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Hepatitis B core antigen (HBcAg) expressed in Escherichia coli is able to self-assemble into large and small capsids comprising 240 (triangulation number T = 4) and 180 (triangulation number T = 3) subunits, respectively. Conventionally, sucrose density gradient ultracentrifugation and SEC have been used to separate these capsids. However, good separation of the large and small particles with these methods is never achieved. In the present study, we employed a simple, fast, and cost-effective method to separate the T = 3 and T = 4 HBcAg capsids by using native agarose gel electrophoresis followed by an electroelution method (NAGE-EE). This is a direct, fast, and economic method for isolating the large and small HBcAg particles homogenously based on the hydrodynamic radius of the spherical particles. Dynamic light scattering analysis demonstrated that the T = 3 and T = 4 HBcAg capsids prepared using the NAGE-EE method are monodisperse with polydispersity values of ∼15% and ∼13%, respectively. ELISA proved that the antigenicity of the capsids was not affected in the purification process. Overall, NAGE-EE produced T = 3 and T = 4 capsids with a purity above 90%, and the recovery was 34% and 50%, respectively (total recovery of HBcAg is ∼84%), and the operation time is 15 and 4 times lesser than that of the sucrose density gradient ultracentrifugation and SEC, respectively.

Display of the nucleocapsid protein (NP) of Nipah virus (NiV) on the hepatitis B core (HBc) particle enhances the antigenicity of the NP. In addition, the NP fused to the amino-teminus (NP401-532 HBc) and immunodominant loop of HBc (HBc79.80NP4O1'S31) was shown to be less sensitive to proteinase K. Some of the proteinase K treated NP401.32HBc and HBc79.80NP,O].s32 remained intact and antigenic. These results indicated two important implications, (1) the use of HBc virus -like particle as a carrier platform for foreign epitopes, (2 ) the use of NP401.532HBc as a diagnostic reagent for NiV infection.

AIMS: To display a liver-specific ligand on the hepatitis B virus core particles for cell-targeting delivery. METHODS AND RESULTS: A liver cell-binding ligand (preS1) was fused at the N-terminal end of the hepatitis B core antigen (HBcAg), but the fusion protein (preS1His(6) HBcAg) was insoluble in Escherichia coli and did not form virus-like particles (VLPs). A method to display the preS1 on the HBcAg particle was established by incorporating an appropriate molar ratio of the truncated HBcAg (tHBcAg) to the preS1His(6) HBcAg. Gold immunomicroscopy showed that the subunit mixture reassembled into icosahedral particles, displaying the preS1 ligand on the surface of VLPs. Fluorescence microscopy revealed that the preS1 ligand delivered the fluorescein-labelled VLPs into the HepG2 cells efficiently. CONCLUSIONS: Chimeric VLPs containing the insoluble preS1His(6) HBcAg and highly soluble tHBcAg were produced by a novel incorporation method. The preS1 ligand was exposed on the surface of the VLPs and was shown to deliver fluorescein molecules into liver cells. SIGNIFICANCE AND IMPACT OF STUDY: The newly established incorporation method can be used in the development of chimeric VLPs that could serve as potential nanovehicles to target various cells specifically by substituting the preS1 ligand with different cell-specific ligands.

The invention provides a method to purify a recombinant virus protein from microbial cells. Hydrophobic interaction Chromatography (HIC) is used to capture recombinant virus protein from both clarified and unclarified microbial cell lysate.

A comparative study on the stability and potential of alginate and pectin based beads for production of poultry probiotic cells using MRS medium in repeated batch fermentation was conducted. The bead cores, made of three types of materials, i.e., ca-alginate, ca-pectinate and ca-alginate/pectinate, were compared. The effect of single and double layer coatings using chitosan and core material, respectively, on the bead stability and cell production were also studied. The pectin based beads were found to be more stable than that of the alginate beads and their stability was further improved by coating with chitosan. The cell concentration in pectin based beads was comparable to that in the alginate beads. On the other hand, pectin based beads gave significantly lower cell concentration in the growth medium for the initial fermentation cycles when compared to the alginate beads. In conclusion, pectin was found to be potential encapsulation material for probiotic cell production owing to its stability and favourable microenvironment for cell growth.

The nucleocapsid (N) protein of Nipah virus (NiV) produced in a recombinant host can replace the use of inactivated virus as a diagnostic reagent because it is safer and affordable. The aim of this study was to express the N protein in Pichia pastoris. The N gene of NiV was cloned into the yeast expression vector, pPICZ B and expressed in P. pastoris. The recombinant N protein of NiV was purified using sucrose density gradient ultracentrifugation and was confirmed with Western blotting using rabbit anti-N antibody. The P. pastoris expressed N protein self-assembled into helical structures as large as 1.5 μm as shown in an electron micrograph. ELISA analysis performed with the swine sera obtained during the viral outbreak proved that the recombinant N protein to be highly antigenic. The NiV N protein produced in P. pastoris serves as an alternative to the recombinant N protein produced in Escherichia coli.

The main objective of this work is to improve the physical properties of lyophilized calcium (Ca)-alginate beads as a carrier material for the stabilization of encapsulated living cells. Improvements in the sphericity, flowability and mechanical strength of the dried beads were attributed to the filler, which provided structure and reinforcement to the Ca-alginate hydrogel networks, as verified by X-ray microtomography and scanning electron microscopy. A quantitative analysis of the micro-images revealed the less porous nature of the alginate-starch beads compared to the control. The beads with filler were also found to be less hygroscopic. The results also show that the cells encapsulated within the beads with reduced porosity and hygroscopicity were clearly more stable during lyophilization and storage than the control. In conclusion, the qualities of the alginate beads were improved by incorporating the solid filler, and the filler had a significant influence on cell viability during lyophilization and storage. © 2010 Elsevier Ltd. All rights reserved.

The aim of this work was to determine the effect of formulation of alginate beads on their mechanical behavior and stiffness when compressed at high speed. The alginate beads were formulated using different types and concentrations of alginate and gelling cations and were produced using an extrusion-dripping method. Single wet beads were compressed at a speed of 40 mm/min, and their elastic limits were investigated, and the corresponding force versus displacement data were obtained. The Young's moduli of the beads were determined from the force versus displacement data using the Hertz's contact mechanics theory. The alginate beads were found to exhibit plastic behavior when they were compressed beyond 50% with the exception of copper–alginate beads for which yield occured at lower deformation. Alginate beads made of higher guluronic acid contents and gelling cations of higher chemical affinity were found to have greater stiffness. Increasing the concentration of alginate and gelling ions also generated a similar effect. At such a compression speed, the values of Young's modulus of the beads were found to be in the range between 250 and 900 kPa depending on the bead formulation.

Negative chromatography antibody purification (N-CAP) using the weak anion exchanger STREAMLINE™ DEAE to extract impurities while retaining the target antibody is proposed as an effective method for the recovery of antibody from rabbit serum. The effects of pH and initial protein concentration on the removal of albumin were investigated. The optimal pH and initial protein concentration for the efficient removal of albumin from rabbit serum were pH 8.0 and 0.5 mg/ml, respectively. Under optimal binding conditions, DEAE successfully removed more than 90% of the albumin from rabbit serum with less than 20% IgG loss. This process offered good polyclonal IgG yield of 80% with a purity of 83% and a purification factor of 5.5. The use of a strong anion exchanger like STREAMLINE™ Q XL for albumin removal was also explored. Under similar optimized conditions, albumin removal by Q XL was as high as 90%. However, IgG recovery and purity were reduced to about 70% and 62%, respectively. Thus, N-CAP using the anion exchanger DEAE removes albumin from rabbit serum and thereby offers an efficient means of purifying polyclonal antibodies.

Nipah Virus (NiV) is an emerging zoonotic paramyxovirus that can be fatal in humans and various types of animals. The phospho (P) protein of NiV plays an important role in RNA synthesis, replication, and genome synthesis. In this study, the NiV P gene was cloned into a pTrcHis2-TOPO vector and the recombinant protein containing a His-tag was produced in Escherichia coli. SDS-PAGE and Western blot analysis using the anti-His antibody confirmed the protein expression. An optimization study of E. coli fermentation showed that the optimal cultivation temperature was 37°C, while the optimal induction time for P protein expression was at 9 h with 1 mM IPTG. Solubility analysis showed that E. coli cultivated at 37°C produced the highest fraction (70%) of soluble P protein. The recombinant P protein was purified from clarified E. coli lysate using an immobilized metal affinity chromatography (IMAC) technique to a purity of 92.67%, with a purification factor of 11.58. The purified P protein strongly reacted with the anti-NiV swine sera collected during a NiV outbreak, suggesting its potential as a diagnostic reagent.

A comparative study on the stability and potential of alginate and pectin based beads for production of poultry probiotic cells using MRS medium in repeated batch fermentation was conducted. The bead cores, made of three types of materials, i.e., ca-alginate, ca-pectinate and ca-alginate/pectinate, were compared. The effect of single and double layer coatings using chitosan and core material, respectively, on the bead stability and cell production were also studied. The pectin based beads were found to be more stable than that of the alginate beads and their stability was further improved by coating with chitosan. The cell concentration in pectin based beads was comparable to that in the alginate beads. On the other hand, pectin based beads gave significantly lower cell concentration in the growth medium for the initial fermentation cycles when compared to the alginate beads. In conclusion, pectin was found to be potential encapsulation material for probiotic cell production owing to its stability and favourable microenvironment for cell growth.

Large quantities of oil palm trunks are available annually during the replanting activities when the oil palm tree passed their economic age, on an average after 25 years are replace with young trees. Basically the oil palm trunks contains about 18- 21% of lignin, 65-80% of holocellulose (a-cellulose and hemicellulose) and quite significant amount starch. This work is aimed to determine the total extractable starch and sugars content from oil palm trunks by using steeping method and dilute acid hydrolysis. The effect of different oil palm trunk powder size on starch, xylose and glucose yield was evaluated. The effect of extraction parameter for each extraction method on the yield of starch and sugars were studied. The highest starch yield was obtained when steeped in the presence of lactic acid, while the highest xylose yield was obtained by 60 min hydrolysis of 60 mesh of oil palm powder with 2% sulfuric acid. For glucose yield, hydrolysis efficiency of 82% was obtained for conversion of oil palm trunk to glucose using two-stage concentrated sulfuric acid hydrolysis. Conclusively oil palm trunk can be considered as a resource of substantial amounts of starch and sugars.

Disposing of solid waste and demand of fossil fuel have become the great challenges in the 21st century. Malaysia as one of the top producers of palm oil and wooden furniture in the world is well positioned to take the challenge of the reuses of its enormous output of lignocellulosic biomass such as oil palm trunk, sawdust of rubberwood and sawdust of mixed hardwood generated from palm oil and furniture industries. Before these lignocellulosic biomasses can be used to produce fuel and major chemicals which are normally derived from petroleum, lignocellulosic materials have to be converted to glucose. Hence, it is a need to investigate the conversion efficiency and to determine the optimum conditions for the conversion of lignocellulosic materials to glucose. This present work is aimed to investigate the potential use of oil palm trunk, rubberwood sawdust and mixed hardwood sawdust as an alternative feedstock for lignocellulosic glucose production. This research also served to identify the optimum two-stage concentrated acid hydrolysis condition that can convert these three lignocellulosic biomasses to glucose efficiently. Two stages concentrated sulfuric acid hydrolysis process using different acid concentration and reaction time were performed on those lignocellulosic biomass samples. The optimum results for oil palm trunk, rubberwood and mixed hardwood sawdust were obtained by using 60% acid concentration reacted for 30 min during 1st stage hydrolysis and subsequently followed by another 60 min reaction time with 30% acid concentration during the 2nd stage hydrolysis. The results, showed that oil palm trunk has a higher glucose conversion yield than those of rubberwood sawdust and mixed hardwood sawdust.

Serum deprivation inhibits cell growth and initiates apoptosis cell death in mammalian cell cultures. Since apoptosis is a genetically controlled cell death pathway, over-expression of anti-apoptotic proteins may provide a way to delay apoptosis. This study investigated the ability of the X-linked inhibitor of apoptosis protein (XIAP) to inhibit apoptosis induced by serum deprivation. Study includes evaluation of the ability of XIAP to prolong culture period and its effect on cell proliferation in serum-deprived media. The full length human XIAP was introduced into CHO-K1 cell lines and the effects of XIAP over-expression on the inhibition of apoptosis induced by serum-deprived conditions were examined. In batch cultures, cells over-expressing XIAP showed decreased levels of apoptosis and a higher number of viable cell under serum-deprived conditions compared to the control cell lines. The viability of control cells dropped to 40% after 2. days of serum deprivation, the XIAP expressing cells still maintained at a viability higher than 90%. Further investigation revealed that the caspase-3 activity of the CHO-K1 cell line was inhibited as a result of XIAP expression. © 2010 The Society for Biotechnology, Japan.

The osmotic shock process for the release of periplasmic recombinant human interferon-α2b from Escherichia coli was optimized using response surface method (RSM). The process parameters such as pH, buffer concentration and sucrose concentration in hypertonic solution, cell concentration to hypertonic solution, contact time of cells with hypertonic solution, temperature of hypertonic solution, cell concentration to hypotonic solution, contact time of cells with hypotonic solution and temperature of hypotonic solution were initially screened using Plackett Burman design. Further optimization was carried out using central composite design (one of the design in RSM) for sucrose concentration in hypertonic solution as well as cell concentration to hypertonic and hypotonic solutions. The optimal cell concentration was 0.05 g/mL in hypertonic solution and 0.2 g/mL in hypotonic solution. The use of hypertonic solution containing 18% sucrose with a combination of 100 mM Tris and 2.5 mM EDTA buffer (pH 8.0 and 25 °C) and cold water (4 °C) as a hypotonic solution gave the optimum release of interferon-α2b. Increased product concentration in the final solution resulted from the optimized process would reduce the downstream steps during purification. The concept of reuse of hypertonic solution was also demonstrated.

Pyroligneous acid which is one of the commercial sources for acetic acid can be produced from high temperature carbonization of lignocellulosic biomass. Acetic acid can be used as a wood preservative to discourage the growth of fungal and molds. However, at higher temperature, organic compounds especially acetic acid in pyroligneous acid degraded except for some phenols. Therefore, effectiveness pyroligneous acid that pyrolysed at different temperature as fungicide and insecticide for used as wood preservative was evaluated. Pyroligneous acids were derived from rubberwood, oil palm trunk and mix hardwood heated at temperature of 300, 400 and 500°C, respectively in an airless container. The yield of pyroligneous acids was calculated and the chemical compounds of the pyroligneous acid were analysed using Fourier Transform InfraRed (FT-IR). For the efficacy of pyroligneous acid tests, rubberwood test blocks were immersed in the pyroligneous acid for 24 h at room temperature. The treated rubberwood test blocks were later tested against mold (Penicillium sp.), white rot fungus (Pycnoporous sanguineus) and subterranean termites, (Coptotermes curvignathus) according to ASTM standard method. The result shows that highest pyroligneous acid yield was found during pyrolysed of lignocellulosic biomass at temperature of 500°C. All the rubberwood test blocks treated with pyroligneous acids were effective against the mold, white rot fungi and termites. Nonetheless, the pyrolysis temperature did not affect the effectiveness of pyroligneous acids against biological agents. Conclusively, pyroligneous acids effective for discourage the growth of mold and white rot fungi as well accelerate the mortality of termites in laboratory condition.

Serum deprivation inhibits cell growth and initiates apoptosis cell death in mammalian cell cultures. Since apoptosis is a genetically controlled cell death pathway, over-expression of anti-apoptotic proteins may provide a way to delay apoptosis. This study investigated the ability of the X-linked inhibitor of apoptosis protein (XIAP) to inhibit apoptosis induced by serum deprivation. Study includes evaluation of the ability of XIAP to prolong culture period and its effect on cell proliferation in serum-deprived media. The full length human XIAP was introduced into CHO-K1 cell lines and the effects of XIAP over-expression on the inhibition of apoptosis induced by serum-deprived conditions were examined. In batch cultures, cells over-expressing XIAP showed decreased levels of apoptosis and a higher number of viable cell under serum-deprived conditions compared to the control cell lines. The viability of control cells dropped to 40% after 2days of serum deprivation, the XIAP expressing cells still maintained at a viability higher than 90%. Further investigation revealed that the caspase-3 activity of the CHO-K1 cell line was inhibited as a result of XIAP expression.

Ethanolic fermentation using Saccharomyces cerevisiae was carried out on three types of hydrolysates produced from lignocelulosic biomass which are commonly found in Malaysia such as oil palm trunk, rubberwood and mixed hardwood. The effect of fermentation temperature and pH of hydrolysate was evaluated to optimize the fermentation efficiency which defined as maximum ethanol yield in minimum fermentation time. The fermentation process using different temperature of 25 °C, 30 °C and 40 °C were performed on the prepared fermentation medium adjusted to pH 4, pH 6 and pH 7, respectively. Results showed that the fermentation time was significantly reduced with the increase of temperature but an adverse reduction in ethanol yield was observed using temperature of 40 °C. As the pH of hydrolysate became more acidic, the ethanol yield increased. Optimum fermentation efficiency for ethanolic fermentation of lignocellulosic hydrolysates using S. cerevisiae can be obtained using 33.2 °C and pH 5.3.

A direct recovery of recombinant nucleocapsid protein of Nipah virus (NCp-NiV) from crude Escherichia coli (E. coli) homogenate was developed successfully using a hydrophobic interaction expanded bed adsorption chromatography (HI-EBAC). The nucleic acids co-released with the recombinant protein have increased the viscosity of the E. coli homogenate, thus affected the axial mixing in the EBAC column. Hence, DNase was added to reduce the viscosity of feedstock prior to its loading into the EBAC column packed with the hydrophobic interaction chromatography (HIC) adsorbent. The addition of glycerol to the washing buffer has reduced the volume of washing buffer applied, and thus reduced the loss of the NCp-NiV during the washing stage. The influences of flow velocity, degree of bed expansion and viscosity of mobile phase on the adsorption efficiency of HI-EBAC were studied. The dynamic binding capacity at 10% breakthrough of 3.2 mg/g adsorbent was achieved at a linear flow velocity of 178 cm/h, bed expansion of two and feedstock viscosity of 3.4 mPa s. The adsorbed NCp-NiV was eluted with the buffer containing a step gradient of salt concentration. The purification of hydrophobic NCp-NiV using the HI-EBAC column has recovered 80% of NCp-NiV from unclarified E. coli homogenate with a purification factor of 12.5.

The application of STREAMLINE Direct HST adsorbent in expanded bed adsorption of lipase from Burkholderia pseudomallei was explored in this study. Scouting of optimum binding and elution condition was performed in batch binding mode. Theaddition of 0.2 M salt in acetate buffer (pH 5) during adsorption has increased thespecificity and quantity of lipase binding onto the adsorbent. The addition of 0.4 M salt in phosphate buffer (pH 7) achieved the highest purification fold (2.5) in elution. The high density of the adsorbent allowed the EBA to be operated at linear velocityas high as 657 cm/h with feedstock containing 4.5% (w/v) wet biomass. The Richardson-Zaki correlation obtained for this EBA system at the presence of 4.5% (w/v) wet biomass is 5.14, a value closed to the laminar flow regime of 4.8, demonstrated that a stable bed is achieved under this operating condition.Meanwhile, a flow velocity of 343 cm/h with bed expansion of 3.2 gave highest dynamic binding capacity (4979.28 U/ml) and productivity (61.52 U/ml.min) for this EBA operation. It also demonstrated that biomass concentration up to 4.5% (w/v) wet weight showed slightly drop of sorption efficiency (0.82) compared to lower biomass concentration (0.94). Further increase of biomass concentration above 4.5% (w/v) wet weight has greatly decreased the equilibrium and dynamic capacity. Application of high density adsorbent tolerated to high density and biomass has reduced the processing time and increased the productivity.

The present invention provides a method to enhance the recovery of recombinant N protein of Nipah virus produced in Escherichia coli, in which by identifying and inhibiting the specific proteases in the cell lysate are achieved by an integrated use of a bioinformatics tool and a protease inhibition experimental setup.

The present invention provides a recombinant matrix protein of Nipah virus containing matrix protein of nipah virus which includes at least ine His tag and MYC epitope. Further, the present invention also provides a recombinant plasmid including recombinant matrix protein of Nipah virus. Also provided is a method for producing recombinant matrix protein of Nipah virus which includes the steps of (a) culturing expression vector of recombinant matrix protein in a medium which allows growth of the expression host, (b)allowing the recombinant matrix protein to be expressed in the expression host, (c) harvesting the expression host from step (b) from the medium, (d) disrupting the expression host harvested from step (c) in a medium for sonication purposes and (e) purifying the recombinant matrix protein obtained from step (d. Also provided is method for purifying recombinant matrix protein of Nipah virus which includes the steps of (a) harvesting and lysing recombinant expression host containing recombinant matrix protein cells, (b) purifying the recombinant matrix protein using nickel affinity column chromatography, (c) dialyzing the purified recombinant matrix protein from step (b) with buffer solution, (d)fractionating the purified recombinant matrix protein from step (c) using centrifugation and (e) collecting and poling the fractioned recombinant matrix protein obtained from step (d) concentration of protein. There is also provided a pharmaceutical composition comprising a therapeutically affective amount of recombinant matrix protein of Nipah virus. Also provided is a diagnostic reagent comprising an effective amount of recombinant matrix protein matrix of Nipah virus.

M13 is a non-lytic filamentous bacteriophage (phage). It has been used widely in phage display technology for displaying foreign peptides, and also for studying macromolecule structures and interactions. Traditionally, this phage has been purified by cesium chloride (CsCl) density gradient ultracentrifugation which is highly laborious and time consuming. In the present study, a simple, rapid and efficient method for the purification of M13 based on anion exchange chromatography was established. A pre-packed SepFast™ Super Q column connected to a fast protein liquid chromatography (FPLC) system was employed to capture released phages in clarified Escherichia coli fermented broth. An average yield of 74% was obtained from a packed bed mode elution using citrate buffer (pH 4), containing 1.5 M NaCl at 1 ml/min flow rate. The purification process was shortened substantially to less than 2 h from 18 h in the conventional ultracentrifugation method. SDS-PAGE revealed that the purity of particles was comparable to that of CsCl gradient density ultracentrifugation method. Plaque forming assay showed that the purified phages were still infectious.

The onset of convection induced by transient mass diffusion in a stationary gas was succesfully predicted with transient instability theory and simulated using a computational fluid dynamics (CFD) scheme. 2D time-dependent simulations were conducted for bottom-up diffusion of a light gas in a stagnant heavy gas. The results of simulations were used to calculate the transient Rayleigh number adopted from the theory of Tan and Thorpe (1996 and 1999). The average transient maximum Rayleigh number from simulations is 707, which is close to the theoretical value of 817 for analogous bottom heating with constant heat flux. The simulated critical times of the onset of convection were in reasonably good agreement with the predicted values from theory.

The biodegradation of oil palm empty fruit bunch (EFB) in liquid fermentation using microorganism complex obtained from palm oil mill effluent (POME) under non-axenic condition was studied. A parallel study on carboxyl methyl cellulose (CMC) as substrate was also conducted as a comparison. The effects of the operating parameters of fermentation, aeration rates on the degradation of EFB and CMC were studied. The degradation was performed at agitation speed of 150 rpm in a 10 litre bioreactor under process parameter for a 10 day period. The evolution of dry matter (DM) and chemical oxygen demand of solids (CODs) were used to measure the performance of the non-sterile liquid fermentation process on the solid degradation of EFB and CMC. The effect of aeration rate on the reduction of DM and CODs differs. For EFB, a 10 fold increase in DM reduction was observed when the aeration rate was raised from 0 to 1.0 vvm. On the other hand, the increase in DM reduction of CMC was lower than that of EFB as a result of the increased aeration rate. The highest CODs reduction for EFB (64.82%) and CMC (20.2%) was achieved at aeration rate of 0.5 vvm. Aeration rate of 0.5 vvm was selected as the rate for optimum biodegradation.

Mechanical and non-mechanical breakages of bacterial cells are usually the preliminary steps in intracellular protein purification. In this study, the recombinant green fluorescent protein (GFP) was purified from intact Escherichia coli cells using preparative PAGE. In this purification process, cells disruption step is not needed. The cellular content of E. coli was drifted out electrically from cells and the negatively charged GFP was further electroeluted from polyacrylamide gel column. SEM investigation of the electrophoresed cells revealed substantial structural damage at the cellular level. This integrated purification technique has successfully recovered the intracellular GFP with a yield of 82% and purity of 95%.

A semi-continuous leachate treatment process was developed in the present study. The fresh leachate was obtained from a municipal solid waste transfer station and palm oil mill effluent (POME) sludge was used as sources of anaerobic microbial complex. The semi-continuous treatment of leachate was operated in two phases; in Phase 1, the pH of the bioreactor was not adjusted, and in Phase 2, the pH of the bioreactor was adjusted by the addition of sodium hydroxide (NaOH). The initial values for both chemical oxygen demand (COD) and biochemical oxygen demand (BOD5) of fresh leachate were extremely high compared with the stabilized landfill leachate. COD reduction rate for the semicontinuous process for Phase 1 and 2 were 37 and 52.7%, respectively. These results clearly showed that pH adjustment is important to enhance the COD removal in leachate treatment. In addition, we have analysed the evolution of volatile fatty acid (VFA) in the entire treatment process. The results indicated that the VFA concentration was a rapid indicator of the reactor’s stability.

Effects of pH, temperature, and level of mixing on the production of fusion M13 phage bearing the peptide sequence (C-WSFFSNI-C) that interacts with HBcAg were investigated in this study. The optimum pH for the phage production was achieved at pH 7, followed by pH 6 and 8. The highest fusion phage titre was obtained at growth temperature of 37°C, followed by 27 and 42°C. The rotational speed at 250 rpm was the optimal mixing level for the phage production. Further increase of rotational speed to 300 rpm has reduced the phage production to a level lower than that obtained at 200 rpm. The results also showed that the propagation of fusion M13 phage has greatly affected the growth of Escherichia coli ER 2738. The viability of the phage produced with the current method was then determined using phage titre and dot-blot assays.

BACKGROUND: Many reports have suggested that mild hypothermic culture conditions improve the specific monoclonal antibody (mAb) productivity of mammalian cells. The effect of active hypothermic growth on the mAb productivity of the hybridoma C2E7 was investigated. Hybridoma growth under hypothermic conditions (32 °C) was stimulated by supplementation of the culture medium with high serum concentrations (up to 30%). RESULTS: Specific and volumetric mAb productivity of a stimulated, active growth, mildly hypothermic hybridoma culture (30% FBS supplemented, 32 °C) were 1.38‐ and 1.34‐fold greater than the control culture (10% FBS supplemented, 37 °C). The enhanced specific mAb productivity under hypothermic conditions was associated with an increase in IgM mRNA levels during both the lag and early exponential phases of hypothermic growth. CONCLUSION: Stimulation of hybridoma growth under mildly hypothermic conditions increased both the specific and volumetric mAb productivity of hybridoma cells.

Hepatitis B core antigen (HBcAg) is an important serological marker used in the diagnosis of hepatitis B virus (HBV) infections. In the current study, a fast and efficient preparative purification protocol for truncated HBcAg from Escherichia coli disruptate was developed. The recombinant HBcAg was first captured by anion exchange expanded bed adsorption chromatography integrated with a cell disruption process. This online capture process has shortened the process time and eliminated the “hold-up” period that may be detrimental to the quality of target protein. The eluted product from the expanded bed adsorption chromatography was subsequently purified using size-exclusion chromatography. The results showed that this novel purification protocol achieved a recovery yield of 45.1% with a product purity of 88.2%, which corresponds to a purification factor of 4.5. The recovered HBcAg is still biologically active as shown by ELISA test.

Accordingly, the present invention provides a recombinant fusion protein which includes at least one His tags and hepatitis B virus (HBV)core antigen. Further , the present invention also provides a method in producing a recombinant fusion protein which includes at least one His tags and hepatitis B virus (HBV) core antigen, the steps include (a) culturing expression host which contains expression vector of the recombinant fusion protein in a medium that allows growth of the expression host, (b) allowing the recombinant fusion protein to be expressed in the expression host, (c) harvesting the expression host from step (b)from the medium, (d) disrupting the expression host harvested from step (c) in a liquid medium for ultrasonication and (e) purifying the recombinant fusion protein of hepatitis B virus core antigen from the liquid medium. Also provided is a method for purifying a recombinant fusion protein which includes a His tag and hepatitis B virus (HBV) core antigen, the steps include (a)loading unclarified lysate of expression host expressing and containing the recombinant fusionprotein into nickle (II)-charged adsorbent, (b) fractioning the recombinant fusion protein using immobilized metal affinity chromatography and (c)collecting the fractionated recombinant fusion protein obtained from step (c) for determined concentration of the hepatitis B virus (HBV) core antigen. There is also provided a pharmaceutical composition comprising a therapetically effective amount of recombinant fusion protein which includes at least on His tag and hepatitis B virus (HBV) core antigen. Also provided is a vaccine comprising a prophylactic effective amount of a recombinant fusion protein which includes a His tag and hepatitis B virus (HBV) core antigen.

The present invention provides a method to purify a recombinant protein from microbial cell immobilized metal affinity chromatography. The method leads to the recovery of the nucleocapsid of Nipah virus in high yields, biologically active and free from other host cell proteins.

The present specification discloses an integrated purification technique for purifying recombinant intracellular protein (GFP) from intact E. coli cells using preparative PAGE column. Under the influence of electric fields, the negatively charged GFP was drifted out from cells without the need of cell disruption process. The electrophoresed cells were observed under SEM and some on the membrane of cells were observed. GFP was then separated from other host cells host protein along polyacrylamide gel and finally eluted electrophoretically from the gel. The invention has successfully recovered the GFP with purity and yeild 95% and 82%, respectively in 100 min operation.

Anaerobic digestion was carried out in this study to treat fresh leachate from municipal solid waste transfer station in a 10 L stirred tank reactor (STR). The treatment process was performed in batch and semi-continuous process. Palm oil mill effluent (POME) sludge was used as an inoculum. A high BOD reduction was achieved in 3 different treatment conditions in this study. A BOD removal of 85, 77 and 90% for the batch (Experiment 1), semi-continuous process without pH adjustment (Experiment 2) and semi-continuous process with pH adjustment (Experiment 3), respectively were recorded. It was observed that there was no significant deficiency in required nutrients for Experiment 1, 2 and 3 in this work. High concentration of volatile fatty acids (VFAs) was detected in Experiment 3, which indicated the instability of bioreactor in which lower methanogenic activity was observed. The levels of acetic acid (HAc) and propionic acid (HPr) appeared to be the VFA species that accumulated and started to cause an imbalance in the reactor. It was found that the use of large amount of sodium hydroxide (NaOH) to adjust the bioreactor pH had caused an inhibition of the metabolic activity of methanogenesis bacteria that involved in the methane production.

The broad species tropism of Nipah virus (NiV) coupled with its high pathogenicity demand a rapid search for a new biomarker candidate for diagnosis. The matrix (M) protein was expressed in Escherichia coli and purified using a Ni-NTA affinity column chromatography and sucrose density gradient centrifugation. The recombinant M protein with the molecular mass (Mr) of about 43 kDa was detected by anti-NiV serum and anti-myc antibody. About 50% of the M protein was found to be soluble and localized in cytoplasm when the cells were grown at 30 degrees C. Electron microscopic analysis showed that the purified M protein assembled into spherical particles of different sizes with diameters ranging from 20 to 50 nm. The purified M protein showed significant reactivity with the swine sera collected during the NiV outbreak, demonstrating its potential as a diagnostic reagent.

The adsorption behaviour and the micro- and mesopore size distributions of commercial palm kernel shell activated carbons (PKSAC) and other commercial activated carbon are characterized. The results showed that PKSAC are predominantly microporous materials, where micropores account 68–79% of total porosity. On the other hand, commercial activated carbons: Norit SX Plus, Calgon 12×40, and Shirasagi “A” activated carbons contained high mesopore fraction ranging from 33 to 52%. The analysis showed that the degree of mesoporosity of PKSAC is increased steadily with the decrease of particle size. This is due to the presence of channels interconnect the smaller pores in the interior of smaller particle size PKSAC. The smaller size PKSAC particle that is highly mesoporous has preformed better on the adsorption of larger molecules such as methylene blue. On the other hand, bigger size PKSAC particle has better performance on the adsorption of smaller adsorbates such as iodine.

Background: Palm kernel shell activated carbon (OPSA) produced by steam gasification at high temperatures generally results in high surface areas of 1146 to 1600 m2 g−1, attributed to the high volume of micropores (0.43 to 0.56 cm3 g−1). The mesoporosity of naturally occurring activated carbons is observed to increase with decreasing particle size. Mechanical grinding was therefore performed to investigate its effect on the mesoporosity and microporosity of OPSA. Results: Mechanical grinding had a strong effect on mesopore volume and average pore diameter, with an increase in mesopore volume from 47 to 66% as particle size decreases. Interestingly, no significant effect on the micropore fraction was observed in ground OPSA particles. Conclusions: The mechanically ground OPSA particles possessed dual adsorption capabilities due to the high microporosity and moderate mesoporosity contained in the structures. This results in interesting porosity behaviour of palm kernel shell activated carbons and the potential to provide materials of distinct sorption capacities with minimal treatment.

A 20 litre packed bed reactor (PBR) with heating and water removal system was designed and fabricated for the esterification of palm oil fatty acid distillate (PFAD) catalyzed by immobilized rice bran lipase (RBL). The PBR was designed based on the characteristics of immobilised RBL and the optimized esterification conditions obtained from method scouting performed in shaked flask. The optimal ratios of immobilised RBL and water removal agent (silica gel) to PFAD for the shaked flask esterification process were 5:1 and 1:2, respectively. The intensified esterification reaction of PBR was operated by circulating the reaction mixture (PFAD and glycerol) in hexane through a packed bed column filled with immobilised RBL. The water generated from esterification reaction was absorbed by silica gel filled in the water removal vessel. The maximum degree of esterification achieved in this developed PBR was 61%. The reaction time required to achieve the maximum degree of esterification was 25% faster than that in the shaked flask.

Leachate from transfer station requires treatment before being discharged into the environment to avoid surface and underground water contamination. Various factors such as waste composition, availability of oxygen and moisture, designing and controlling of transfer station operations have been shown to affect the composition of the leachate. The high COD, BOD, ammonia nitrogen (NH3-N) and heavy metals contents of fresh leachate are the main problems faced by leachate treatment operators. The result of the present study indicated that this process reduced the COD content by 43%.The average removal efficiencies of BOD5, TS, TSS, and VSS were 80, 49, 37 and 39 %, respectively.

Expanded bed adsorption chromatography (EBAC) is a single pass operation that has been used as primary capture step in various protein purifications. The most common problem in EBAC is often associated with successful formation of a stable fluidized bed during the absorption stage, which is critically dependent on parameters such as liquid velocity, bed height, particle (adsorbent) size and density as well as design of column and type of flow distributor. In this study, residence time distribution (RTD) test using acetone as non-binding tracer acetone was performed to evaluate liquid dispersion characteristics of the EBAC system. A high B(o) number was obtained indicating the liquid dispersion in the system employed is very minimal and the liquid flow within the bed was close to plug flow, which mimics a packed bed chromatography system. Evaluation on the effect of flow velocities and bed height on the performance of Streamline DEAE using feedstock containing heat-treated crude Escherichia coli homogenate of different biomass concentrations was carried out in this study. The advantages and disadvantages as well as the problems encountered during recovery of HBcAg with aforementioned parameters are also discussed in this paper.

The effect of mild hypothermic (32 degrees C) conditions on cell growth, cell-cycle progress, and antibody production of hybridoma C2E7 cells was investigated in the present study. The growth of hybridoma cells was slower during the mild hypothermic condition compared to that at 37 degrees C; this led to about 10% decrease in maximum viable cell density and volumetric antibody productivity. However, under mild hypothermic growth conditions, the culture viability was substantially improved and the specific antibody productivity was enhanced compared to that at 37 degrees C. The average specific productivity for the entire batch culture at 32 degrees C is about 5% higher than that at 37 degrees C. Cell-cycle analysis data showed that there was no growth arrestment during the mild hypothermic growth of hybridoma cells. The G1-phase cells were increased, while the S-phase cells were decreased gradually as the culture time progressed. Further analysis showed that the specific antibody productivity of hybridoma cells was correlated to the fraction of S-phase cells.

The onset of incipient fluidization is investigated theoretically and simulated by a computational fluid dynamics (CFD) procedure. The onset of incipient instability in a particle bed is preceded by stable gas diffusion in the interstices and is caused by a critical momentum force that may overcome the inertia of the particles. The critical momentum force is provided by the critical superficial gas velocity Uc in the form of critical mass flux of diffusion. It is found that the first movement of particles may be predicted by a critical transient Rayleigh number determined by a critical superficial velocity equals to the minimum fluidization velocity, Umf. The onset of incipient fluidization was found to occur at a critical transient Rayleigh number of 3.1, which is close to the lowest theoretical value for buoyancy convection in a porous medium bounded by free surfaces. Consequently the onset times of incipient fluidization may be predicted accurately. The finding has been found to be supported by the present CFD study, past experiments and simulations in the literature.