Purification and Characterization of Hyaluronate Lyase from <Emphasis Type="Italic">Arthrobacter globiformis</Emphasis> A152

A hyaluronate lyase was obtained by cultivating Arthrobacter globiformis strain A152. The enzyme was purified to homogeneity from the supernatant by ammonium sulfate fractionation, Q Sepharose Fast Flow, and Sephadex G-100 chromatography. The purification resulted in a 32.78-fold increase in hyaluronate lyase activity with specific activity of 297.2 U/mg. The molecular weight of the enzyme determined by SDS-PAGE was approximately 73.7 kDa. Using hyaluronic acid (HA) as a substrate, the maximal reaction rate (V_max) and the Michaelis–Menten constant (K_m) of hyaluronate lyase were found to be 4.76 μmol/min/ml and 0.11 mg/ml, respectively. The optimum pH and temperature values for hyaluronate lyase activity were pH 6.0 and 42 °C, respectively. This enzyme was stable at pH 4–10, 5–7, and 5–7 at 4, 37, and 42 °C, respectively. Investigation about temperature effects on hyaluronate lyase displayed that it was stable at 30–37 °C and also showed high activity at 37 °C. The enzymatic activity was enhanced by Ca²⁺ and was strongly inhibited by Cu²⁺ and SDS. These properties suggested that the hyaluronate lyase in this study could bring promising prospects in medical and industry applications.     

Expression and Characterization of a Novel Antifungal Exo-β-1,3-glucanase from <Emphasis Type="Italic">Chaetomium cupreum</Emphasis>

A novel β-1,3-glucanase gene, designated Ccglu17A, was cloned from the biological control fungus Chaetomium cupreum Ame. Its 1626-bp open reading frame encoded 541 amino acids. The corresponding amino acid sequence showed highest identity (67 %) with a glycoside hydrolase family 17 β-1,3-glucanase from Chaetomium globosum. The recombinant protein Ccglu17A was successfully expressed in Pichia pastoris, and the enzyme was purified to homogeneity with 10.1-fold purification and 47.8 % recovery yield. The protein’s molecular mass was approximately 65 kDa, and its maximum activity appeared at pH 5.0 and temperature 45 °C. Heavy metal ions Fe²⁺, Mn²⁺, Cu²⁺, Co²⁺, Ag⁺, and Hg²⁺ had inhibitory effects on Ccglu17A, but Ba²⁺ promoted the enzyme’s activity. Ccglu17A exhibited high substrate specificity, almost exclusively catalyzing β-1,3-glycosidic bond cleavage in various polysaccharoses to liberate glucose. The enzyme had a Km of 2.84 mg/mL and Vmax of 10.7 μmol glucose/min/mg protein for laminarin degradation under optimal conditions. Ccglu17A was an exoglucanase with transglycosylation activity based on its hydrolytic properties. It showed potential antifungal activity with a degradative effect on cell walls and inhibitory action against the germination of pathogenic fungus. In conclusion, Ccglu17A is the first functional exo-1,3-β-glucanase to be identified from C. cupreum and has potential applicability in industry and agriculture.     

Electricity Production and Characterization of High-Strength Industrial Wastewaters in Microbial Fuel Cell

Microbial fuel cells (MFCs) convert electrochemical energy into electrical energy immediately and have a big potential usage for the same time wastewater treatment and energy recovery via electro-active microorganisms. However, MFCs must be efficiently optimized due to its limitations such as high cost and low power production. Finding new materials to increase the cell performance and reduce cost for MFC anodes is mandatory. In the first step of this study, different inoculation sludges such as anaerobic gum industry wastewater, anaerobic brewery wastewater and anaerobic phosphate were tested, and MFC that was set up with anaerobic gum industry wastewater inoculation sludge exhibited the highest performance. In the second step of this study, various wastewaters such as chocolate industry, gum industry and slaughterhouse industry were investigated for anode bacteria sources. Several electrochemical techniques have been employed to elucidate how wastewaters affect the MFCs’ performance. Among all the mentioned wastewaters, the best performance was achieved by the MFCs fed with slaughterhouse wastewater; this device produced a maximum power density of 267 mW·m^?2.     

Size-Selective Harvesting of Extracellular Vesicles for Strategic Analyses Towards Tumor Diagnoses

Extracellular vesicles (EV), typified by exosomes or microvesicles, are expected to be effective diagnostic markers for cancers. The sizes of the vesicles range from 20 to 1000 nm, but the size-dependent variations of the contents of EVs are still poorly understood. We succeeded in the size-selective harvesting of the vesicles by utilizing the molecular weight-dependent characteristics of a variety of polyethylene glycols (PEG) as precipitating reagents and analyzed the antigens displayed on the surfaces of the vesicles and the miRNAs included in the vesicles from each size group. As a result, the relatively larger (<100 nm) particles precipitated by PEG5k clearly exhibited the greatest amount of epithelial cell adhesion molecule (EpCAM), from both breast cancer (MCF-7) and colon cancer (HCT116) cells, and a larger quantity of microRNA (miRNA) specific to breast cancer cells (miRNA155 for MCF-7) seemed to be contained in the PEG-precipitated particles. The results demonstrated that the quantities of both the tumor-specific miRNA and protein were similarly distributed among the several classes of the size-sorted EVs and that the size-selective harvesting of EVs may be informative for strategic analyses towards the diagnoses of cancers.     

Structure Characterization and Lead Detoxification Effect of Carboxymethylated Melanin Derived from <Emphasis Type="Italic">Lachnum</Emphasis> Sp.

In the present study, an intracellular melanin, named LIM205, was separated from Lachnum YM205 mycelia and was purified on a Sephadex G-15 column. The molecular weight of LIM205 was determined as 522 Da, and its molecular formula was speculated as C₂₈H₁₄N₂O₇S. The possible chemical structure of LIM205 was determined according to the results of Fourier transform infrared (FT-IR), ¹H NMR, ¹³C NMR, and pyrolysis/GC-MS analysis. With the aim to increase its water solubility, its carboxymethylated derivative, named CLIM205, was formed by the substitution of hydrogen atoms in LIM205 with one, two, and three carboxymethylate groups. FT-IR, UV, and ESI-MS analysis demonstrated that the carboxymethylate groups were conjugated onto LIM205. The lead detoxification activities of LIM205 and CLIM205 had also been investigated. In vivo test showed that both LIM205 and CLIM205 reduced the tissue lead concentration, enhanced lead excretion, and reversed lead-induced alterations in superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) concentrations in mice, with CLIM205 showed better efficacy. The study indicates that LIM205 and CLIM205 have significant lead detoxification effect which will contribute to solve related problems.     

Research on the Solid State Fermentation of Jerusalem Artichoke Pomace for Producing <Emphasis Type="Italic">R</Emphasis>,<Emphasis Type="Italic">R</Emphasis>-2,3-Butanediol by <Emphasis Type="Italic">Paenibacillus polymyxa</Emphasis> ZJ-9

R,R-2,3-butanediol (R,R-2,3-BD) was produced by Paenibacillus polymyxa ZJ-9, which was capable of utilizing inulin without previous hydrolysis. The Jerusalem artichoke pomace (JAP) derived from the conversion of Jerusalem artichoke powder into inulin extract, which was usually used for biorefinery by submerged fermentation (SMF), was utilized in solid state fermentation (SSF) to produce R,R-2,3-BD. In this study, the fermentation parameters of SSF were optimized and determined in flasks. A novel bioreactor was designed and assembled for the laboratory scale-up of SSF, with a maximum yield of R,R-2,3-BD (67.90 g/kg (JAP)). This result is a 36.3% improvement compared with the flasks. Based on the same bath of Jerusalem artichoke powder, the total output of R,R-2,3-BD increased by 38.8% for the SSF of JAP combined with the SMF of inulin extraction. Overall, the utilization of JAP for R,R-2,3-BD production was beneficial to the comprehensive utilization of Jerusalem artichoke tuber.     

Prospecting Agro-waste Cocktail: Supplementation for Cellulase Production by a Newly Isolated Thermophilic <Emphasis Type="Italic">B. licheniformis</Emphasis> 2D55

Bacteria isolated from thermophilic environment that can produce cellulase as well as utilise agro-waste biomass have a high potential for developing thermostable cellulase required in the biofuel industry. The cost for cellulase represents a significant challenge in converting lignocellulose to fermentable sugars for biofuel production. Among three potential bacteria examined, Bacillus licheniformis 2D55 (accession no. KT799651) was found to produce the highest cellulolytic activity (CMCase 0.33 U/mL and FPase 0.09 U/mL) at 18–24 h fermentation when grown on microcrystalline cellulose (MCC) as a carbon source in shake flask at 50 °C. Cellulase production process was further conducted on the untreated and NaOH pretreated rice straw (RS), rice husk (RH), sugarcane bagasse (BAG) and empty fruit bunch (EFB). Untreated BAG produced the highest FPase (0.160 U/mL), while the highest CMCase (0.150 U/mL) was supported on the pretreated RH. The mixture of untreated BAG and pretreated RH as agro-waste cocktail has remarkably improved CMCase (3.7- and 1.4-fold) and FPase (2.5- and 11.5-fold) compared to the untreated BAG and pretreated RH, respectively. The mechanism of cellulase production explored through SEM analysis and the location of cellulase enzymes of the isolate was also presented. Agro-waste cocktail supplementation provides an alternative method for an efficient production of cellulase.     

Co-Fermentation of Cheese Whey and Crude Glycerol in EGSB Reactor as a Strategy to Enhance Continuous Hydrogen and Propionic Acid Production

This study evaluated the production of hydrogen and propionic acid in an expanded granular sludge bed (EGSB) reactor by co-fermentation of cheese whey (CW) and crude glycerol (CG). The reactor was operated at hydraulic retention time (HRT) of 8 h by changing the CW/CG ratio from 5:1 to 5:2, 5:3, 5:4, and 5:5. At the ratio of 5:5, HRT was reduced from 8 to 0.5 h. The maximum hydrogen yield of 0.120 mmol H₂ g COD^?1 was observed at the CW/CG ratio of 5:1. Increasing the CG concentration repressed hydrogen production in favor of propionic acid, with a maximum yield of 6.19 mmol HPr g COD^?1 at the CW/CG ratio of 5:3. Moreover, by reducing HRT of 8 to 0.5 h, the hydrogen production rate was increased to a maximum value of 42.5 mL H₂ h^?1 L^?1at HRT of 0.5 h. The major metabolites were propionate, 1,3-propanediol, acetate, butyrate, and lactate.     

Kinetics of Enzymatic Synthesis of Cinnamyl Butyrate by Immobilized Lipase

This work illustrates the enzymatic synthesis of cinnamyl butyrate by esterification of butyric acid and cinnamyl alcohol. Experiments were performed to study the various operating parameters such as molar ratio, enzyme concentration, temperature, and speed of agitation. Also, the suitable kinetic model for esterification reaction was predicted and the various kinetic parameters were determined. It has been observed that the experimental results agree well with the simulated results obtained by following the ping-pong bi-bi mechanism with dead-end inhibition by both the substrate acid and alcohol. The highest 90% conversion of butyric acid was observed after 12 h at the following reaction conditions: substrate molar ratio 1:2 (butyric acid/cinnamyl alcohol), temperature 50 °C, enzyme loading 2% (with respect to the weight of the substrates), and agitation speed 250 rpm. Diffusional mass transfer limitations between substrate and enzyme surface do not show significant effect on reaction kinetics. Enzyme reusability study reveals that it retains 85% of its catalytic activity after five consecutive cycles.