigated by microbeam X-ray diffraction with Fresnel Zone Plate technique in synchrotron radiation (SPring-8). Radial scanning of spherulites were performed in 2 or 4 μm steps with 0.5 μm beam diameter of fine microfocus beam. The X-ray diffraction patterns for P(3HB) homopolymer spherulites showed the change of crystal orientation along the radial direction (a-axis), while the b- and c-axes rotated around a-axis. The intensities in microbeam X-ray diagrams of spherulites for P(3HB) copolymers changed periodically as function of the distance along the radial direction. The periodicity measured by X-ray diffraction was matched with narrow band spacing (15-25 μm) observed by polarized optical microscopy.     

Scaling laws and internal structure for characterizing electrospun poly[(R)-3-hydroxybutyrate] fibers

Using chloroform/dimethylformamide (CF/DMF) co-solvent, electrospinning of poly[(R)-3-hydroxybutyrate] (PHB) solutions was carried out at ambient temperature. The effects of the applied voltage (V), flow-rate (Q), and solution viscoelastic properties on the Taylor cone, electrified jet, and fiber morphology were investigated. In addition, the electric field developed by the needle-plate electrode configuration was calculated using a finite element analysis to reveal the tip-to-collector (H) effect. Among the processing parameters (V, Q and H), it was found that Q played a key role in determining the jet diameter (d_j) and electrospun fiber diameter (d_f), and scaling laws existed between them, i.e., d_j-Q^0.61 and d_f-Q^0.33. The diameter reduction ratios of D_o/d_j (D_o is the needle diameter) and d_j/d_f were measured as 50-120 and 5-10, respectively; it suggested that major jet stretching took place in the straight electrified jet region, and further chain orientation could be gained by the subsequent process of jet whipping. By changing PHB concentrations from 5 to 15 wt%, the solution viscosity (η_o) was increased from 100 to 4900 cP, whereas the surface tension and solution conductivity remained unchanged; it provided a good model solution to exclusively reveal the η_o effect on the electrospinning process. Our results showed that the η_o-dependence of d_j and d_f also followed simple scaling laws: d_j-η_o^0.06, and d_f-η_o^0.39, with a prefactor depending on the processing variables, mainly the flow-rate. Regardless of the PHB concentrations used, the obtained PHB fibers showed a similar crystallinity fraction of ca. 0.63 and possession of major α-crystals together with a small amount of β-crystals with zigzag chain conformation.     

Solid-state NMR study on the structure and mobility of the noncrystalline region of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

The noncrystalline structures of poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) copolymers were studied by variable temperature solid-state wide-line ¹H NMR and solid-state high-resolution ¹³C NMR spectroscopy. It is found that at room temperature there exists a rich and rigid component in the noncrystalline region of PHB and PHBV. The content of this component decreases with the increase in 3-hydroxyvalerate content in PHBV and with the increase in temperature. The brittleness of PHB may be partly attributed to the rigidness of the noncrystalline region at room temperature, while the improvement of the properties of PHBV may come from the enhanced mobility of the noncrystalline region.     

Segregation morphology of poly(3-hydroxybutyrate)/poly(vinyl acetate) and poly(3-hydroxybutyrate-co-10% 3-hydroxyvalerate)/poly(vinyl acetate) blends as studied via small angle X-ray scattering

Segregation morphology of poly(3-hydroxybutyrate) (PHB)/poly(vinyl acetate) (PVAc) and poly(3-hydroxybutyrate-co-10% 3-hydroxyvalerate) (P(HB-co-10% HV)/PVAc blends crystallized at 70 °C have been investigated by means of small angle X-ray scattering (SAXS). Morphological parameters including the crystal thickness (l_c) and the amorphous layer thickness (l_a) were deduced from the one-dimensional correlation function (γ(z)). Blending with PVAc thickened the PHB crystals but not the P(HB-co-10% HV) crystals. On the basis of the composition variation of l_a, and the volume fraction of lamellar stacks (?_s) revealed that PHB/PVAc blends created the interlamellar segregation morphology when the weight fraction of PVAc (w_PVAc)≤0.2 and the interlamellar and interfibrillar segregation coexisted when w_PVAc>0.2, while P(HB-co-10% HV)/PVAc blends yielded the interfibrillar segregation morphology at all blend compositions. For both PHB/PVAc and P(HB-co-10% HV)/PVAc blends, the distance of PVAc segregation was promoted by increasing PVAc composition and the distance of PVAc segregation in P(HB-co-10% HV)/PVAc blends was greater than in PHB/PVAc at a given PVAc composition. The crystal growth rate played a key role in controlling the segregation of PVAc.     

The chemomechanical properties of microbial polyhydroxyalkanoates

Microbially produced polyhydroxyalkanoates (PHAs) are fully biodegradable biopolyesters that have attracted much attention recently as alternative polymeric materials that can be produced from biorenewable and biowaste resources. The properties of these biological polymers are affected by the same fundamental principles as those of fossil-fuel derived polyolefins, with a broad range of compositions available based on the incorporation of different monomers into the PHA polymer structure, and with this broad range tailoring subsequent properties. This review comprehensively covers current understanding with respect to PHA biosynthesis and crystallinity, and the effect of composition, microstructure and supramacromolecular structures on chemomechanical properties. While polymer composition and microstructure are shown to affect these properties, the review also finds that a key driver for determining polymer performance properties is compositional distribution. From this review it follows that PHA–PHA blend compositions are industrially important, and the performance properties of such blends are discussed. A particular need is identified for further research into the effect of chemical compositional distribution on macromolecular structure and end-use properties, advanced modeling of the PHA accumulation process and chain growth kinetics for better process control.     

The domain structure and mobility of semi-crystalline poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate): A solid-state NMR study

Solid-state NMR techniques have been employed to investigate the domain structure and mobility of the bacterial biopolymeric metabolites such as poly(3-hydroxybutyrate) (PHB) and its copolymers poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) containing 2.7 mol% (PHBV2.7) and 6.5 mol% (PHBV6.5) 3-hydroxyvalerate. Both single-pulse excitation with magic-angle spinning (SPEMAS) and cross-polarization magic-angle spinning (CPMAS) ¹³C NMR results showed that these biopolymers were composed of amorphous and crystalline regions having distinct molecular dynamics. Under magic-angle spinning, ¹H T_1ρ and ¹³C T₁ showed two processes for each carbon. Proton relaxation-induced spectral editing (PRISE) techniques allowed the neat separation of the ¹³C resonances in the crystalline regions from those in the amorphous ones. The proton spin-lattice relaxation time in the tilted rotating frame, , measured using the Lee-Goldburg sequence with frequency modulation (LGFM) as the spin-locking scheme, was also double exponential and significantly longer than ¹H T_1ρ. The difference between for the amorphous and crystalline domains was greater than that of ¹H T_1ρ. Our results showed that the differences could be exploited in LGFM-CPMAS experiments to separate the signals from two distinct regions. ¹H spin-diffusion results showed that the domain size of the mobile components in PHB, PHBV2.7 and PHBV6.5 were about 13, 24 and 36 nm whereas the ordered domain sizes were smaller than 76, 65 and 55 nm, respectively. The results indicated that the introduction of 3-hydroxyvalerate into PHB led to marked molecular mobility enhancement in the biopolymers.     

Hydrolytic degradation of poly[(R)-3-hydroxybutyric acid] in the melt

Poly[(R)-3-hydroxybutyric acid] [R-P(3HB)] was hydrolyzed in high-temperature and high-pressure water at the temperature range of 180-300 °C and for a period of 360 min. The formation, racemization, and decomposition of 3-hydroxybutyric acids (3HBs) and molecular weight change of R-P(3HB) were investigated. The highest yield of (R)-3-hydroxybutyric acid (R-3HB), ca. 80%, was obtained at 200 °C in the hydrolytic degradation periods of 240-360 min. Too-high hydrolytic degradation temperature such as 300 °C induced the decomposition and racemization of formed 3HBs, resulting in decreased yield of R-3HB. The hydrolytic degradation of R-P(3HB) proceeds homogeneously and randomly via a bulk erosion mechanism. The molecular weight of R-P(3HB) decreased exponentially without formation of low-molecular-weight specific peaks originating from crystalline residues. The hydrolytic degradation rates in the melt estimated from M_n changes were lower for R-P(3HB) than for poly(l-lactide) (PLLA) in the temperature range of 180-220 °C. The activation energy for the hydrolytic degradation (ΔE_h) of R-P(3HB) in the melt (180-250 °C) was 30.0 kcal mol⁻¹, which is higher than 12.2 kcal mol⁻¹ for PLLA in the melt in the temperature range (180-250 °C). This study reveals that hydrolytic degradation of PHB in the melt is an effective and simple method to obtain (R)-3HB and to prepare R-P(3HB) having different molecular weights without containing the specific low-molecular-weight chains, because of the removal of the effect caused by crystalline residues.     

Thermal and mechanical properties of poly(3-hydroxybutyrate-co-3-hydroxyvalerate)/cellulose nanowhiskers composites

Bacterial polyester poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) was reinforced with cellulose nanowhiskers (CNW) in 1-5 wt.% concentrations using a solvent casting method. The CNW was prepared from microcrystalline cellulose (MCC) using sulfuric acid hydrolysis. The influence of CNW on the PHBV crystallization, thermal, dynamic mechanical and mechanical properties were evaluated using polarized optical microscope (POM), differential scanning calorimeter (DSC), dynamic mechanical analysis (DMA), tensile and bulge tests, respectively. POM test results demonstrated that CNW was an effective PHBV nucleation agent. Tensile strength, Young’s modulus and toughness of PHBV increased with the increasing concentration of CNW. DMA results showed an increased tan δ peak temperature and broadened transition peak, indicating restrained PHBV molecular mobility in the vicinity of the CNW surface. Storage modulus of the PHBV also increased with the addition of CNW, especially at the temperatures higher than the PHBV glass transition temperature. These results indicated that the CNW could substantially increase the mechanical properties of PHBV and this increase could be attributed to the strong interactions between these two phases.     

Controlled synthesis and characterizations of amphiphilic poly[(R,S)-3-hydroxybutyrate]-poly(ethylene glycol)-poly[(R,S)-3-hydroxybutyrate] triblock copolymers

Well-defined biodegradable amphiphilic triblock copolymers consisting of atactic poly[(R,S)-3-hydroxybutyrate] (PHB) and poly(ethylene glycol) (PEG) as the side hydrophobic block and middle hydrophilic block were synthesized via ring opening polymerization of (R,S)-β-butyrolactone from PEG macroinitiators and characterized using NMR, GPC, FT-IR, XRD, DSC and TG analyses. The controlled synthesis was made possible by the facile synthesis of pure PEG macroinitiators through a TEMPO-mediated oxidation. Constituting 40-70 wt% of the copolymer content, PHB blocks grown were amorphous while PEG formed crystalline phase when segment was sufficiently long. While hindering PEG crystallization, atactic PHB mixed well with amorphous PEG to give single T_g in all the copolymers. The copolymers exhibited two-step thermal degradation profile starting with PHB degradation from 210 to 300 °C, then PEG from 350 to 450 °C.     

Miscibility and crystallization behavior of biodegradable blends of two aliphatic polyesters. Poly(3-hydroxybutyrate-co-hydroxyvalerate) and poly(ε-caprolactone)

Biodegradable polymer blends of poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV) and poly(ε-caprolactone) (PCL) blends were prepared with the ratio of PHBV/PCL ranging from 80/20-20/80 by co-dissolving the two polyesters in chloroform and casting the mixture. Differential scanning calorimetry (DSC) and optical microscopy (OM) were used to investigate the miscibility and crystallization of PHBV/PCL blends. Experimental results indicated that PHBV showed no miscibility with PCL for PHBV/PCL blends as evidenced by the existence of unchanged composition independent glass transition temperature and the biphasic melt. Crystallization of PHBV and PCL was studied with DSC and analyzed by the Avrami equation by using two-step crystallization in the PHBV/PCL blends. The crystallization rate of PHBV at 70 °C decreased with the increase of PCL in the blends, while the crystallization mechanism did not change. In the case of the isothermal crystallization of PCL at 42 °C, the crystallization rate increased with the addition of PHBV, and the crystallization mechanism changed, too, indicating that the crystallization of PHBV at 70 °C had an apparent influence on the crystallization of PCL at 42 °C.     

Miscibility and crystallization behaviour of biodegradable blends of two aliphatic polyesters. Poly(3-hydroxybutyrate-co-hydroxyvalerate) and poly(butylene succinate) blends

Blends of poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV) and poly(butylene succinate) (PBSU), both biodegradable semicrystalline polyesters, were prepared with the ratio of PHBV/PBSU ranging from 80/20 to 20/80 by co-dissolving the two polyesters in chloroform and casting the mixture. Differential scanning calorimetry (DSC) and optical microscopy (OM) were used to study the miscibility and crystallization behaviour of PHBV/PBSU blends. Experimental results indicate that PHBV is immiscible with PBSU as shown by the almost unchanged glass transition temperature and the biphasic melt. Crystallization of PHBV/PBSU blends was studied by DSC using two-step crystallization and analyzed by the Avrami equation. The crystallization rate of PHBV decreases with the increase of PBSU in the blends while the crystallization mechanism does not change. In the case of the isothermal crystallization of PBSU, the crystallization mechanism does not change. The crystallization rate of PBSU in the blends is lower than that of neat PBSU; however, the change in the crystallization rate of PBSU was not so big in the blends. The different content of the PHBV in the blends does not make a significant difference in the crystallization rate of PBSU.     

Multiple melting behavior of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) investigated by differential scanning calorimetry and infrared spectroscopy

The multiple melting behavior of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (P(HB-co-HHx)) (HHx = 12 mol%) isothermally crystallized from the melt state has been characterized by differential scanning calorimetry (DSC) and Fourier transform infrared (FTIR) spectroscopy. The influence of different experimental variables (such as crystallization temperature, time, and heating rate) on the multiple melting behavior of P(HB-co-HHx) was investigated by using DSC. Moreover, it has been further examined by monitoring intensity changes of the characteristic IR bands during the subsequent heating process. For the isothermally crystallized P(HB-co-HHx) samples, triple melting peaks were observed upon heating. The weak lowest-temperature DSC endotherm I always appears at the position just above the crystallization temperature, and shifts to a higher temperature linearly with the logarithm of the crystallization time. The combination of DSC and IR results suggested that the occurrence of peak I was a result of the melting of crystals formed upon long-time annealing. As for the other two main melting endothermic peaks, endotherm II corresponds to the melting of crystals formed during the primary crystallization, and endotherm III is ascribed to the melting peak of the crystals formed by recrystallization during the heating process.     

Characterization on mixed-crystal structure and properties of poly(butylene adipate-co-terephthalate) biodegradable fibers

Biodegradable ideal random copolymer poly(butylene adipate-co-terephthalate) (PBAT), with 44 mol% butylene terephthalate (BT), was melt-spun into fibers with take-up velocity up to 5 km/min. The structure development and properties of the as-spun fibers were investigated through birefringence, WAXD, SAXS, DSC and tensile test. Despite of the ideal randomness and composition (1:1) of PBAT copolymer, PBAT fiber showed well-developed PBT-like crystal structure, while its melting temperature (ca. 121 °C) was over 100 °C lower than that of PBT. Based on the quantitative analyses on the lattice spacing, the crystallinity and the fraction of crystallizable BT sequences, the crystal structure of PBAT was characterized to be formed by mixed-crystallization of BT and BA units, where BA units were incorporated into BT lattice. This mixed-crystal structure was found to undergo PBT-like reversible crystal modification with the application and removal of tensile stress. This crystal modification was found to occur in a higher strain region compared with that of PBT fibers.     

Investigation of water diffusion in poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) by generalized two-dimensional correlation ATR-FTIR spectroscopy

Water diffusion process in biodegradable poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx, HHx = 12 mol%) was investigated by generalized 2D correlation time-resolved ATR-FTIR spectroscopy based on the analysis of v(OH) stretching and δ(OH) bending bands of water as well as v(CO) and v(C-O-C) stretching bands of PHBHHx. Three states of water were figured out during water diffusion process. They are bulk water, bound water and free water. The water diffusion mechanism was suggested as: water molecules firstly diffuse into the micro-voids in bulk water form or are dispersed on the surface in free water form, and then penetrate into the polymer matrix in hydrogen bound water with the hydrophilic groups of PHBHHx. Moreover, water molecules diffuse into the loose amorphous phase and then into compact crystalline phase. Water diffusion coefficient in PHBHHx was thus evaluated as 7.8 ± 0.7 × 10⁻⁸ cm² s⁻¹ for the PHBHHx with crystallinity of 16.2 ± 0.3% at 293 K.     

Different thermal behaviors of microbial polyesters poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)

Various methods were employed to study the thermal behaviors of a novel microbial polyhydroxyalkanoate (PHA) terpolyester, namely, poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) (PHBVHHx) compared with poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx). PHBVHHx showed higher crystallization rate and degree of crystallinity. PHBVHHx exhibited also different multiple melting behaviors from PHBHHx. The WAXD results demonstrated that the crystal lattice of PHBVHHx was more compact than that of PHBHHx, suggesting stronger interaction between chain stems. DSC and in-situ heating WAXD studies revealed that PHBVHHx showed a partial melting-lamellar thickening-remelting process during heating, while PHBHHx demonstrated a melting-rapid formation of new crystals-remelting process. It is proposed that the simultaneous introduction of 3-hydroxyvalerate and 3-hydroxyhexanoate monomers into poly(3-hydroxybutyrate) improves the mobility of chain stems along the chain direction, leading to easier intralamellar slip during heating or drawing, further resulting in improvement of mechanical properties, which was supported by the DMA tests. Consequently, we establish a relationship between the thermal behavior and the mechanical properties of biodegradable plastics, which we believe is applicable to other polymers as well.     

Effect of hydrogen bonding on the crystallization behavior of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)/silica hybrid composites

Biodegradable poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHB-HHx)/hydrophobically modified silica hybrid composites were prepared using simple melt compounding and the effect of hydrogen bonding on their crystallization behavior was observed. The intermolecular hydrogen bonding between PHB-HHx and silica increased gradually with the increase of silica content of the hybrid composites. However, the extent of intermolecular hydrogen bonding was not directly proportional to the silica content. Although, the crystallization rates of the PHB-HHx/silica hybrids decreased as the strength of intermolecular hydrogen bonding increased, the constant value of the Avrami exponent indicates that the presence of silica does not alter the nucleation mechanism or the geometry of the crystal growth of the PHB-HHx hybrids. The calculated crystallization activation energy increased with the addition of silica, suggesting that silica retards the overall crystallization rate of the PHB-HHx hybrid composites as a result of the existence of intermolecular hydrogen bonding. The relationship between the extent of intermolecular hydrogen bond and crystallization rate is described by the empirical second-order equation.     

Surface structure of folded-chain crystals of poly(R-3-hydroxybutyrate) of different chain length

The structure of the crystalline-amorphous interface of poly(R-3-hydroxybutyrate) (PHB) of different molar mass is evaluated by analysis of the rigid amorphous fraction and by analysis of the degree of reversible melting and crystallization. The rigid amorphous fraction of low-molar-mass PHB of 5 kDa is only 5-10%, and at best half of that of high-molar-mass PHB of almost 500 kDa, despite identical crystallinity. This result is paralleled by observation of distinctly larger degree of reversible melting and crystallization in PHB of high molar mass. The larger rigid amorphous fraction and higher degree of reversible melting and crystallization in PHB of high molar mass, consistently and independently, prove enhanced covalent coupling of crystals and amorphous structure, and/or de-coupling of segments of macromolecules which traverse between phases, respectively. The distinct isolation of crystals in PHB of low molar mass is discussed in terms of absence of wide loops/folds, long-chain cilia, and tie-molecules.     

Influence of the swelling history on the swelling kinetics of stimuli-responsive poly[(N-isopropylacrylamide)-co-(methacrylic acid)] hydrogels

The influence of the swelling history on the swelling behavior of poly[(N-isopropylacrylamide)-co-(methacrylic acid)] P[(N-iPAAm)-co-(MAA)] random copolymers hydrogels synthesized by free radical polymerization in solution of N-iPAAm and MAA comonomers crosslinked with tetraethylene glycol dimethyl acrylate (TEGDMA) has been studied. The swelling behavior under pH 7 at 18, 29, 39 and 49 °C of this series of copolymers, previously soaked either at pH 2 or 7 has been investigated. The swelling kinetics of these two series of samples displays different behavior as function of the composition and temperature. However, the equilibrium swelling values only show slight dependences on the previous soaking pH and temperature. When samples are soaked at pH 7, then the swelling at pH 7 follows a first order kinetics, irrespective of the copolymer composition or the temperature at which the experiment has been carried out. In this case, the swelling process is very fast and depends only slightly on temperature. The first order rate constant increases with the MAA content in the hydrogel. Furthermore, the swelling rate of copolymer hydrogels soaked at pH 2, show strong dependence on composition and temperature. They follow an autocatalytic swelling kinetics due to the disruption of hydrogen bond arrangements. An initial slow water uptake is followed by an acceleration process, in which water molecules inside the gel help the next water molecules to come in. Two rate constants, a first-order rate constant and an autocatalytic one have been obtained from the kinetics analysis. They have revealed different temperature dependence which may be due to a balance between hydrophobic and hydrogen bond interactions. The temperature dependence of the swelling kinetics is stronger and more complex for copolymers treated under pH 2 than for copolymers soaked under pH 7.     

Structure and dynamic mechanical properties of poly(ethylene terephthalate-co-4,4′-bibenzoate) fibers

Poly(ethylene terephthalate-co-4,4′-bibenzoate) (PETBB) fibers containing 5, 15, 35, 45, 55, and 65 mol% bibenzoate (BB) were melt spun. Fiber structure has been determined using wide angle X-ray diffraction, birefringence, and FTIR spectroscopy. When drawn to their respective maximum draw ratios, the structures and properties of high BB containing fibers (PETBB45, 55 and 65) are significantly different than those of PET and low BB containing fibers (PETBB5, 15, and 35). For example, 90% of the ethylene glycol units in high BB containing fibers are in the trans conformation, while only 80% of these units are in trans conformation in PET and low BB containing fibers. Overall orientation of the high BB containing fibers is higher (orientation factor f > 0.85) than those of PET and low BB containing fibers (f < 0.6). Orientation of the crystalline regions is quite high (f_cr ∼ 0.95) for both groups of fibers, while orientation of the amorphous regions (f_am) of high BB containing fibers is higher (∼0.8) than those of the PET and low BB containing fibers (∼0.4). High BB containing fibers exhibit much higher storage modulus and modulus retention with temperature than low BB containing fibers. Glass transition temperature determined from the dynamic loss tangent peak decreased with increasing BB content, while this transition completely disappeared in the high BB containing fibers. The magnitude of the secondary transition, observed at about −50 °C, decreased with increasing BB content. Another secondary transition, not observed in PET, was observed at about 70 °C in high BB containing fibers. These dynamic mechanical results have been rationalized in terms of the observed structural parameters.     

Production of (R)-3-Quinuclidinol by E. coli Biocatalysts Possessing NADH-Dependent 3-Quinuclidinone Reductase (QNR or bacC) from Microbacterium luteolum and Leifsonia Alcohol Dehydrogenase (LSADH)

We found two NADH-dependent reductases (QNR and bacC) in Microbacterium luteolum JCM 9174 (M. luteolum JCM 9174) that can reduce 3-quinuclidinone to optically pure (R)-(−)-3-quinuclidinol. Alcohol dehydrogenase from Leifsonia sp. (LSADH) was combined with these reductases to regenerate NAD⁺ to NADH in situ in the presence of 2-propanol as a hydrogen donor. The reductase and LSADH genes were efficiently expressed in E. coli cells. A number of constructed E. coli biocatalysts (intact or immobilized) were applied to the resting cell reaction and optimized. Under the optimized conditions, (R)-(−)-3-quinuclidinol was synthesized from 3-quinuclidinone (15% w/v, 939 mM) giving a conversion yield of 100% for immobilized QNR. The optical purity of the (R)-(−)-3-quinuclidinol produced by the enzymatic reactions was >99.9%. Thus, E. coli biocatalysis should be useful for the practical production of the pharmaceutically important intermediate, (R)-(−)-3-quinuclidinol.