Composite coal fly ash solid acid catalyst in synergy with chloride for biphasic preparation of furfural from corn stover hydrolysate.

A solid acid catalyst SO42-/SnO2-Al2O3-CFA was synthesized based on industrial waste coal fly ash (CFA) as carrier and applied in the conversion of oxalic acid pretreated corn stover hydrolysate to produce furfural. Physical properties of the solid acid catalyst were characterized by SEM, FTIR, XRD, BET, EDAX, and NH3-TPD. Highly wrinkled structure of SO42-/SnO2-Al2O3-CFA could provide more specific surface area for the covalent linkage between SiO2 and SnO2. Factors influencing the efficacy of SO42-/SnO2-Al2O3-CFA were systematically explored. The highest furfural yield of 84.7% was reached in NH4Cl-toluene biphasic system at 180 °C for 30 min. The recyclability of SO42-/SnO2-Al2O3-CFA and toluene could be achieved for five batches with stable performance in transformation of xylose-rich corn stover hydrolysate. This study provided a novel solid acid catalyst with promising potential in the synthesis of furfural from corn stover.

Detoxification of furfural residues hydrolysate for butanol fermentation by Clostridium saccharobutylicum DSM 13864.

The toxicity of furfural residues (FRs) hydrolysate is a major obstacle in its application. This work focused on the detoxification of FRs hydrolysate and its application in butanol fermentation. Combination of activated carbon and resin 717 was appropriate for the detoxification of hydrolysate. Mixed sterilization of FRs hydrolysate and corn steep liquor (CSL) was better than the separate ones, since proteins in CSL could adsorb and remove toxic components during sterilization. The results further confirmed that simultaneous sterilization of activated carbon + resin and fermentation medium was more efficient for detoxification and butanol production, in which 76.4% of phenolic compounds and 99.3% of Maillard reaction products were removed, 8.48 g/L butanol and 12.61 g/L total solvent were obtained. This study provides feasible and economic approaches for the detoxification of FRs hydrolysate and its application in butanol production.

Enhanced curdlan production with nitrogen feeding during polysaccharide synthesis by Rhizobium radiobacter.

Curdlan is a secondary metabolite synthesized by Agrobacterium sp. and some other bacteria. A newly isolated exopolysaccharide-producing strain was identified to be Rhizobium radiobacter CGMCC 12099. The polysaccharide product was confirmed to be curdlan with a molecule weight of 1.4×10(5)Da, and its molecular structure was determined by HPLC and infrared spectrum. Although nitrogen source is necessary for cell reproduction, curdlan production is largely dependent on nitrogen limitation, as well as cell vitality. Here, a nitrogen feeding strategy was investigated to elevate the curdlan production by R. radiobacter. The optimal concentration and addition time of (NH4)2HPO4 were investigated. The results showed that the enhanced cell density was correlated to the amount of (NH4)2HPO4 added. Also, nitrogen addition in earlier fermentation stage was beneficial to the cell growth and curdlan production. Furthermore, continuously feeding strategy was employed by feeding (NH4)2HPO4 at a constant rate of 1.24g/h at 35(th)h of fermentation for 9h, achieving a final curdlan production of 65.27g/L, productivity of 0.544g/L/h and glucose conversion rate of 38.89%. The curdlan production was improved by 2.1 times compared with that without nitrogen addition. This study provides a feasible and cheap nitrogen feeding strategy to enhance curdlan production.

Arginine deiminase: recent advances in discovery, crystal structure, and protein engineering for improved properties as an anti-tumor drug.

Arginine deiminase (ADI) is an important arginine-degrading enzyme with wide applications, in particular as an anti-cancer agent for the therapy of arginine-auxotrophic tumors. In recent years, novel ADIs with excellent properties have been identified from various organisms, and crystal structures of ADI were investigated. To satisfy the requirements of potential therapeutic applications, protein engineering has been performed to improve the activity and properties of ADIs. In this mini-review, we systematically summarized the latest progress on identification and crystal structure of ADIs, and protein engineering strategies for improved enzymatic properties, such as pH optimum, K m and k cat values, and thermostability. We also outlined the PEGylation of ADI for improved circulating half-life and immunogenicity, as well as their performance in clinical trials. Finally, perspectives on extracellular secretion and property improvement of ADI were discussed.

Characterization and Soluble Expression of D-Hydantoinase from Pseudomonas fluorescens for the Synthesis of D-Amino Acids.

An active D-hydantoinase from Pseudomonas fluorescens was heterogeneously overexpressed in Escherichia coli BL21(DE3) and designated as D-PfHYD. Sequence and consensus analysis suggests that D-PfHYD belongs to the dihydropyrimidinase/hydantoinase family and possesses catalytic residues for metal ion and hydantoin binding. D-PfHYD was purified to homogeneity by nickel affinity chromatography for characterization. D-PfHYD is a homotetramer with molecular weight of 215 kDa and specific activity of 20.9 U mg(-1). D-PfHYD showed the highest activity at pH 9.0 and 60 °C. Metal ions such as Mn(2+), Fe(2+), and Fe(3+) could activate D-PfHYD with 20 % improvement. Substrate specificity analysis revealed that purified D-PfHYD preferred aliphatic to aromatic 5'-monosubstituted hydantoins. Among various strategies tested, chaperone GroES-GroEL was efficient in improving the soluble expression of D-PfHYD. Employing 1.0 g L(-1) recombinant E. coli BL21(DE3)-pET28-hyd/pGRO7 dry cells, 100 mM isobutyl hydantoin was converted into D-isoleucine with 98.7 % enantiomeric excess (ee), isolation yield of 78.3 %, and substrate to biocatalyst ratio of 15.6. Our results suggest that recombinant D-PfHYD could be potentially applied in the synthesis of D-amino acids.

Simultaneous saccharification and fermentation of dilute alkaline-pretreated corn stover for enhanced butanol production by Clostridium saccharobutylicum DSM 13864.

Simultaneous saccharification and fermentation (SSF) process was applied for biobutanol production by Clostridium saccharobutylicum DSM 13864 from corn stover (CS). The key influential factors in SSF process, including corn steep liquor concentration, dry biomass and enzyme loading, SSF temperature, inoculation size and pre-hydrolysis time were optimized. In 5-L bioreactor with SSF process, butanol titer and productivity of 12.3 g/L and 0.257 g/L/h were achieved at 48 h, which were 20.6% and 21.2% higher than those in separate hydrolysis and fermentation (SHF), respectively. The butanol yield reached 0.175 g/g pretreated CS in SSF, representing 50.9% increase than that in SHF (0.116 g/g pretreated CS). This study proves the feasibility of efficient and economic production of biobutanol from CS by SSF.

Enhancing cellulose accessibility of corn stover by deep eutectic solvent pretreatment for butanol fermentation.

In this study, an effective corn stover (CS) pretreatment method was developed for biobutanol fermentation. Deep eutectic solvents (DESs), consisted of quaternary ammonium salts and hydrogen donors, display similar properties to room temperature ionic liquid. Seven DESs with different hydrogen donors were facilely synthesized. Choline chloride:formic acid (ChCl:formic acid), an acidic DES, displayed excellent performance in the pretreatment of corn stover by removal of hemicellulose and lignin as confirmed by SEM, FTIR and XRD analysis. After optimization, glucose released from pretreated CS reached 17.0 g L(-1) and yield of 99%. The CS hydrolysate was successfully utilized in butanol fermentation by Clostridium saccharobutylicum DSM 13864, achieving butanol titer of 5.63 g L(-1) with a yield of 0.17 g g(-1) total sugar and productivity of 0.12 g L(-1)h(-1). This study demonstrates DES could be used as a promising and biocompatible pretreatment method for the conversion of lignocellulosic biomass into biofuel.

Cloning, Expression, and Characterization of budC Gene Encoding meso-2,3-Butanediol Dehydrogenase from Bacillus licheniformis.

The budC gene encoding a meso-2,3-butanediol dehydrogenase (BlBDH) from Bacillus licheniformis was cloned and overexpressed in Escherichia coli BL21(DE3). Sequence analysis reveals that this BlBDH belongs to short-chain dehydrogenase/reductase (SDR) superfamily. In the presence of NADH, BlBDH catalyzes the reduction of diacetyl to (3S)-acetoin (97.3% ee), and further to (2S,3S)-2,3-butanediol (97.3% ee and 96.5% de). Similar to other meso-2,3-BDHs, it shows oxidative activity to racemic 2,3-butanediol whereas no activity toward racemic acetoin in the presence of NAD(+). For diacetyl reduction and 2,3-butanediol oxidation, the pH optimum of BlBDH is 5.0 and 10.0, respectively. Unusually, it shows relatively high activity over a wide pH range from 5.0 to 8.0 for racemic acetoin reduction. BlBDH shows lower K m and higher catalytic efficiency toward racemic acetoin (K m = 0.47 mM, k cat /K m = 432 s(-1)·mM(-1)) when compared with 2,3-butanediol (K m = 7.25 mM, k cat /K m = 81.5 s(-1)·mM(-1)), indicating its physiological role in favor of reducing racemic acetoin into 2,3-butanediol. The enzymatic characterization of BlBDH provides evidence for the directed engineering of B. licheniformis for producing enantiopure 2,3-butanediol.

Succinic acid production from corn stover by simultaneous saccharification and fermentation using Actinobacillus succinogenes.

Simultaneous saccharification and fermentation (SSF) technique was applied for succinic acid production by Actinobacillus succinogenes in a 5-l stirred bioreactor with corn stover as the raw material. The process parameters of SSF, including corn stover pretreatment condition, substrate concentration, enzyme loading and fermentation temperature were investigated. Results indicated that pretreating corn stover with diluted alkaline was beneficial for the succinic acid production, and succinic acid yield could be significantly increased when adding the cellulase supplemented with cellobiase. The maximal succinic acid concentration and yield could reach 47.4 g/l and 0.72 g/g-substrate, respectively. The corresponding operation conditions were summarized as follows: SSF operation at 38 °C for 48 h, diluted alkaline pretreated corn stover as substrate with concentration of 70 g/l, enzyme loading of 20FPU cellulase and 10 U cellobiase per gram substrate. This result suggested an industrial potential of succinic acid production by using SSF and corn stover.

Fermentative production of succinic acid from straw hydrolysate by Actinobacillus succinogenes.

In this work, straw hydrolysates were used to produce succinic acid by Actinobacillus succinogenes CGMCC1593 for the first time. Results indicated that both glucose and xylose in the straw hydrolysates were utilized in succinic acid production, and the hydrolysates of corn straw was better than that of rice or wheat straw in anaerobic fermentation of succinic acid. However, cell growth and succinic acid production were inhibited when the initial concentration of sugar, which was from corn straw hydrolysate (CSH), was higher than 60 g l(-1). In batch fermentation, 45.5 g l(-1) succinic acid concentration and 80.7% yield were attained after 48 h incubation with 58 g l(-1) of initial sugar from corn straw hydrolysate in a 5-l stirred bioreactor. While in fed-batch fermentation, concentration of succinic acid achieved 53.2 g l(-1) at a rate of 1.21 g l(-1) h(-1) after 44 h of fermentation. Our work suggested that corn straw could be utilized for the economical production of succinic acid by A. succinogenes.

Economical succinic acid production from cane molasses by Actinobacillus succinogenes.

In this work, production of succinic acid by Actinobacillus succinogenes CGMCC1593 using cane molasses as a low cost carbon source was developed. In anaerobic bottles fermentation, succinic acid concentration of 50.6+/-0.9 g l(-1) was attained at 60 h using an optimum medium containing molasses pretreated with sulfuric acid, resulting in a succinic acid yield of 79.5+/-1.1% and sugar utilization of 97.1+/-0.6%. When batch fermentation was carried out in a 5-l stirred bioreactor with pretreated molasses, 46.4 g l(-1) of succinic acid was attained at 48 h and faster cells growth was also observed. Fed batch fermentation was performed to minimize the substrate (sugar) inhibition effect, giving 55.2 g l(-1) of succinic acid and 1.15 g l(-1)h(-1) of productivity at 48 h. The present study suggests that the inexpensive cane molasses could be utilized for the economical and efficient production of succinic acid by A. succinogenes.

Anatomical Factors Influencing the Stimulation Intensity of the Functional Electrical Stimulation of the Supraspinatus Muscle

OBJECTIVE: This study was designed to evaluate the contribution of anatomical factors to the stimulation intensity needed for functional electrical stimulation (FES) of shoulder girdle muscles, especially the supraspinatus. METHOD: Anatomical dimensions, including the length of the arm and scapular spine, were measured in twenty three normal subjects. Depth and thickness of the supraspinatus and trapezius muscle were measured ultrasonographically. FES was applied for supraspinatus muscles, and the minimal intensity required to induce contraction was recorded. Correlations of intensity with the anatomical dimensions were investigated statistically. RESULTS: The thickness of the supraspinatus muscle and the length of the scapular spine showed statistically significant correlations with the minimal intensity for FES of supraspinatus muscles. No other anatomical measurements showed significant correlation. CONCLUSION: The intensity required for FES was affected by the thickness and length of muscles, rather than other anatomical variables. The results of this study suggest that one of the major factors contributing to the determination of the intensity of FES is the size of muscles. If the intensity could be estimated before stimulation, based on the size of muscle, unnecessary discomfort of the patients would be avoided.

Expression of Caveolin-3 in the Muscle Cell and Tissue

OBJECTIVE: Caveolae are the microdomain of the plasma membrane that have been implicated in signal transduction and caveolin is a principal component of the caveolae. Caveolin-3, a family of caveolin related protein, is expressed only in muscle tissue. Here we examined the expression of caveolin-3 in the course of myobalst differentiation and within the muscle tissue. METHOD: L6 cell, rat skeletal myoblast, was cultured in the low mitogen medium and caveolin-3 expression was observed both by immunocytochemistry and western blot analysis. Localization of caveolin-3 within the muscle tissue was investigated and compared to that of dystrophin. RESULTS: While caveolin-3 was not expressed in the proliferating myolast, caveolin-3 was expressed in the differentiated myoblast. Caveolin-3 and dystrophin were co-expressed in the membrane of muscle tissue and integrated density of caveolin-3 was elevated in the area of muscle injury. In the Duchenne muscular dystrophy, caveolin-3 was expressed in the membrane of muscle tissue, but dystrophin was not. CONCLUSION: Caveolin-3 was induced during the myobalst differentiation and its expression was increased during the muscle regeneration. Caveolin-3 was physically associated with dystrophin as a complex, but not absolutely required for the biogenesis of dystrophin complex.

Diagnostic Value of MR Imaging in Differentiation of Meniscal Tear Patterns

PURPOSE: To evaluate the diagnostic accuracy of magnetic resonance (MR) imaging in the differentiation ofmeniscal tear patterns of the knee. MATERIALS AND METHODS: MR images of 93 patients with meniscal tear wereincluded in this study. On the basis of arthroscopic findings, the configuration of meniscal tears was classifiedas horizontal (n=44), longitudinal (n=34), transverse (n=11), or oblique (n=5). Oblique sagittal and coronal MRimages were obtained and com-pared with the arthroscopic findings. RESULTS: Among 94 cases ofarthroscopically-proven meniscal tears, 35 of 44 horizontal and 27 of 34 longitudi-nal configurations werecorrectly interpreted on MR images. Sensitivity and specificity for horizontal configu-ration were 80 % and 80 %,respectively, while the corresponding values for longitudinal configuration were 79 % and 95 %. On MR images, tworadial configurations were correctly interpreted from 11 confirmed tears and only one oblique configuration fromfive confirmed tears. CONCLUSION: MR imaging was useful for the differentiation of horizontal and longitudinaltears, but inaccurate in cases involving radial or oblique tears.