Search

Conventional statistical investigations have primarily focused on the comparison of the simple one-dimensional characteristics of protein cavities, such as number, surface area, and volume. These studies have failed to discern the crucial distinctions in cavity properties between thermophilic and mesophilic proteins that contribute to protein thermostability. In this study, the significance of cavity properties, i.e., flexibility and location, in protein thermostability was investigated by comparing structural differences between homologous thermophilic and mesophilic proteins. Three dimensions of protein structure were categorized into three regions (core, boundary, and surface) and a comparative analysis of cavity properties using this structural index was conducted. The statistical analysis revealed that cavity flexibility is closely related to protein thermostability. The core cavities of thermophilic proteins were less flexible than those of mesophilic proteins (averaged B' factor values, −0.6484 and −0.5111), which might be less deleterious to protein thermostability. Thermophilic proteins exhibited fewer cavities in the boundary and surface regions. Notably, cavities in mesophilic proteins, across all regions, exhibited greater flexibility than those in thermophilic proteins (>95% probability). The increased flexibility of cavities in the boundary and surface regions of mesophilic proteins, as opposed to thermophilic proteins, may compromise stability. Recent protein engineering investigations involving mesophilic xylanase and protease showed results consistent with the findings of this study, suggesting that the manipulation of flexible cavities in the surface region can enhance thermostability. Consequently, our findings suggest that a rational or computational approach to the design of flexible cavities in surface or boundary regions could serve as an effective strategy to enhance the thermostability of mesophilic proteins. [ABSTRACT FROM AUTHOR]

Corynebacterium glutamicum was engineered to produce glutaric acid by metabolic engineering approaches starting from the heterologous introduction of the glutaric acid biosynthesis pathway by the expression of Pseudomonas putida davTDBA genes. Two conversion modules i.e., L -lysine conversion module and glutaric acid production module, were constructed on the basis of dual-vector systems to support high-level glutaric acid biosynthesis from glucose with reduced byproducts accumulation. Fed-batch fermentation of the final engineered strain harboring two conversion modules along with Escherichia coli dapBmut (dapBC115G,G116C) expression resulted in the production of 65.6 g L−1 of glutaric acid in a 5 L fermenter. Furthermore, pilot-scale fed-batch fermentation was carried out in a 500 L fermenter to prepare large amounts of glutaric acid, resulting in the production of 56.0 g L−1 of glutaric acid. Glutaric acid produced by the pilot-scale fermentation of C. glutamicum was purified and then used for the synthesis of dioctyl glutarate (DOG) which was further applied in PVC film plasticization and genuine leather coating. It was found that the physical properties of the PVC film plasticized by DOG were comparable to those of the PVC film plasticized by dioctyl succinate (DOS) and dioctyl adipate (DOA). Also, a nitrocellulose (NC) coating agent consisting of DOG could support similar flexibilities and abrasion resistance of leather to those treated with the NC coating agent consisting of DOS and DOA. These results suggest that bio-based DOG developed in this study can be considered as a promising plasticizer having comparable plasticization efficiencies with other aliphatic dicarboxylic acid ester plasticizers. [ABSTRACT FROM AUTHOR]

This paper describes an optimization of planetary mill pretreatment and saccharification processes for improving biosugar production. Pitch pine (Pinus rigida) wood sawdust waste was used as biomass feedstock and the process parameters optimized in this study were the buffering media, the milling time, the enzyme quantity, and the incubation time. Glucose yields were improved when acetate buffer was used rather than citrate buffer. Initially, with each process variable tests, the optimal values were 100 minutes of milling, an enzyme concentration of 16 FPU/g-biomass, and a 12-hour enzymatic hydrolysis. Typically, interactions between these experimental conditions and their effects on glucose production were next investigated using RSM. Glucose yields from the Pinus rigida waste exceeded 80% with several of the conditions tested, demonstrating that milling can be used to obtain high levels of glucose bioconversion from woody biomass for biorefinery purposes. [ABSTRACT FROM AUTHOR]

Abstract: Commonly sold carbonated beverages typically contain 110g/L of sugar. Consequently, this study evaluated the use of four products, including two colas, one diet cola and one lemon–lime flavored beverage from two large distributors world-wide and one local distributor, as fermentative sugar sources. Zymomonas mobilis ZM4 cultures were able to ferment the sugars present, which included sucrose, fructose and glucose, consuming all three completely within about 10h after addition. The results from each of the beverages tested were similar, except for the diet beverage, with a final ethanol concentrations of around 25g/L, an average productivity of approximately 2g*L−1h−1 and average yields of 0.43g ethanol/g glucose. Using a diet beverage with a fructose–glucose mixture added extraneously, we demonstrated that the beverages were not inhibitory or toxic to the cultures. Subsequent experiments with three clostridial strains showed that these beverages can also be used within fermentative processes to generate butanol and butyric acid. As with Z. mobilis ZM4, Clostridium acetobutylicum ATCC 824 and Clostridium beijerinckii NCIMB 8052 were capable of fermenting all three sugars and had final butanol concentrations of 5.3 and 8.7g/L, respectively. In contrast, Clostridium tyrobutyricum ATCC 25755 is unable to utilize sucrose as a carbon source. Although limited to only the monosaccharides fructose and glucose, the butyric acid concentration was 15.4g/L and had a yield of 0.43g butyrate/g glucose, which is only slightly below the theoretical maximum of 0.47. This study illustrates the potential of using wasted or expired carbonated beverages in the biofuels industry. [Copyright &y& Elsevier]

Abstract: Real-time quantitative PCR analyses of Escherichia coli str. BL21(DE3) exposed to 0.5g/L ferulic and coumaric acid showed that the inaA gene was significantly induced (7.7- and 3.6-fold higher, respectively). Consequently, a transcriptional fusion of the inaA promoter with the luxCDABE operon was constructed and characterized with several compounds identified within hydrolysates. Tests demonstrated that the phenolics were major inducers, while acetic acid and furfural had only a minor or no effect on the inaA expression respectively. Additional tests with mutant E. coli strains found that a marA partially abolished the response while a marB knock-out led to a 2–3-fold higher basal level expression as evidenced by the bioluminescent levels of the cultures. However, a significant induction was seen even in the marA mutant, suggesting some other control mechanism is involved in regulating inaA expression during an exposure to the hydrolysate compounds. Finally, E. coli str. BL21(DE3)/pSP4 was used to analyze a spruce hydrolysate sample. Real-time quantitative PCR showed a 2.8-fold induction of the inaA expression level while the bioluminescence from the exposed culture was 22-fold higher than the control, demonstrating the possible application of this reporter strain to analyze hydrolysates for the presence of fermentation-inhibiting phenolics. [Copyright &y& Elsevier]

Lignin, an aromatic polymer found in plants, has been studied for years in many biological fields. Initially, when biofuel was produced from lignocellulosic biomass, lignin was regarded as waste generated by the biorefinery and had to be removed, because of its inhibitory effects on fermentative bacteria. Although it has since proven to be a natural resource for bio-products with considerable potential, its utilization is confined by its complex structure. Hence, the microbial degradation of lignin has attracted researchers' interest to overcome this problem. From this perspective, the studies have primarily focused on fungal systems, such as extracellular peroxidase and laccase from white- and brown-rot fungi. However, recent reports have suggested that bacteria play an increasing role in breaking down lignin. This paper, therefore, reviews the role of bacteria in lignin and lignin-related research. Several reports on bacterial species in soil that can degrade lignin and their enzymes are included. In addition, a cellulolytic anaerobic bacterium capable of solubilizing lignin and carbohydrate simultaneously has recently been identified, even though the enzyme involved has not been discovered yet. The assimilation of lignin-derived small molecules and their conversion to renewable chemicals by bacteria, such as muconic acid and polyhydroxyalkanoates, including genetic modification to enhance their capability was discussed. This review also covers the indirect use of bacteria for lignin degradation, which is concerned with whole-cell biosensors designed to detect the aromatic chemicals released from lignin transformation.

Background: Plant-based cellulose presents the best source of renewable sugars for biofuel production. However, the lignin associated with plant cellulose presents a hurdle as hydrolysis of this component leads to the production of inhibitory compounds, such as ferulic acid., Results: The impacts of ferulic acid, a phenolic compound commonly found in lignin hydrolysates, on the growth, solvent production, and transcriptional responses of Clostridium beijerinckii NCIMB 8052 were determined. Addition of ferulic acid to growing cultures resulted in a decrease in the growth and solvent production by 30% and 25%, respectively, when compared to the control cultures. To better understand the toxicity of this compound, microarray analyses were performed using samples taken from these cultures at three different growth states. Several gene ontology terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were identified showing significant change at each status, including ATP-binding cassette (ABC) transporters, two component system, and oxidoreductase activity. Moreover, genes related with efflux systems and heat shock proteins were also strongly up-regulated. Among these, expression of the groESL operon was induced by more than fourfold and was consequently selected to improve C. beijerinckii tolerance to ferulic acid. Real-time quantitative PCR (RT-qPCR) analysis confirmed that C. beijerinckii harboring the plasmid, pSAAT-ptb_Gro, had a two- to fivefold increased groESL operon expression during growth of these cultures. Moreover, this strain was more tolerant to ferulic acid as the growth of this recombinant strain and its bioconversion of glucose into solvents were both improved., Conclusions: Using transcriptomics, we identified numerous genes that are differentially expressed when C. beijerinckii cultures were exposed to ferulic acid for varying amounts of time. The operon expressing groESL was consistently up-regulated, suggesting that this gene cluster may contribute to strain tolerance. This was confirmed as recombinant cultures showed both an enhanced growth and solvent yield in the presence of 0.5 g/L ferulic acid.

We describe patterning of bacterial biofilms using polymer-based aqueous two-phase system (ATPS) microprinting protocols. The fully aqueous but selectively bacteria-partitioning nature of the ATPS allows spatially distinct localization of suspensions of bacteria such as Pseudomonas aeruginosa and Escherichia coli with high precision. The ATPS patterned bacterial suspensions form spatially distinct biofilms over time. Due to the fully aqueous and gentle noncontact printing procedures employed, coculture biofilms composed of multiple types of bacteria could be printed not only adjacent to each other but also directly over another layer of existing biofilm. In addition, the ATPS environment also allows free diffusion of small molecules between spatially distinct and localized bacterial suspensions and biofilms. This enables biofilms to chemically affect or be affected by neighboring biofilms or planktonic cells, even if they consist of different strains or species. We show that a β-lactamase producing biofilm confers ampicillin resistance to neighboring nonresistant planktonic cells, as seen by a 3,600-fold increase in survival of the ampicillin-sensitive strain. These examples demonstrate the ability of ATPS-based biofilm patterning methods to enable unique studies on commensalistic effects between bacterial species.

Lignin hydrolysates contain many different chemical species, including ferulic acid, coumaric acid, vanillic acid, vanillin, syringaldehyde and furfural. From the perspective of biofuels, these compounds are problematic and can cause downstream loss of product if not removed prior to beginning the fermentative process. In contrast, a search for these compounds within the literature turns up many papers where the same compounds have beneficial properties pertaining to human health, including as antioxidants and in cancer prevention, or are involved in bacterial cell-to-cell signaling. Consequently, this article reviews the dual nature of these and other compounds found in lignin hydrolysates, highlighting both their detrimental and beneficial activities.

Using an aqueous two-phase system comprised of dextran and polyethylene glycol, this article describes the stable spatial patterning of sub-microlitre droplets of bacterial suspensions. Microdroplets of different types of bacterial populations are positioned and maintained adjacent to each other without significant dispersion even though the bacteria are in suspension and not surface bound. Small molecules, in contrast, diffuse relatively freely between the two aqueous phases. The usefulness of these capabilities is demonstrated by generating a small array of suspensions containing different Escherichia coli strains engineered to respond fluorescently or luminescently to different chemical stimuli. When a chemical stimulus is presented, this droplet array produces a pattern of bacterial "illumination" that reflects the type of chemical to which the array was exposed.