Songping Zhang

Vegetable-oil-based polymers as future polymeric biomaterials.

Vegetable oils are one of the most important classes of bio-resources for producing polymeric materials. The main components of vegetable oils are triglycerides - esters of glycerol with three fatty acids. Several highly reactive sites including double bonds, allylic positions and the ester groups are present in triglycerides from which a great variety of polymers with different structures and functionalities can be prepared. Vegetable-oil-based polyurethane, polyester, polyether and polyolefin are the four most important classes of polymers, many of which have excellent biocompatibilities and unique properties including shape memory. In view of these characteristics, vegetable-oil-based polymers play an important role in biomaterials and have attracted increasing attention from the polymer community. Here we comprehensively review recent developments in the preparation of vegetable-oil-based polyurethane, polyester, polyether and polyolefin, all of which have potential applications as biomaterials.

Nanoparticle-supported multi-enzyme biocatalysis with in situ cofactor regeneration.

Although there have been a long history of studying and using immobilized enzymes, little has been reported regarding the nature of immobilized cofactors. Herein we report that cofactor NAD(H) covalently attached to silica nanoparticles successfully coordinated with particle-immobilized enzymes and enabled multistep biotransformations. Specifically, silica nanoparticle-attached glutamate dehydrogenase (GLDH), lactate dehydrogenase (LDH) and NAD(H) were prepared and applied to catalyze the coupled reactions for production of alpha-ketoglutarate and lactate with the cofactor regenerated within the reaction cycle. It appeared that particle-particle collision driven by Brownian motion of the nanoparticles provided effective interactions among the catalytic components, and thus realized a dynamic shuttling of the particle-supported cofactor between the two enzymes to keep the reaction cycles continuing. Total turnover numbers (TTNs) as high as 20,000h(-1) were observed for the cofactor. It appeared to us that the use of particle-attached cofactor promises a new biochemical processing strategy for cofactor-dependent biotransformations.

Microbial fuel cell based biosensor for in situ monitoring of anaerobic digestion process.

A wall-jet microbial fuel cell (MFC) was developed for the monitoring of anaerobic digestion (AD). This biofilm based MFC biosensor had a character of being portable, short hydraulic retention time (HRT) for sample flow through and convenient for continuous operation. The MFC was installed in the recirculation loop of an upflow anaerobic fixed-bed (UAFB) reactor in bench-scale where pH of the fermentation broth and biogas flow were monitored in real time. External disturbances to the AD were added on purpose by changing feedstock concentration, as well as process configuration. MFC signals had good correlations with online measurements (i.e. pH, gas flow rate) and offline analysis (i.e. COD) over 6-month operation. These results suggest that the MFC signal can reflect the dynamic variation of AD and can potentially be a valuable tool for monitoring and control of bioprocess.

Degradation of high concentration 2,4-dichlorophenol by simultaneous photocatalytic-enzymatic process using TiO2/UV and laccase.

Removal of 2,4-dichlorophenol (2,4-DCP) by TiO2/UV photocatalytic, laccase, and simultaneous photocatalytic-enzymatic treatments were investigated. Coupling of native laccase with TiO2/UV showed a negative synergetic effect due to the rapid inactivation of laccase. Immobilizing laccase covalently to controlled porous glass (CPG) effectively enhanced the stability of laccase against TiO2/UV induced inactivation. By coupling CPG-laccase with the TiO2/UV the degradation efficiency of 2,4-DCP was significantly increased as compared with the results obtained when immobilized laccase or TiO2/UV were separately used. Moreover, the enhancement was more remarkable for the degradation of 2,4-DCP with high concentration, such that for the degradation of 5mM 2,4-DCP, 90% removal percentage was achieved within 2h with the coupled degradation process. While for the TiO2/UV and CPG-laccase process, the removal percentage of 2,4-DCP at 2h were only 26.5% and 78.1%, respectively. The degradation kinetics were analyzed using a intermediate model by taking into account of the intermediates formed during the degradation of 2,4-DCP. The high efficiency of the coupled degradation process therefore provided a novel strategy for degradation of concentrated 2,4-DCP. Furthermore, a thermometric biosensor using the immobilized laccase as biorecognition element was constructed for monitoring the degradation of 2,4-DCP, the result indicated that the biosensor was precise and sensitive.

Temperature sensitivity of cellulase adsorption on lignin and its impact on enzymatic hydrolysis of lignocellulosic biomass.

Unproductive enzyme adsorption is an important factor in addition to steric hindrance of lignin that limits the enzymatic hydrolysis of lignocellulosic biomass. While both are important factors, enzymatic hydrolysis of pretreated biomass is most likely conducted in the presence of certain amount of lignin residues that may not necessarily present accessibility hindrance, but can competitively absorb the enzyme. This paper presents a study with purified lignin samples to elucidate the role of unproductive enzyme adsorption. It appeared that lignin adsorbed cellulase quickly at 4 °C with adsorption equilibrium reached within 1h, similar to that observed for crystalline cellulose. Increasing temperature to 50 °C (typical hydrolytic reaction condition) facilitated the rate of cellulase adsorption on cellulose with a peak of adsorption reached at 0.25 h; however, adsorption on lignin was surprisingly slower and took over 12h to reach equilibrium, which was accompanied with a 10-fold increase in adsorption capacity. Despite the high adsorption capacity of lignin (which is comparable to that of cellulose) at 50 °C, the presence of added lignin imposed only minimal impact on the enzyme apparent activity, most likely due to the slow adsorption kinetics of lignin.

Simultaneous production of 1,3-dihydroxyacetone and xylitol from glycerol and xylose using a nanoparticle-supported multi-enzyme system with in situ cofactor regeneration

Cofactor-dependent biotransformations often require consumption of a secondary substrate for cofactor regeneration. Alternatively, two synthetic reactions may be coupled together through cofactor regeneration cycles. Simultaneous production of value-added products from glycerol and xylose was realized in this work through an enzymatic NAD(H) regeneration cycle involving two enzymes. Glycerol dehydrogenase (GDH) catalyzed the production of 1,3-dihydroxyacetone (DHA) from glycerol, while xylose reductase (XR) enabled the reduction of xylose to xylitol using the protons released from glycerol. Both enzymes were immobilized with P(MMA-EDMA-MAA) nanoparticles. Interestingly, the immobilized multi-enzyme system showed much improved productivity and stability as compared to native enzymes, such that the total turnover number (TTN) reached 82 for cofactor regeneration while the yield reached 160g/g-immobilized GDH for DHA production.

Tethering of nicotinamide adenine dinucleotide inside hollow nanofibers for high-yield synthesis of methanol from carbon dioxide catalyzed by coencapsulated multienzymes.

Enzymatic conversion of carbon dioxide (CO2) to fuel or chemicals is appealing, but is limited by lack of efficient technology for regeneration and reuse of expensive cofactors. Here we show that cationic polyelectrolyte-doped hollow nanofibers, which can be fabricated via a facile coaxial electrospinning technology, provide an ideal scaffold for assembly of cofactor and multienzymes capable of synthesizing methanol from CO2 through a cascade multistep reaction involving cofactor regeneration. Cofactor and four enzymes including formate, formaldehyde, alcohol, and glutamate dehydrogenases were in situ coencapsulated inside the lumen of hollow nanofibers by involving them in the core-phase solution for coaxial electrospinning, in which cationic polyelectrolyte was predissolved. The polyelectrolyte penetrating across the shell of the hollow nanofibers enabled efficient tethering and retention of cofactor inside the lumen via ion-exchange interactions between oppositely charged polyelectrolytes and cofactor. With carbonic anhydrase assembled on the outer surface of the hollow nanofibers for accelerating hydration of CO2, these five-enzymes-cofactor catalyst system exhibited high activity for methanol synthesis. Compared with methanol yield of only 36.17% using free enzymes and cofactor, the hollow nanofiber-supported system afforded a high value up to 103.2%, the highest reported value so far. It was believed that the linear polyelectrolytes acted as spacers to enhance the shuttling of cofactor between enzymes that were coencapsulated within near vicinity, thus improving the efficiency of the system. The immobilized system showed good stability in reusing. About 80% of its original productivity was retained after 10 reusing cycles, with a cofactor-based cumulative methanol yield reached 940.5%.

Enabling multi-enzyme biocatalysis using coaxial-electrospun hollow nanofibers: redesign of artificial cells

Highly efficient immobilization of multi-enzyme systems involving cofactor regeneration represents one of the greatest challenges in bioprocessing. Particulate artificial cells with enzymes and cofactors encapsulated within microcapsules have long been the major type of multi-enzyme biocatalysts. In the present work, a novel hollow nanofiber-based artificial cell that performs multi-step reactions involving efficient coenzyme regeneration was fabricated in situ by a facile co-axial electrospinning process. To that end, a mixture of glycerol and water containing the dissolved multi-enzyme system for the bile acid assay, which included 3α-hydroxysteroid dehydrogenase (3α-HSD), diaphorase (DP) and NADH was fed as the core phase solution, and a N,N-dimethylacetylamide solution of 30 wt% polyurethane was fed as the shell phase solution during the co-axial electrospinning. The relationship between the structures of the hollow nanofibers and the activity and stability of the encapsulated enzymes was studied. At core and shell phase electrospinning solution flow rates of 0.07 and 0.5 mL h-1, activity recoveries as high as 76% and 82% were obtained for the encapsulated 3α-HSD and DP. The hollow nanofiber-based artificial cells were successfully used for the bile acid assay, yielding good linearity for bile acid concentrations ranging from 0-200 μM. Compared with the solution-based multi-enzyme system, the hollow nanofiber-based multi-enzyme system presented a lumped activity recovery of 75%. In addition, the hollow nanofiber provided the multi-enzyme system confined inside the nano-domain of the hollow fibers with a unique stabilizing mechanism, such that more than a 170-fold increase in half-life at 25 °C was obtained for the encapsulated 3α-HSD and DP. This study is expected to greatly promote and broaden the application of multi-enzyme systems in industry, biosensor, biomedical, and many other related research fields.

Effect of molecular mobility on coupled enzymatic reactions involving cofactor regeneration using nanoparticle-attached enzymes.

Cofactor-dependent multi-step enzymatic reactions generally require dynamic interactions among cofactor, enzyme and substrate molecules. Maintaining such molecular interactions can be quite challenging especially when the catalysts are tethered to solid state supports for heterogeneous catalysis for either biosynthesis or biosensing. The current work examines the effects of the pattern of immobilization, which presumably impacts molecular interactions on the surface of solid supports, on the reaction kinetics of a multienzymic system including glutamate dehydrogenase, glucose dehydrogenase and cofactor NAD(H). Interestingly, particle collision due to Brownian motion of nanoparticles successfully enabled the coupled reactions involving a regeneration cycle of NAD(H) even when the enzymes and cofactor were immobilized separately onto superparamagnetic nanoparticles (124 nm). The impact of particle motion and collision was evident in that the overall reaction rate was increased by over 100% by applying a moderate alternating magnetic field (500 Hz, 17 Gs), or using additional spacers, both of which could improve the mobility of the immobilized catalysts. We further observed that integrated immobilization, which allowed the cofactor to be placed in the molecular vicinity of enzymes on the same nanoparticles, could enhance the reaction rate by 1.8 fold. These results demonstrated the feasibility in manipulating molecular interactions among immobilized catalyst components by using nanoscale fabrication for efficient multienzymic biosynthesis.

Hybrid Magnetic Cross-Linked Enzyme Aggregates of Phenylalanine Ammonia Lyase from Rhodotorula glutinis

Novel hybrid magnetic cross-linked enzyme aggregates of phenylalanine ammonia lyase (HM-PAL-CLEAs) were developed by co-aggregation of enzyme aggregates with magnetite nanoparticles and subsequent crosslinking with glutaraldehyde. The HM-PAL-CLEAs can be easily separated from the reaction mixture by using an external magnetic field. Analysis by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) indicated that PAL-CLEAs were inlayed in nanoparticle aggregates. The HM-PAL-CLEAs revealed a broader limit in optimal pH compared to free enzyme and PAL-CLEAs. Although there is no big difference in Km of enzyme in CLEAs and HM-PAL-CLEAs, Vmax of HM-PAL-CLEAs is about 1.75 times higher than that of CLEAs. Compared with free enzyme and PAL-CLEAs, the HM-PAL-CLEAs also exhibited the highest thermal stability, denaturant stability and storage stability. The HM-PAL-CLEAs retained 30% initial activity even after 11 cycles of reuse, whereas PAL-CLEAs retained 35% of its initial activity only after 7 cycles. These results indicated that hybrid magnetic CLEAs technology might be used as a feasible and efficient solution for improving properties of immobilized enzyme in industrial application.