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Endo-1,4-β-xylanase catalyzes the random hydrolysis of β-1,4-D-xylosidic bonds in xylan, resulting in the formation of oligomers of xylose. This study aims to demonstrate the promise of endoxylanases from alkaliphilic Bacillus halodurans for the production of xylooligosaccharides (XOS) from oil palm empty fruit bunch (EFB) at high pH. Two enzyme preparations were employed: recombinant endoxylanase Xyn45 (GH10 xylanase) and nonrecombinant endoxylanases, a mixture of two extracellular endo-1,4-β-xylanases Xyn45 and Xyn23 (GH11 xylanase) produced by B. halodurans. EFB was first treated with an alkaline solution. Then, the dissolved xylan-containing fraction was retained, and a prepared enzyme was added to react at pH 8 to convert xylan into XOS. Compared with the use of only Xyn45, the combined use of Xyn45 and Xyn23 resulted in a higher yield of XOS, suggesting the synergistic effect of the two endoxylanases. The yield of XOS obtained from EFB was as high as 46.77% ± 1.64% (w/w), with the xylobiose-to-xylotriose ratio being 6:5. However, when the enzyme activity dose was low, the product contained more xylotriose than xylobiose. Four probiotic lactobacilli and bifidobacteria grew well on a medium containing XOS from EFB. The presence of XOS increased cell mass and reduced pH, suggesting that XOS promoted the growth of probiotics.

In this study, expired egg white was used as a template, and a sol–gel method was employed to prepare pure-phase TiO2 nano-powder and mixed-phase powders doped with NaF and NaI. The influences of different calcination temperatures, doping elements, and doping amounts during the preparation process on the photocatalytic performance and activity of the prepared TiO2 powders were studied. The results of the experiments showed that the F-doped TiO2 had the highest photocatalytic activity when the doping amount was 1.2%, as examined by EDS, where the sintering temperature was 500 °C. F-doped TiO2 nanoparticles were also synthesized by the sol–gel method using tetrabutyl titanate and NaF mixed with expired egg white protein as the precursor. The F-TiO2 photocatalyst was characterized using FE-SEM, HR-TEM, EDS, XPS, and UV-Vis, and the photocatalytic activity was evaluated by photodegradation of methylene blue under visible light. The results showed that doping with F reduced the energy band gap (3.04 eV) of TiO2, thereby increasing the photocatalytic activity in the visible-light region. The visible-light wavelength range and photocatalytic activity of the catalyst were also affected by the doping amount.

This study aims to compare and analyze the chemical structures of bacterial biosurfactants produced by an indigenous Bacillus subtillis strain cultured in minimal salt medium (MSM) enriched with fresh cooking oil and used cooking oils that are cheaper and easy to obtained in huge quantities. Optimization of biosurfactants was successfully conducted in two MSMs enriched with 2% (v/v) fresh cooking oil and 2% (v/v) waste cooking oil respectively. The initial pH of growth medium was fixed at pH 7.64 and the whole fermentation process was maintained in a constant temperature of 30 °C. Chemical structures analysis of biosurfactants produced at 1L scale in bioreactor were conducted using spectroscopic methods such as Nuclear Magnetic Resonance (NMR) and Fourier-Transform Infrared spectroscopy (FTIR). The chemical compound isolated from the biosurfactant produced from the waste cooking oil was pentyl 2-ureidobut-3-enoate (1) while (1-hydroxyethyl)amino)methyl heptanoate (2) was isolated from the biosurfactant produced from fresh cooking oil. This study demonstrated the ability of Bacillus subtilis to produce a low-cost biosurfactant characterized as lipopeptide.

Fluoride contamination in water due to natural and anthropogenic activities has been documented as serious problems worldwide commanding a major threat to the environment. Present study focuses to synthesis bacterial-surfactin ( Bacillus subtilis ) mediated nano-hydroxyapatite (HAp), novel adsorbents for defluoridation. HAp particle size and morphology were controlled by varying temperature of 90–150 °C and pH of 7–11, respectively. The TEM and SEM micrographs reveal that the short-rod particle is observed 20–30 nm at 90 °C and pH 11. The ratio between the length (nm) and width (nm) of nanoparticle are decreased from 4.17 to 1.65 with increasing pH (7–11). The selected area diffraction (SAD) of particles are indicated uniform rod-like monocrystals. The XRD and FTIR observations were indicated the synthesized HAp nanoparticles were well-crystallized with purity phase and high quality. The study reflected that the fluoride removal from contaminated water by HAp was increased significantly (R 2 = 99) with the increasing adsorbent concentration, temperature and time, with two-step adsorption process as the first portion a rapid adsorption occurs during first 90 min after which equilibrium is slowly achieved. The adsorption process is closer to Freundlich isotherm (R 2 > 98) than to Langmuir isotherm (R 2 ≈ 92), indicating HAp as a good adsorbent (n > 3). Above 97% of fluoride removal were noticed at a HAp dose of 0.06 g/10 mL. The adsorption kinetics more fit with pseudo-second-order (R 2 = 99) in compare to pseudo-first-order (R 2 ≈ 91). The slope and intercept of Arrhenius equation indicated the activation/adsorption energy (E a ) of 3.199 kJ/mol and frequency factor (A) of 1.78 1/s. Adsorption thermodynamic parameters (free energy (ΔG 0) and entropy (ΔS > 0)) indicates the spontaneous and endothermic reactions of the adsorption process. Thus, newly synthesized HAp nanoparticles exhibit as a good adsorbent for fluoride removal, theoretically and experimentally being applicable for environmental pollution control.

The polarity distribution of water-soluble organic matter (WSOM) is believed to play an important role in determining the water uptake properties of aerosol particles. The 1-octanol−water partitioning method has been proposed as a method for classifying WSOM by polarity. However, the broad partitioning functions of the method make it difficult to estimate polarity distribution. In this study, a data inversion technique was developed to determine the polarity distributions of WSOM. The inversion technique was evaluated against artificially generated polarity distributions with corresponding uncertainties. In addition, the technique was combined with positive matrix factorization (PMF) analysis of the aerosol mass spectrometric data, allowing an estimation of the polarity distribution of the PMF factors. The technique was applied to WSOM generated by smoldering mosquito coils. As the 1-octanol−water partitioning coefficients of WSOM correlate well with water solubility, future application of this method to the investigation of hygroscopic growth and cloud condensation nucleus (CCN) activity will help to quantitatively connect the chemical characteristics and water uptake properties of WSOM. Copyright © 2020 American Association for Aerosol Research

Polarity distribution of water-soluble organic matter (WSOM) is an important factor in determining the hygroscopic and cloud nucleation abilities of organic aerosol particles. We applied a novel framework to quantitatively classify WSOM based on the 1-octanol-water partition coefficient (KOW), which often serves as a proxy of polarity. In this study, WSOM was generated in a laboratory biomass-burning experiment by smoldering of Indonesian peat and vegetation samples. The fractionated WSOM was analyzed using a UV-visible spectrophotometer, spectrofluorometer, and time-of-flight aerosol chemical speciation monitor. Several deconvolution methods, including positive matrix factorization, parallel factor analysis, and least-squares analysis, were applied to the measured spectra, resulting in three classes of WSOM. The highly polar fraction of WSOM, which predominantly exists in the range of log KOW < 0, is highly oxygenated and exhibits similar optical properties as those of light-absorbing humic-like substances (HULIS, termed after the humic substances due to the similarity in chemical characteristics). WSOM in the least-polar fraction, which mainly distributes in log KOW > 1, mostly consists of hydrocarbon-like and high molecular weight species. In between the most- and least-polar fraction, WSOM in the marginally polar fraction likely contains aromatic compounds. The analyses have also suggested the existence of HULIS with different polarities. Comparison with previous studies indicates that only WSOM in the highly polar fraction (log KOW < 0) likely contributes to water uptake. Nanyang Technological University National Research Foundation (NRF) We acknowledge Patrick Martin for permitting us to use the UV–vis and fluorescence spectrometers. We thank Yongli Zhou, Ashleen S. Y. Tan, and Liudongqing Yang for assistance with the UV–vis and fluorescence measurement, Xin Wang for designing the graphical abstract, and Pavel Adamek for English editing. We also thank Dr. Haris Gunawan for supporting sample collection in Indonesia. This research was supported by the National Research Foundation Singapore under its Singapore NRF Fellowship scheme (National Research Fellow Award, NRF2012NRF-NRFF001-031), NRF Campus for Research Excellence and Technological Enterprise (CREATE) Programme, the Earth Observatory of Singapore (EOS), Nanyang Technological University, and the Research Institute for Humanity and Nature (RIHN) (Project ID 14200117).

Water-soluble organic matters (WSOMs) play an important role in determining magnitudes of climatic and environmental impacts of organic aerosol particles because of their contributions to hygroscopic growth and cloud formation. These processes are dependent on water solubility as well as distribution of this property in a particle, yet no method has been available to quantify such characteristics. In this study, we developed a theoretical framework to classify WSOM by 1-octanol-water partitioning that has a strong correlation with water solubility. 1-octanol-water partitioning coefficient also has a strong correlation with a traditional solid phase extraction method, facilitating interpretation of data from the technique. The theoretical analysis demonstrated that the distributions of WSOM classified by 1-octanol-water partitioning depend on (1) the volume ratio of 1-octanol and aqueous phases, and (2) extraction steps. The method was tested by using organic aerosol particles generated by smoldering of a mosquito coil, which serves as a surrogate for biomass burning particles. The WSOM extracted from the mosquito coil burning particles was classified by 1-octanol-water partitioning at different volume ratios. These solutions, including both the 1-octanol and aqueous phases, were nebulized to generate particles for measurements using an online aerosol mass spectrometer. The mass spectra indicated that highly oxygenated species tend to be highly soluble, while high molecular weight compounds are less soluble. Linear combinations of these mass spectra allowed the estimation of the mass fractions of WSOM partitioned to 1-octanol and aqueous phases, thereby facilitating the evaluation of the mass fractions of cloud condensation nuclei (CCN) active materials. NRF (Natl Research Foundation, S’pore) Accepted version

Xylitol production from xylose by the yeast Candida magnoliae TISTR 5663 was enhanced by supplementing the fermentation medium with furfural (300mg/L) and glucose (3g/L with an initial mass ratio of glucose to xylose of 1:10) together under oxygen limiting conditions. In the presence of furfural and glucose, the final concentration of xylitol was unaffected relative to control cultures but the xylitol yield on xylose increased by about 5%. Supplementation of the culture medium with glucose alone at an initial concentration of 3g/L, stimulated the volumetric and specific rates of xylose consumption and the rate of xylitol production from xylose. In a culture medium containing 30g/L xylose, 300mg/L furfural and 3g/L glucose, the volumetric production rate of xylitol was 1.04g/L h and the specific production rate was 0.169g/g h. In the absence of furfural and glucose, the volumetric production rate of xylitol was ∼35% lower and the specific production rate was nearly 30% lower. In view of these results, xylose-containing lignocellulosic hydrolysates contaminated with furfural can be effectively used for producing xylitol by fermentation so long as the glucose-to-xylose mass ratio in the hydrolysate does not exceed 1:10 and the furfural concentration is ≤300mg/L.

Cassava waste pulp (CWP)–enzymatic hydrolysate was co-fermented with molasses (CWP-EH/molasses mixture) with the aim to optimize ethanol production by Saccharomyces cerevisiae TISTR 5606 (SC 90). The optimal fermentation conditions for ethanol production using this mixture were 245 g/L initial total sugar supplemented with KH2PO4 (8 g/L), at 30 °C for 48 h of fermentation under an oxygen-limited condition with agitation at 100 rpm, producing an ethanol concentration of 70.60 g/L (0.31 g ethanol/g total sugar). The addition of cassava tuber fiber (solid residue of CWP after enzymatic hydrolysis) at 30 g/L dry weight to the CWP-EH/molasses mixture increased ethanol production to 74.36 g/L (0.32 g ethanol/g total sugar). Co-fermentation of CWP-EH with molasses had the advantage of not requiring any supplementation of the fermentation mixture with reduced nitrogen.

The production of ethanol from the fast-growing perennial C4 grass Miscanthus floridulus by simultaneous saccharification and fermentation (SSF) was investigated. M. floridulus biomass was composed of 36.3% glucan, 22.8% hemicellulose, and 21.3% lignin (based on dried mass). Prior to SSF, harvested stems of M. floridulus were pretreated separately by alkali treatment at room temperature, alkali treatment at 90 °C, steam explosion, and acid-catalyzed steam explosion. The delignification rates were determined to be 73.7%, 61.5%, 42.7%, and 63.5%, respectively, by these four methods, and the hemicellulose removal rates were 51.5%, 85.1%, 70.5%, and 97.3%, respectively. SSF of residual solids after various pretreatments was performed with dried yeast (Saccharomyces cerevisiae) and cellulases (Accellerase 1000) by using 10% water-insoluble solids (WIS) of the pretreated M. floridulus as the substrate. The ethanol yields from 72-h SSF of M. floridulus biomass after these pretreatments were 48.9 ± 3.5, 78.4 ± 1.0, 46.4 ± 0.1, and 69.0 ± 0.1% (w/w), respectively, while the ethanol concentrations after 72-h SSF were determined to be 15.4 ± 1.1, 27.5 ± 0.3, 13.9 ± 0.1, and 30.8 ± 0.1 g/L, respectively. Overall, the highest amount of ethanol (0.124 g/g-dried raw material) was generated from dried raw material of M. floridulus after alkaline pretreatment at 90 °C. The acid-catalyzed steam explosion pretreatment also resulted in a high ethanol yield (0.122 g/g-dried raw material). Pretreatment resulting in high lignin and hemicellulose removal rates could make biomass more accessible to enzyme hydrolysis and lead to higher ethanol production.

Magnetic nanoparticles bound with glutathione (GSH) are useful for diagnostics, enzyme immobilization, and affinity precipitation by using the strong and specific interaction of GSH with glutathione S-transferase (GST)-fused proteins. Our studies revealed that GSH-bound magnetic nanoparticles could be obtained using the covalent bond linkage of GSH and nanoparticles to promote the stability of bound GSH. To yield this conjugate, superparamagnetic iron oxide nanoparticles (SPIONs) were prepared and modified using tetraethoxysilane (TEOS) and 3-aminopropyltriethoxysilane (APTES), which introduced amino groups that were then activated with maleic anhydride (MA) for covalent binding of GSH. After MA was used to activate the amino-grafted SPION for 24 h, the yield of GSH conjugation increased over 4 days from 37 % to 74 % of the original amine density on the surface as the incubation of GSH with MA-activated SPION. These GSH-bound magnetic nanoparticles, designated as SPION@silica-GSH with approximately 103 nmol GSH/mg particles, were ready for coupling with GST-fused protein through the GSH-GST affinity interaction. A GST-tagged small fragment of ubiquitin-like-specific protease 1 (sfULP1) was used as the model protein for immobilization on SPION@silica-GSH. ULP1 is a small ubiquitin-like modifier (SUMO) protease. Results indicated that this immobilized GST-sfULP1 could retain 87 % ± 5 % enzyme activity of free protease before immobilization and could catalyze the cleavage of the SUMO-fused peptide (SUMO-GLP-1) to obtain glucagon-like peptide-1, a peptide hormone for type 2 diabetes therapy.

Organic matter (OM) accounts for a significant fraction of submicron particulate in the atmosphere. The impact of OM on climate and human health is largely contributed by the water-soluble OM (WSOM). Interplay of WSOM and water leads to hygroscopic growth and cloud formation processes. Magnitude of the organic hygroscopicity can be determined by the polarity distribution of WSOM, yet experimental method to systematically examine the relationship is not available. This thesis presents a novel framework to classify WSOM according to its 1-octanol−water partition coefficient (KOW), which often serves as a metric of polarity and water solubility. Fractionization of OM by the KOW approach offers information about the degree of oxidation, which has been viewed as a critical indicator in governing the thermodynamic properties of OM. A theoretical method was developed to classify WSOM by varying the volume ratio of 1-octanol and aqueous phases and extraction steps. An inversion algorithm was further developed to derive KOW distribution of WSOM. The method was verified by OM from proxy of biomass burning particles, and applied to Indonesian biomass burning particles. Optical and chemical characteristics of the extracted WSOM were measured by UV−visible spectrophotometer, spectrofluorometer, and aerosol mass spectrometer. Deconvolution methods including positive matrix factorization, parallel factor analysis, and least-squares analysis were applied to the measured spectra, categorizing WSOM into three classes. The highly polar fraction of WSOM (log KOW < 0) is dominated by highly oxygenated species; the marginally polar fraction (0 ≤ log KOW ≤ 1) likely contains aromatic compounds; while the least polar fraction (log KOW ≥ 1) mostly consists of hydrocarbon-like species. Comparison with hygroscopicity measurements indicates that only WSOM in the highly polar fraction (log KOW < 0) can significantly contribute to water uptake. Doctor of Philosophy

The marine microalgae Nannochloropsis oculata is a promising source of biofuel because of its high lipid content. For achieving high productivity of oil from microalgae, a high cell concentration before harvesting is beneficial. The present study investigated fed-batch cultures of N. oculata fed with vitamins and nutrient solutions and found that the biomass yield of N. oculata in the fed-batch culture was 1.25 times higher than that in batch culture. Fed-batch cultivation, especially at high illumination, decreased the inhibitory effect of high carbon dioxide (CO 2 ) concentration on the microalgal growth. The specific growth rate was directly proportional to the light intensity in the CO 2 environment. A light intensity of 40,000 Lux was able to achieve high specific growth rates in fed-batch cultivation at a CO 2 volume fraction of 2%–15%. The tolerance of N. oculata to CO 2 was enhanced by the daily feeding of nutrients in the fed-batch cultivation. At 2% CO 2 , a final cell density of about OD 682 = 11.4 was achieved in the fed-batch culture in 30 days. Furthermore, a cell density of 14.4 g L −1 was obtained by outdoor fed-batch cultivation in 27 days.

BH2, a monoclonal antibody prepared against the denatured human leukocytic antigen-B27 heavy chain (HLA-B27 HC), can immunoprecipitate the misfolded HLA-B27 HC complexed with Bip in the endoplasmic reticulum and recognize the homodimerized HLA-B27 HC that is often observed on the cell membrane of patients suffered from ankylosing spondylitis (AS). However, the recognition specificity of BH2 toward the other molecules of HLA-B type and toward the different types of HLA molecules remained uncharacterized. In this study, we carried out the HLA-typing by using the Luminex Technology to characterize the recognition specificity of BH2 and analyzed the binding domain of HLA-B27 HC by BH2. Our results indicated that BH2 preferably binds to molecules of HLA-B and -C rather than HLA-A and the binding site is located within the α2 domain of HLA-B27 HC.

This report describes a novel way to generate a highly effective hydrophobic cement surface via a carbonation route using sodium stearate. Carbonation reaction was carried out at different temperatures to investigate the hydrophobicity and morphology of the calcium carbonate formed with this process. With increasing temperatures, the particles changed from irregular shapes to more uniform rod-like structures and then aggregated to form a plate-like formation. The contact angle against water was found to increase with increasing temperature; after 90 °C there was no further increase. The maximum contact angle of 129° was obtained at the temperature of 60 °C. It was also found that carbonation increased the micro hardness of the cement material. The micro hardness was found to be dependent on the morphology of the CaCO3 particles. The rod like structures which caused increased mineral filler produced a material with enhanced strength. The 13C cross polarization magic-angle spinning NMR spectra gave plausible explanation of the interaction of organic-inorganic moieties.

Acetic acid, a potential growth inhibitor, commonly occurs in lignocellulosic hydrolysates. The growth of Cupriavidus necator DSM 545 and production of poly(3-hydroxybutyrate) (PHB) by this bacterium in a glucose-based medium supplemented with various initial concentrations of acetic acid are reported. The bacterium could use both glucose and acetic acid to grow and produce PHB, but acetic acid inhibited growth once its initial concentration exceeded 0.5 g/L. As acetic acid is an unavoidable contaminant in hydrolysates used as sugar sources in commercial fermentations, a mathematical model was developed to describe its impact on growth and the production of PHB. The model was shown to satisfactorily apply to growth and PHB production data obtained in media made with acetic-acid-containing hydrolysates of Napier grass and oil palm trunk as carbon substrates.

The relationship between hygroscopic properties and chemical characteristics of Indonesian biomass burning (BB) particles, which are dominantly generated from peatland fires, was investigated using the humidified tandem differential mobility analyzer. In addition to peat, acacia (a popular species at plantation) and fern (a pioneering species after disturbance by fire) were used for experiments. Fresh Indonesian peat burning particles are almost non-hygroscopic (mean hygroscopicity parameter, κ

A co-expression system was established in Escherichia coli for enhancing the cellular expression of heat shock transcription factor, sigma 32 (σ(32)). A Shine-Dalgarno sequence and the rpoH gene of E. coli, which encodes σ(32), were cloned into a bacterial plasmid containing a gene fusion encoding a doubly tagged N-acetyl-d-neuraminic acid aldolase (GST-Neu5Ac aldolase-5R). After the IPTG induction, a substantially higher level of sigma 32 was observed up to 3 h in the co-expression cells, but an enhancement in the solubility of target protein was manifest only in the first hour. Nevertheless, the co-expression of sigma 32 led to higher level of Neu5Ac aldolase enzymatic activity in both the soluble and insoluble (inclusion body) fractions. The Neu5Ac aldolase activity of the supernatant from the lysate of cells co-expressing GST-Neu5Ac aldolase-5R and recombinant σ(32) was 3.4-fold higher at 3 h postinduction than that in cells overexpressing GST-Neu5Ac aldolase-5R in the absence of recombinantly expressed σ(32). The results of acrylamide quenching indicated that the conformational quality of the fusion protein was improved by the co-expression of recombinant σ(32). Thus, the increased level of intracellular σ(32) might have created favorable conditions for the proper folding of recombinant proteins through the cooperative effects of chaperones/heat shock proteins expressed by the E. coli host, which resulted in smaller inclusion bodies, improved conformational quality and a higher specific activity of the overexpressed GST-Neu5Ac aldolase-5R protein.

The production of ethanol from sugarcane bagasse by simultaneous saccharification and fermentation (SSF) using thermotolerant Kluveromyces marxianus var. marxianus and commercial cellulase of Accellerase 1000 was investigated. Sugarcane bagasse pretreated with NaOH at the room temperature for 24 h resulted in an increase in cellulose content to 55.2% (w/w). With 10% of water-insoluble-solids (WIS) at pH 5.0 supplemented with 0.2 ml cellulase/g-WIS and 1 g/L of thermotolerant yeast, the SSF of pretreated sugarcane bagasse conducted in flasks led to a respective theoretical ethanol yield of 85.1%, 92.2% and 76.2% under 37, 42 and 45 °C. As the rotary drum reactor was scaled up to 100L and loaded with 10 kg alkali-pretreated sugarcane bagasse, the SSF at 42 °C for 72 h along with same doses of WIS, cellulase and yeast resulted in a final ethanol concentration and the theoretical ethanol yield of 24.6 g/L and 79%, respectively. These observations indicated that the performance and the chosen SSF operating conditions for the scale-up drum reactor was as effective as those attained from flask runs. As demonstrated, the use of rotary drum reactor for cellulosic ethanol production under SSF operating conditions is simple to scale up and shows commercial potential.

Folic acid (FA) was coupled onto titanium dioxide nanoparticles to provide a specificity of cell targeting. Modification was confirmed by infrared absorption spectra and zeta potential that suggested a formation of linkage between carboxylic acid of FA and titanium dioxide. The surface attachment of folic acid did not cause a significantly change in particles size, but led to a reduction of photocatalytic activity of TiO 2 . The FA-modified TiO 2 nanoparticles could be internalized by cells at a much faster rate than the unmodified TiO 2 , due to the mediation of folate receptor on the cancer cells. The UV irradiation caused death of HeLa cells pretreated with FA-modified TiO 2 more effectively than that of HeLa cells treated with unmodified TiO 2 . Results suggest that to modify TiO 2 with folic acid using appropriately the FA-to-TiO 2 mass ratio of 0.2 could yield nanoparticles having higher cytotoxicity under photoexcitation. Photocatalytic TiO 2 nanoparticles could not only damage to cellular components including plasma membranes leading to cell nucrosis but also induce the programming cell death. Results from flow cytometry-based analysis indicated that the mechanism of cell death was a combination of necrosis and apoptosis.

Two SDS-resistant endochitinases, designated as ASCHI53 and ASCHI61, were isolated from Aeromonas schubertii in a soil sample from southern Taiwan. MALDI-TOF mass measurement indicates the molecular weights of 53,527 for ASCHI53 and 61,202 for ASCHI61. N-terminal and internal amino acid sequences were obtained, and BLAST analysis of the sequences and MS/MS peptide sequencing showed that they were novel proteins. Degradation of chitin by these two endochitinases gave rise to hexameric chitin oligosaccharide, a compound known to have several potent biomedical functions. ASCHI53 and ASCHI61 retained, respectively, 65% and 75%, of their chitinase activity in the presence of 5% SDS and 100% of their activity in the presence of 10% β-mercaptoethanol. These results demonstrate that they are SDS-resistant endochitinases and probably have a rigid structure. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009

A magneto-transformation method was developed for transferring plasmid DNA into Escherichia coli. Superparamagnetic nanoparticles (Fe3O4) having the saturation magnetization up to 68 emu/g were prepared by the method of co-precipitation at alkaline and thermal conditions, coated with polyethyleneimine (PEI), and then complexed with negatively charged DNA molecules. Plasmid DNA (pGEX-1λT) attached to the PEI-coated magnetic nanoparticles could be transformed into E. coli DH1 by the application of a high magnetic field in pulses. Experimental results suggested that magnetic nanoparticles could well assist DNA delivery into bacterial cells. Factors that could significantly influence the transformation efficiency included the multiple pulsing in magnetic field and concentrations of DNA and magnetic nanoparticles. The highest transformation efficiency could be achieved by pulsing for three times in the 2.15 T magnetic field. The survival declined drastically at the additional pulses. Magneto-transformation of cells with 25 μg DNA and 16 μg magnetic nanoparticles resulted in transformants number in the range from 1600 to 2480, corresponding to efficiency from 64 to 99 cfu/μg DNA. Although the yield of transformants increased with input DNA, the transformation efficiency decreased with DNA dosage. Superparamagnetic nanoparticles played a crucial role in the bacterial transformation in strong magnetic field. Increasing the amount of input magnetic nanoparticles decreased both the survival and transformation efficiency. The higher dosage of nanoparticles led to more dead cells.

A locally isolated stain Aeromonas schubertii was cultured and induced by powdered chitin for the production of chitinases. Extracellular proteins were purified by ammonium sulfate precipitation, dialysis to remove salts, and then preparative isoelectric focusing (IEF) to yield several chitinases. The purified enzymes were analyzed by SDS–PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) with and without glycol chitin and were found to be SDS-resistant. The chitinase present in the highest abundance was the one with an estimated molecular weight of 75 kDa. The Michaelis constant and turnover number were determined to be 0.29 mM and 1 s−1, respectively, for this enzyme using colloidal chitin azure as the substrate. However, the ethanol treatment of this enzyme could significantly increase its chitinolytic activity. Other chitinases obtained in the same IEF fraction were determined to have molecular weights of ca. 30, 38, and 110 kDa. Since the proteins with highest chitinase activity were collected from IEF fraction tube with pH value of 4.8, those chitinase were believed to be acidic. An activity assay method using colloidal chitin azure as the substrate was recommended since it possessed a broader range of linearity in comparison with conventional reducing sugar equivalent method.

The administration of dimethylnitrosamine (DMN) into rabbit induced liver fibrosis/cirrhosis and finally caused a lethal hepatic failure. Blood collected from the rabbit was centrifuged and the supernatant was analyzed by two-dimensional gel electrophoresis (2-DE) for the study of proteome in serum. Compared with 2-DE gel of serum from healthy rabbit, a significant reduction in the number of protein spots having molecular weights (MWs) below 21 kDa was observed in the gels of the serum from the rabbit treated with DMN, while the secretion of albumin was kept at a high level. Separated spots in the two-dimensional gel were cut, digested with trypsin, and analyzed by MALDI-TOF mass spectrometry. Serum amyloid A-3 protein precursor (SAA3) and other serum amyloid A (SAA) protein precursors were identified by matching the peptide masses with those in database. In the SAA family of acute-phase/inflammatory response proteins, SAA3 is mainly synthesized in the liver. The SAA3 secreted level in the serum decreased with time after DMN administration as the result of hepatic dysfunctions.

Human mesenchymal stem cells (MSCs) modified by targeting DNA hypermethylation of genes in the Salvador/Warts/Hippo pathway were induced to differentiate into neuronal cells in vitro. The differentiated cells secreted a significant level of brain-derived neurotrophy factor (BDNF) and the expression of BDNF receptor tyrosine receptor kinase B (TrkB) correlated well with the secretion of BDNF. In the differentiating cells, CREB was active after the binding of growth factors to induce phosphorylation of ERK in the MAPK/ERK pathway. Downstream of phosphorylated CREB led to the functional maturation of differentiated cells and secretion of BDNF, which contributed to the sustained expression of pERK and pCREB. In summary, both PI3K/Akt and MAPK/ERK signaling pathways play important roles in the neuronal differentiation of MSCs. The main function of the PI3K/Akt pathway is to maintain cell survival during neural differentiation; whereas the role of the MAPK/ERK pathway is probably to promote the maturation of differentiated MSCs. Further, cellular levels of protein kinase C epsilon type (PKC-e) and kinesin heavy chain (KIF5B) increased with time of induction, whereas the level of NME/NM23 nucleoside diphosphate kinase 1 (Nm23-H1) decreased during the time course of differentiation. The correlation between PKC-e and TrkB suggested that there is cross-talk between PKC-e and the PI3K/Akt signaling pathway.

The production of fructooligosaccharides (FOSs) from sucrose catalyzed by β- d -fructofuranosidase was achieved with the use of immobilized mycelia of Aspergillus japonicus in gluten. When 1 g mycelia-immobilized particles having a cell content of 20% (w/w) were incubated with 100 ml of sucrose solution with an initial concentration of 400 g/liter, the total produced FOSs were determined to be about 61%, w/w of total sugars in the mixture after a batch reaction for 5 h. The reaction velocity increased with the cell content in the gluten matrix and reached the maximum value when the cell content was as high as 20% (w/w). As the mycelia-immobilized gluten particles were packed in to a column reactor for continuous production of FOSs, a productivity of 173 g per hour per liter of reactor volume was achieved at a flow-rate of 0.8 ml/min. The mass fraction of FOSs increased from 0.20 to 0.54 w/w as the flow-rate decreased from 1 to 0.1 ml/min, corresponding to the residence time increasing from 0.35 to 3.5 h. The mycelia were well entrapped in the gluten matrix and the cell-immobilized preparations were stable in long-term operation. Gluten was found to be adequate for using as the base material for immobilization mycelia-associated enzymes.

Iminodiacetic acid (IDA) and octyl moieties were covalently bound on nonporous particles, which were prepared from dispersion polymerization of methyl methacrylate and glycidyl methacrylate. After being charged with copper ions, the IDA-bound particles could specifically adsorb deoxyribonuclease I (DNase I) through the affinity interaction between protein and immobilized metal ion. A mixed-ligand (metal-chelate and octyl-bound) support was obtained after hydrophobic (octyl) groups were also introduced to the particle surface. The affinity adsorption of DNase I on the copper-IDA chelate was influenced by interaction between the protein and the bound octyl group. Both the affinity and the hydrophobic interactions could be well described by the Langmuir isotherms. The equilibrium adsorption constants were estimated separately to be 0.96 and 0.50 liter g(-1) for affinity and hydrophobic bindings, respectively. For binding on mixed-ligand support, the adsorption constant was 0.45 liter g(-1). It was evident that both affinity and hydrophobic interactions are involved in the adsorption of proteins onto mixed-ligand particles. Desorption of the inactive proteins from the support was possible by increasing the hydrophobicity of the solution. Copyright 2001 Academic Press.

Alteration of the pH of the mobile phase is the most common method to perform elution of the bound components from an affinity column. The values of the adsorption rate constant and the dissociation constant between the protein to be separated from a mixture and the immobilized ligand vary during the chromatographic process. A mathematical model used to predict the performance of pH elution on a high-performance affinity chromatographic column, packed with non-porous adsorbents, has been solved numerically with the application of the finite element method. The effects of binding capacity, sample load, adsorption rate constant and equilibrium dissociation constant under adsorption and elution conditions on the split-peak phenomenon and the shape and spreading of the elution peak were investigated. Simulation results showed that the time required to elute all bound solutes is mainly determined by the dissociation constant which is adjustable by altering the pH of the mobile phase. The dispersive effect on the elution peak is largely influenced by the rate constant under adsorption and elution conditions, while for the non-retained fraction (split-peak), it is governed by the rate and dissociation constants under adsorption conditions only. Influence of sample loading on the non-retained fraction and the elution peak also is significant, especially for columns with small binding capacity. As the affinity column is employed for the purpose of analyzing, a calibration graph based on the height of the elution peak is easily obtained provided that the effects of adsorption rate constant and dissociation constant are all taken into account.

In the production of ethanol by Zymomonas mobilis ATCC 10988, using sucrose as the substrate, we added immobilized invertase to the fermentation broth to promote sucrose hydrolysis. The rapid accumulation of glucose in the medium caused the inhibition of levan formation and an enhancement of the growth rate at the initial stage of fermentation. The formation of sorbitol could also be reduced by addition of immobilized invertase, because the accumulation of glucose and fructose was observed to be even lower than without immobilized invertase. After the addition of 0.2 g immobilized invertase (activity, 2, 100 U g−1, using 0.8 m sucrose at pH 5 and 30°C) to a 1.2-l batch fermenter containing 200 g l−1 sucrose, the formation of byproducts was significantly reduced and the ethanol yield was increased from 0.29 to 0.40 g g−1. High ethanol yields were also obtained from continuous cultures. When 0.2 g immobilized invertase was added to the fermenter, the dilution rate for maximal ethanol productivity was determined to be 0.2 h−1. The coupled microbe-immobilized-invertase system proved to be better than the two-stage (separate hydrolysis and fermentation) system in the yield of ethanol produced.

TheMichelia champaca callus was induced from rachises of theM. champaca flowers on 1/2 MS medium containing 3% sugar and 0.9% agar. The medium was also supplemented with 2,4-dichlorophenoxy acetic acid (2,4-D) and 6-benzylaminopurine (BAP) as the growth regulators. It was observed that the calli ofM. champaca could be induced on media containing 0–50 μM 2,4-D and 0.1–1 μM BAP. The calli were grown well on media on media containing 0.1–1.0 μM BAP and 2,4-D up to 50 μM. As 1 μM BAP was used, a lower concentration of 2,4-D was associated with a fast initiation of calli, but the culture of these calli turned brown quickly. To the contrary, a higher concentration of 2,4-D led to a slower rate of callus formation and the culture hardly turned brown. The optimum pH for the cell culture was about 5.6 as 1 μM of 2,4-D and BAP were present in the medium.

A procedure for the formulation of biocontrol fungi involving the immobilization of wet biomass within gluten was developed. The release of active fungi from formulated preparations to soil was governed by the soil pH, the moisture content in soil, and the entrapped biomass content. After incorporation to loamy soils having initial moisture content of 5-20%, all formulated agents could generate 10(6)-10(7) colony forming units g(-1) soil in the second week. The use of formulated preparations reduced the amount of biomass required, compared with non-formulated fungi as biocontrol agents.

Recent efforts in the investigation of chromatographic characterization of molecularly imprinted polymers (MIPs) have focused mainly on the nature of heterogeneous binding sites. More data on the thermodynamics than on the kinetic features of MIP columns have been published. The present article addresses the sources of peak broadening and tailing, which are the main drawbacks often associated with imprinted polymers in chromatography for practical applications. With use of the theory of nonlinear chromatography, the peak properties of a MIP column, including the retention and peak broadening and tailing, can be well interpreted. Efforts to improve chromatographic efficiency using MIPs prepared by approaches different from the conventional method, including covalent imprinting and the format of uniformly sized spherical microbeads, are reviewed and discussed. This review leads to the conclusion that nonlinear chromatography theory is useful for characterizing chromatographic features of MIP columns, since a MIP is essentially an affinity-based chromatographic stationary phase. We expect more theoretical and experimental studies on the kinetic aspects of MIP columns, especially the factors influencing the apparent rate constant, as well as the analysis of the influences of mobile-phase composition on the chromatographic performance. In addition to revealing the affinity interaction by molecular recognition, slow nonspecific interactions which may be inherited from the imperfect imprinting and may be involved in the rebinding of the template to MIPs also need to be characterized.

Background Overexpression of recombinant proteins usually triggers the induction of heat shock proteins that regulate aggregation and solubility of the overexpressed protein. The two-dimensional gel electrophoresis (2-DE)-mass spectrometry approach was used to profile the proteome of Escherichia coli overexpressing N-acetyl-D-glucosamine 2-epimerase (GlcNAc 2-epimerase) and N-acetyl-D-neuraminic acid aldolase (Neu5Ac aldolase), both fused to glutathione S-transferase (GST) and polyionic peptide (5D or 5R). Results Overexpression of fusion proteins by IPTG induction caused significant differential expression of numerous cellular proteins; most of these proteins were down-regulated, including enzymes connected to the pentose phosphate pathway and the enzyme LuxS that could lead to an inhibition of tRNA synthesis. Interestingly, when plasmid-harboring cells were cultured in LB medium, gluconeogenesis occurred mainly through MaeB, while in the host strain, gluconeogenesis occurred by a different pathway (by Mdh and PckA). Significant up-regulation of the chaperones ClpB, HslU and GroEL and high-level expression of two protective small heat shock proteins (IbpA and IbpB) were found in cells overexpressing GST-GlcNAc 2-epimerase-5D but not in GST-Neu5Ac aldolase-5R-expressing E. coli. Although most of the recombinant protein was present in insoluble aggregates, the soluble fraction of GST-GlcNAc 2-epimerase-5D was higher than that of GST-Neu5Ac aldolase-5R. Also, in cells overexpressing recombinant GST-GlcNAc 2-epimerase-5D, the expression of σ32 was maintained at a higher level following induction. Conclusions Differential expression of metabolically functional proteins, especially those in the gluconeogenesis pathway, was found between host and recombinant cells. Also, the expression patterns of chaperones/heat shock proteins differed among the plasmid-harboring bacteria in response to overproduction of recombinant proteins. In conclusion, the solubility of overexpressed recombinant proteins could be enhanced by maintaining the expression of σ32, a bacterial heat shock transcription factor, at higher levels during overproduction.

The interface between adjoining layers in a multi-layer medium is usually considered to have zero thickness. Actually, although very thin, the interface may accumulate mass due to the so-called secondary phenomena occurring at the interface in a diffusion process. In this work, a regular perturbation technique is used to find an interface condition which takes into account the immobilization reaction which may occur with diffusion in a multi-layer medium. By the correct adjustment of parameters, the new interface condition can be reduced to the classical one. An example is given to illustrate the application of the new interface condition and an experimental method is suggested to determine the physical properties of a reaction membrane.