Characterization of Glucokinase Catalysis from a Pseudo-Dimeric View Abstract Glucose phosphorylation by glucokinase exhibits a sigmoidal dependency on substrate concentration regardless of its simple structure. Dimorph mechanism suggested the existence of two enzymatic states with different catalytic properties, which has been shown to be plausible by structural analysis. However, the dimorph mechanism gives rise to a complicated or non-explicit non-closed mathematical form. It is neither feasible to apply the dimorph mechanism in effector characterizations. To improve the area of glucokinase study with stronger theoretical support and less complication in computation, we proposed the investigation of the enzyme from a pseudo-dimeric angle. The proposed mechanism started from the idealization of two monomeric glucokinase as a dimeric complex, which significantly simplified the glucose phosphorylation kinetics, while the differences in enzyme reconfiguration caused by variable substrates and effectors have been successfully characterized. The study presented a simpler and more reliable way in studying the properties of glucokinase and its effectors, providing guidelines of effector developments for hyperglycemia and hypoglycemia treatment.

In Vitro Fabrication and Biocompatibility Assay of a Biomimetic Osteoblastic Niche Abstract A novel HAp-CS/Gel biomimetic osteoblastic niche was fabricated by freeze-drying, and its mechanical strength and biocompatibility were characterized. HAp-CS/Gel scaffolds in various ratios of 100:5, 100:10, and 100:20 (CS/Gel to HAp) were prepared by freeze-drying prior to chemical cross-linking followed by infrared spectrum analysis, EDS, FITR, SEM, fluorescence microscopy, MTT, and ALP experiments. Results from the infrared spectrum analysis showed that HAp doping remained the surface morphology and the architecture of scaffold with interconnected pores in the size range of 135 to 150 μm. The HAp doping ratio of 100:20 was found to be optimal based on its high porosity of 90%, better water uptake folds of 19.1. In addition, EDS and FITR analyses demonstrated that HAps were uniformly distributed on the surface of a scaffold with aggregates and particles, which has sufficient roughness for cell attachment and proliferation of osteoblasts. Under SEM and fluorescent microscopy, osteoblasts seeded onto the scaffold showed evenly distributed viable cells, which is believed to form a biomimetic niche. In the present study, we further demonstrate that osteoblasts can maintain their function and grow well on the scaffold through MTT and ALP tests. Thus, the scaffold has favorable physical properties and biocompatibility to support the proliferation and differentiation of osteoblasts and further to support the constructs of biomimetic osteoblastic niche.

Biotransformation of Isoeugenol into Vanillin Using Immobilized Recombinant Cells Containing Isoeugenol Monooxygenase Active Aggregates Abstract For efficiently enhancing the activity of isoeugenol monooxygenase, a whole cell overproducing active aggregate IEM720-18A was successfully fabricated via the fusion of amphiphilic short peptide 18A (EWLKAFYEKVLEKLKELF) and isoeugenol monooxygenase and then efficiently expressed in E. coli BL21 (DE3). Such resulting bacteria, E. coli BL21 (DE3) harboring pET30a-IEM720-18A was applied in the biotransformation of isoeugenol to vanillin with the optimization of cultivation conditions. Our results revealed that the vanillin concentration reached to the highest level (14.5 mmol/L) under the optimized reaction conditions including 1.5-g cells containing active aggregate of IEM720-18A, 10% (v/v) dimethyl sulfoxide (DMSO), 100 mmol/L isoeugenol, 50 mmol/L glycine-sodium hydroxide buffer (pH 10.5) in 10 mL reaction volume, and 200 rpm at 25 °C for 36 h. Moreover, the active aggregate IEM720-18A was immobilized with 100 mmol/L glutaraldehyde at 4 °C to improve the operational stability. The highest activity could be achieved when the reactions were carried out at 25 °C and the relative activity of the immobilized enzyme maintained over 60% after seven recycles. Our study provides a new approach to the biotransformation of isoeugenol into vanillin.

Capacitive Sensor to Monitor Enzyme Activity by Following Degradation of Macromolecules in Real Time Abstract A capacitive sensor was developed to analyze the presence and enzymatic activity of a model protease from standard solutions by following the degradation of the substrate in real time. The enzyme was chosen based on its specific digestion of the hinge region of immunoglobulin G (IgG). Real-time enzyme activity was monitored by measuring the change in capacitance (∆C) based on the release of IgG fragments after enzymatic digestion by the enzyme. The results indicated that the developed capacitive system might be used successfully for label-free and real-time monitoring of enzymatic activity of different enzymes in a sensitive, rapid, and inexpensive manner in biotechnological, environmental, and clinical applications.

A Case Study on Converting Organic Farm Waste Vegetables to Biogas Using a Cartridge Design Anaerobic Digester Abstract Anaerobic digestion of multiple waste vegetables collected from an organic farm in Central Illinois was carried out using a new cartridge design anaerobic digestion system. Waste vegetables, including carrot, cucumber, bell pepper, onion, lettuce, and potato, were chopped and then mixed together to be used as the digestion feedstock. Three cartridges in the digestion chamber were rotated every week. Results showed that the system was stable, in terms of biogas and methane yields, ammonium-nitrogen concentration, and pH value, throughout the 90-day operation. On average, the daily and accumulative methane yield were 23.38 L/day/kg-VS and 490.98 L/kg-VS (21-day retention time), respectively. Rotation of cartridge significantly affected methane yield, methane concentration in biogas, and hydrogen sulfide concentration in biogas. Especially, the average hydrogen sulfide concentration decreased from 1145 ppm, to 695 ppm, and then to 539 ppm, in biogas samples taken on 2 days, 4 days, and 7 days after rotation. No liquid waste was generated throughout the test. A rough estimate of the potential biogas yield shows that if all the waste vegetables and crop residues collected from this farm were used in this new anaerobic digestion system, US$4711 in energy can be saved in a year.

Effect of Novel Pretreatment of Steam Explosion Associated with Ammonium Sulfite Process on Enzymatic Hydrolysis of Corn Straw Abstract Effective pretreatment process to improve enzymatic saccharification and decrease inhibitors generation is a key operation involved in the lignocellulosic bioconversion. The pretreatment of steam explosion associated with ammonium sulfite (SEAS) process was carried out to investigate the effect on enzymatic hydrolysis and fermentation production as a combinatorial pretreatment. Results showed that after pretreatment (1.0 MPa, 30 min, 20%w/w ammonium sulfite added), the phenolic inhibitors derived from lignin significantly removed (37.8%), which transformed to chemical humic acid (humic acid and fulvic acid) mostly. Sugar conversion (glucan (77.8%) and xylan (73.3%)) and ethanol concentration (40.8 g/L) of combinatorial pretreated samples were increased by 24.7% and 33.8%, respectively, compared with steam explosion (SE) pretreated samples. FT-IR and elemental analysis results indicated that the lignin structure changed and aromatization degree increased after SEAS pretreatment. In addition, the ratio of C/N decreased and compost maturity degree increased with the holding time. The effect on the growth of wheat seedlings of soluble fulvic acid solution from combinatorial pretreatment was investigated, where below 1% (w/w) concentration did contribute to growth. Therefore, one-step chemical pretreatment process could be provided for inhibitors removal, enzymatic saccharification increase, and chemical humic acid formation as well.

Enhanced Low Molecular Weight Poly-γ-Glutamic Acid Production in Recombinant Bacillus subtilis 1A751 with Zinc Ion Abstract Poly-γ-glutamic acid (γ-PGA) is a novel biodegradable polyamide material. Microbial fermentation is the only way to produce γ-PGA, but the molecular weight of γ-PGA varied depending on different strains and culture conditions used. The molecular weight of γ-PGA is a main factor affecting the utilization of γ-PGA. It is urgent to find an efficient way to prepare γ-PGA with specific molecular weight, especially low molecular weight. Bacillus subtilis ECUST is a glutamate-dependent strain that produces γ-PGA. In this study, a recombinant B. subtilis harboring the γ-PGA synthase gene cluster pgsBCAE of our preciously identified γ-PGA–producing B. subtilis ECUST was constructed. Assay of γ-PGA contents and properties showed that recombinant B. subtilis 1A751-pBNS2-pgsBCAE obtained the ability to synthesize γ-PGA with low molecular weight (about 10 kDa). The excessive addition of glutamate inhibited the γ-PGA synthesis, while the addition of Zn2+ could promote the synthesis of γ-PGA by increasing the transcription of pgsB but had no effect on the molecular weight of synthesized γ-PGA. Under optimized conditions, γ-PGA produced by recombinant B. subtilis 1A751-pBNS2-pgsBCAE increased from initial 0.54 g/L to 3.9 g/L, and the glutamate conversion rate reached 78%. Recombinant B. subtilis 1A751-pBNS2-pgsBCAE has the potential for efficient preparation of low molecular weight γ-PGA.

Bioconversion of Poplar Wood Hemicellulose Prehydrolysate to Microbial Oil Using Cryptococcus curvatus Abstract Poplar wood hemicellulose prehydrolysate was used for microbial oil production using an oleaginous microorganism Cryptococcus curvatus. Initially, the effect of substrate concentration and nitrogen content was investigated on synthetic media. Then poplar wood prehydrolysate without detoxification was used as substrate in the fermentation. The result showed that this strain is capable of consuming both hexose and pentose sugars, a challenge in fermentation of hemicellulosic streams. It was able to accumulate 36.98% of lipid and the fermentation resulted in 13.78 g/L of biomass and 5.13 g/L of lipid under optimum conditions after 164 h of fermentation. The lipid product obtained was characterized in terms of their fatty acid profiles. Overall, this study shows that it is possible to produce microbial oil from a sustainable renewable feedstock like poplar wood hemicellulose prehydrolysate. This robust strain used has the ability to grow on industrially produced hemicellulose which can help in the development of an integrated biorefinery, where all the three components of lignocellulosic biomass are utilized.

Activity-Structure Study on the Peptide Fraction of AG2: a Potent In Vitro Transfection Agent Abstract Gemini-based amphiphiles are candidates for biomedical applications. In fact, most of the gemini compounds described in the literature have been prepared to be used as new synthetic vectors in gene transfection. Our group carried out an activity-structure study starting from the structure of the gemini [AG2-C18/]2, which is an effective in vitro transfection reagent. We synthesized a series of novel amphiphilic amino acid derivatives of low molecular weight, named AGn-Cm (N), in which the same apolar region (m) of oleic or palmitic acid was maintained and the peptide region was modified by amino acid insertions, deletions, and substitutions. We also determined the transfection efficiency, critical micelle concentration, particle size, and ζ-potential for these derivatives. Amphiphiles AG10-C16 and AG10-C18 were more active at a lower N/P ratio than AG2-C18. These amphiphiles showed no activity when lysine was replaced by ornithine, and the activity of all derivatives increased when there were more ornithine residues and a W/O = 1 ratio in the peptide region. It can be said that for AG10-C16, these two structural requirements on the amino acid portion predominated over the type of aliphatic chain used.

Partitioning of Recombinant Pseudomonas putida POS-F84 Proline Dehydrogenase in Aqueous Two-Phase Systems: Optimization Using Response Surface Methodology Abstract Empirical modeling the partition behavior and recovery of a recombinant Pseudomonas putida POS-F84 proline dehydrogenase (ProDH) in aqueous two-phase systems (ATPS) was carried out by response surface methodology (RSM). Polyethylene glycol 1000 (PEG-1000) concentration, sodium carbonate concentration, and pH, which were the most important factors, were chosen for modeling the partition feature of enzyme. The adequacy of the models was investigated by means of variance analysis. Also, to confirm the efficiency of the ATPS in partition and purification of recombinant ProDH, purity and enzymatic activity was studied. After numerical optimization, an optimal ATPS composed of 14.33% PEG-1000 and 11.79% sodium carbonate at pH 7.48 was achieved. Yield, purification factor, and recovery were 81.41%, 60.82, and 270.82%, respectively. Purified recombinant ProDH was found as a single protein band into the upper PEG-rich phase and the specific activity was calculated to be 46.23 ± 2.1 U/mg. Collectively, our data showed that the RSM could be an appropriate and powerful tool to define the best ATPS system for recovery and purification of P. putida ProDH.