Addition of nitrogen sources as supplementary nutrient into MSM medium to enhance biodegradation by stimulating the growth four isolates, Acinetobacter faecalis, Staphylococcus sp., Pseudomonas putida and Neisseria elongata isolated from petroleum contaminated groundwater, wastewater aeration pond and biopond at the oil refinery Terengganu Malaysia was investigated. The organic nitrogen sources tested not only supported growth but also enhances biodegradation of 1% Tapis crude oil. All four isolates showed good growth especially when peptone was employed as the organic nitrogen compared to growth in the basal medium. Gas chromatography showed that more then 91, 93, 94 and 95% degradation of total hydrocarbon was observed after 5 days of incubation by isolates Pseudomonas putida, Neisseria elongate, Acinetobacter faecalis and Staphylococcus sp., respectively.
The present study aimed to provide an insight of C. jejuni ATCC 33560 phenotype profiles (carbon sources and sensitivity to osmolytes and pH) using Phenotypic MicroArray (PM) system in response to optimal and suboptimal temperature. C. jejuni ATCC 33560 showed utilization carbon sources from amino acids and carboxylates but not from sugars. C. jejuni ATCC 33560 is sensitive to NaCl at 2% and above but showed survival in a wide range of food preservatives (sodium lactate, sodium phosphate, sodium benzoate, ammonium sulphate and sodium nitrate). When incubated at suboptimal temperature, no phenotype loss was observed in carbon source plates. Phenotype loss of C. jejuni ATCC 33560 was observed in sodium chloride (1%), sodium sulphate (2-3%), sodium formate (1%), sodium lactate (7-12%), sodium phosphate pH7 (100mM and 200mM), ammonium sulphate pH8 (50mM), sodium nitrate (60mM, 80mM and 100mM), sodium nitrite (10mM), and growth in pH5. The phenotypic profile from present study will provide a better insight related to survival of C. jejuni ATCC 33560.
Surface antigens are the most abundant proteins found on the surface of the parasite Toxoplasma gondii. Surface antigen 1 (SAG1) and Surface antigen 2 (SAG2) remain the most important and extensively studied surface proteins. These antigens have been identified to play a role in host cell invasion, immune modulation, virulence attenuation. Recombinant SAG1/2 was cloned and expressed in yeast Pichia pastoris. We describe here optimization of critical parameters involved in high yield expression of the recombinant SAG1/2. Our results suggest that recombinant SAG1/2 were best expressed at 30ºC, pH 6 and 1% methanol as the carbon source by X33 Pichia cells. Additional optimizations included the downstream process such as ammonium sulphate precipitation and dialysis. The fusion protein was purified using Ni-NTA purification system with 80% recovery. The purified protein was 100% specific and sensitive in detection of toxoplasmosis.
Oil palm empty fruit bunch graft poly (acrylic acid-co-acrylamide) superabsorbent composite (OPEFB-g-(PAA-co-PAM) SAPC) was synthesized by graft copolymerization of the acrylic acid (AA) and acrylamide (AM) comonomer onto OPEFB fibre using ammonium persulfate (APS) and N,N-methylene bisacarylamide (MBA) as an initiator and crosslinker, respectively. The absorbency in various chloride salt solutions indicated that the absorbency decreased with increasing ionic strength of the salt solutions. Moreover, the absorbency under load (AUL) of SAPC was investigated at various applied loading and results show that, AUL decreased with increasing applied loading. Infrared Spectroscopy (IR) and Thermogravimetric Analysis (TGA) were carried out to confirm the chemical structure and thermal properties of the synthesized superabsorbent, respectively.
Factors influencing poly(3-hydroxybutyrate) P(3HB) production by Cupriavidus necator CCUG52238(T) utilizing oil palm frond (OPF) juice were clarified in this study. Effects of initial medium pH, agitation speed, and ammonium sulfate (NH(4))(2)SO(4) concentration on the production of P(3HB) were investigated in shake flasks experiments using OPF juice as the sole carbon source. The highest P(3HB) content was recorded at pH 7.0, agitation speed of 220 rpm, and (NH(4))(2)SO(4) concentration at 0.5 g/L. By culturing the wild-type strain of C. necator under the aforementioned conditions, the cell dry weight (CDW) and P(3HB) content obtained were 9.31 ± 0.13 g/L and 45 ± 1.5 wt.%, respectively. This accounted for 40% increment of P(3HB) content compared to the nonoptimized condition. In the meanwhile, the effect of dissolved oxygen tension (DOT) on P(3HB) production was investigated in a 2-L bioreactor. Highest CDW (11.37 g/L) and P(3HB) content (44 wt.%) were achieved when DOT level was set at 30%. P(3HB) produced from OPF juice had a tensile strength of 40 MPa and elongation at break of 8% demonstrated that P(3HB) produced from renewable and cheap carbon source is comparable to those produced from commercial substrate.
Chitin was successfully grafted with polystyrene by free radical mechanism using ammonium persulfate (APS) initiator. The reaction was carried out in aqueous medium. The effect of pH, chitin:monomer weight ratio, APS, reaction time and reaction temperature were investigated. The results showed that the optimum conditions for grafting of polystyrene were found as follows: pH 7, chitin:monomer weight ratio of 1:3, 0.4 g of APS, reaction temperature of 60 °C and reaction time 2 h. The graft copolymer was characterized by Fourier transform infrared spectroscopy, thermogravimetric analysis (TGA) and differential scanning electron microscopy (DSC). Gel permeation chromatography (GPC) analysis carried out on the hydrolyzed graft copolymer showed that the Mn and Mw were 6.3395×10(4) g/mol and 1.69283×10(5) g/mol, respectively, with polydispersity index of 2.7.
Bromelain is one of the vegetal proteases found in pineapple plant. It has numerous applications in food and pharmaceuticals. This review discussed different bromelain purification techniques which will assist in determining the effect of processing conditions on the purification efficacy. There are four purification techniques to be discussed, namely; reverse micellar system, aqueous two phase extraction, cation exchange chromatography and ammonium sulphate precipitation. Of the four techniques, cation exchange chromatography had shown the best bromelain purification technique with purification fold of 10.0 followed by reverse micellar system containing CTAB/ isooctane/ hexanol/ butanol, ATPE containing PEG polymer, ammonium sulphate precipitation and ATPE containing PEO-PPO-PEO with purification fold of 5.2, 4.0, 2.81 and 1.25, respectively.
Proteases in ginger rhizome have the potentials in industrial applications. This study was conducted to extract and characterize the proteolytic enzyme from ginger (Zingiber officinale Roscoe). Ginger protease (GP) was extracted from ginger rhizome by homogenization with 100 mM potassium phosphate buffer pH 7.0 containing 10 mM cysteine and 5 mM EDTA which were found to be the most efficient extraction buffer and stabilizers. After centrifugation at 10,500 x g, protein in the crude extract was precipitated using 60% ammonium sulfate following which the precipitate was redissolved in 50 mM potassium phosphate buffer pH 7.0, dialyzed and then lyophilized. The extraction method yielded 0.94% (w/w of fresh weight) of GP with a specific activity of 27.6 ± 0.1 Unit/mg protein where 1 Unit is defined as the amount of protease causing an increase in absorbance by 1 unit per minute using azocasein as the substrate. Results show that the GP was completely inhibited by heavy metal cations i.e. Cu2+and Hg2+, and a thiol blocking agent or inhibitor, n-ethyl maleimide (NEM), indicating that GP is most probably a cysteine protease. The enzyme has an optimum temperature at 60⁰C and the optimum pH ranged between pH 6 to 8. Monovalent cations (K+ and Na+) have no significant effect on activity of GP, but divalent and trivalent cations showed moderate inhibitory effect. Detergents such as sodium dodecyl sulfate increased the activity of GP while Tween 80 and Tween 20 slightly reduced the activity.
Unripe and ripe bilimbi (Averrhoa bilimbi. L) were ground and the extracted juices were partially purified by ammonium sulfate precipitation at the concentrations of 40 and 60% (w/v). The collected proteases were analysed for pH, temperature stability, storage stability, molecular weight distribution, protein concentration and protein content. Protein content of bilimbi fruit was 0.89 g. Protease activity of both the unripe and ripe fruit were optimum at pH 4 and 40ºC when the juice were purified at 40 and 60% ammonium sulfate precipitation. A decreased in protease activity was observed during the seven days of storage at 4°C. Molecular weight distribution indicated that the proteases protein bands fall between 10 to 220 kDa. Protein bands were observed at 25, 50 and 160 kDa in both the unripe and ripe bilimbi proteases purified with 40% ammonium sulfate, however, the bands were more intense in those from unripe bilimbi. No protein bands were seen in proteases purified with 60% ammonium sulfate. Protein concentration was higher for proteases extracted with 40% ammonium sulfate at both ripening stages. Thus, purification using 40% ammonium sulfate precipitation could be a successful method to partially purify proteases from bilimbi especially from the unripe stage.
A molybdenum-reducing bacterium from Antarctica has been isolated. The bacterium converts sodium molybdate or Mo⁶⁺ to molybdenum blue (Mo-blue). Electron donors such as glucose, sucrose, fructose, and lactose supported molybdate reduction. Ammonium sulphate was the best nitrogen source for molybdate reduction. Optimal conditions for molybdate reduction were between 30 and 50 mM molybdate, between 15 and 20°C, and initial pH between 6.5 and 7.5. The Mo-blue produced had a unique absorption spectrum with a peak maximum at 865 nm and a shoulder at 710 nm. Respiratory inhibitors such as antimycin A, sodium azide, potassium cyanide, and rotenone failed to inhibit the reducing activity. The Mo-reducing enzyme was partially purified using ion exchange and gel filtration chromatography. The partially purified enzyme showed optimal pH and temperature for activity at 6.0 and 20°C, respectively. Metal ions such as cadmium, chromium, copper, silver, lead, and mercury caused more than 95% inhibition of the molybdenum-reducing activity at 0.1 mM. The isolate was tentatively identified as Pseudomonas sp. strain DRY1 based on partial 16s rDNA molecular phylogenetic assessment and the Biolog microbial identification system. The characteristics of this strain would make it very useful in bioremediation works in the polar and temperate countries.
Cross-linked enzyme aggregate (CLEA) is easily prepared from crude enzyme and has many advantages to the environment and it is considered as an economic method in the context of industrial biocatalysis compared to free enzyme. In this work, a highly active and stable CLEA-lipase from cocoa pod husk (CPH) which is a by-product after removal of cocoa beans, were assayed for their hydrolytic activity and characterized under the optimum condition successfully. Face centered central composite design (FCCCD) under response surface methodology (RSM) was used to get the optimal conditions of the three significant factors (concentration of ammonium sulfate, concentration of glutaraldehyde and concentration of additive) to achieve higher enzyme activity of CLEA. From 20 runs, the highest activity recorded was around 9.407U (83% recovered activity) under the condition of using 20% saturated ammonium sulfate, 60mM glutaraldehyde as cross-linker and 0.17mM bovine serum albumin as feeder. Moreover, the optimal reaction temperature and pH value in enzymatic reaction for both crude enzyme and immobilized were found to be 45°C at pH 8 and 60°C at pH 8.2, respectively. A systematic study of the stability of CLEA and crude enzyme was taken with regards to temperature (25-60°C) and pH (5-10) value and in both factors, CLEA-lipase showed more stability than free lipase. The Km value of CLEA was higher compared to free enzyme (0.55mM vs. 0.08mM). The CLEA retained more than 60% of the initial activity after six cycles of reuse compared to free enzyme. The high stability and recyclability of CLEA-lipase from CPH make it efficient for different industrial applications.
Threadfin bream (Nemipterus japonicas) muscle was hydrolysed using protease extracted from
bilimbi (Averrhoa bilimbi L.) fruit. This study was performed in order to compare the efficiency of bilimbi protease in producing threadfin bream protein hydrolysate with the commercial protease; alcalase 2.4 L. Initially, protease was extracted and then purified using 40% ammonium sulfate precipitation method. The proteolytic activity of the crude extract and purified protease was determined. Precipitation using 40% ammonium sulfate resulted in bilimbi protease specific activity of 2.36 U/mg and 23.13% recovery. Threadfin bream hydrolysate was prepared based on the pH-stat method by hydrolysis for 2 hrs. Hydrolysis using bilimbi protease produced 34.76% degree of hydrolysis (DH) and 3.75% yield while hydrolysis using alcalase resulted in 86.6% DH with 22.78% yield. Alcalase hydrolysate showed higher solubility than bilimbi protease hydrolysate at pH 7 with 70.87 and 32.16% solubility, respectively. Results also showed that protein content of threadfin bream hydrolysate produced using alcalase was higher (86.86%) than those produced using bilimbi protease (22.12%). However, both hydrolysates showed low moisture content between 3.93 to 7.00%. The molecular weight distribution analysis using SDS–PAGE indicated the distribution of smaller peptides especially in alcalase hydrolysate. Overall, the results showed that alcalase is more efficient enzyme choice than bilimbi protease for preparing threadfin bream hydrolysates. However, both hydrolysates could play an important role thus contribute to the food industry.
Proteases were extracted from starfruit at maturity Index 2 (unripe, light green) and Index 7 (very ripe, orange) and partially purified using acetone and 40% ammonium sulfate precipitations. Higher yield and proteolytic activity were observed for proteases purified using acetone than 40% ammonium sulfate. As for maturity index, yield and protein concentration of proteases from Index 2 were higher than those from Index 7. SDS-PAGE result showed intense bands for acetone proteases while a distinct band at 50 kDa was observed in all the proteases. Enzyme activity decreased during the seven days storage at 4°C with minimum relative activity of 70% achieved for acetone proteases at day seven. This study suggested that acetone precipitation is more effective method for purifying starfruit protease based on the yield and proteolytic activity compared to using 40% ammonium sulphate precipitation. In order to obtain higher protein concentration and proteolytic activity, starfruit at the unripe stage, Index 2 is a better raw material than Index 7 to be used for protease production.
Polyphenol oxidase (PPO) catalyzes the conversion of phenolic compounds into o-quinones which will lead to food browning. This phenomenon causes huge implications on food industries, as it degrades food quality over time. By combining both ammonium sulphate precipitation and gel filtration chromatography, PPO was partially purified up to 5.26-fold with 11.23% yield. The enzyme activity was 5120 EU/mL using 4-methylcatechol as substrate. Maximal PPO activity was found at 30oC, pH 5.0 for 4-methylcatechol and 40°C, pH 6.0 for catechol. The PPO showed a higher affinity towards 4-methylcatechol but higher thermal stability when reacting with catechol. The Km and Vmax values were 5.00 mM, 2000 EU/ml for 4-methylcatechol and 10.79 mM, 526.32 EU/ml for catechol. Energy for inactivation (Ea) obtained using 4-methylcatechol and catechol were 12.57 kJ/mol and 14.23 kJ/mol from respective substrates. Sodium disulfite was a better inhibitor where 79.17% of PPO inhibition was achieved. The isolation and characterization of round brinjal PPO serves as a guideline to predict the behavior of enzyme, leading to effective prevention of its browning during processing and storage.
Molybdenum is an emerging pollutant. Bioremediation of this heavy metal is possible by the
mediation of Mo-reducing bacteria. These bacteria contain the Mo-reducing enzymes that can
conver toxic soluble molybdenum into molybdenum blue; a less soluble and less toxic form of the
metal. To date only the enzyme has been purified from only one bacterium. The aim of this study is
to purify the Mo-reducing enzyme from a previously isolated Mo-reducing bacterium Bacillus
pumilus strain Lbna using ammonium sulphate fractionation followed by ion exchange and then
gel filtration. Two clear bands were obtained after the gel filtration step with molecular weights
of 70 and 100 kDa. This indicates that further additional purification methods need to be used
to get a purified fraction. Hence, additional steps of chromatography such as hydroxyapatite or
chromatofocusing techniques can be applied in the future.
The synthesis of high quality graphene via economic way is highly desirable for practical applications. In this study, graphene flake was successfully synthesized on Cu/MgO catalyst derived from recovered Cu via etching in ammonium persulfate solution. Recovered Cu acted as efficient active metal in Cu/MgO catalyst with good crystal structure and composition according to XRD and XRF results. FESEM, EDX, HRTEM, Raman spectroscopy and SAED analysis were carried out on the synthesized graphene. The formation of single, bilayer and few layer of graphene from Cu/MgO catalyst derived from recovered Cu was feasible.
Marasmius cladophyllus was examined for its ability to degradatively decolourise the recalcitrant dye Remazol Brilliant Blue R (RBBR) and screened for the production of ligninolytic enzymes using specific substrates. Monitoring dye decolourisation by the decrease in absorbance ratio of A592/A500 shows that the decolourisation of RBBR dye was associated with the dye degradation. Marasmius cladophyllus produces laccase and lignin peroxidase in glucose minimal liquid medium containing RBBR. Both enzyme activities were increased, with laccase activity recorded 70 times higher reaching up to 390 U L-1 on day 12. Further in vitro RBBR dye decolourisation using the culture medium shows that laccase activity was correlated with the dye decolourisation. Fresh RBBR dye continuously supplemented into the decolourised culture medium was further decolourised much faster in the subsequent round of the RBBR dye decolourisation. In vitro dye decolourisation using the crude laccase not only decolourised 76% of RBBR dye in just 19 hours but also decolourised 54% of Orange G and 33% of Congo red at the same period of time without the use of any exogenous mediator. This rapid dye decolourisation ability of the enzymes produced by M. cladophyllus thus suggested its possible application in the bioremediation of dye containing wastewater.
In this novel study, we report on the use of two molybdenum-reducing bacteria with the ability to utilise the herbicide glyphosate as the phosphorus source. The bacteria reduced sodium molybdate to molybdenum blue (Mo-blue), a colloidal and insoluble product, which is less toxic. The characterisation of the molybdenum-reducing bacteria was carried out using resting cells immersed in low-phosphate molybdenum media. Two glyphosate-degrading bacteria, namelyBurkholderia vietnamiensisAQ5-12 andBurkholderiasp. AQ5-13, were able to use glyphosate as a phosphorous source to support molybdenum reduction to Mo-blue. The bacteria optimally reduced molybdenum between the pHs of 6.25 and 8. The optimum concentrations of molybdate for strainBurkholderia vietnamiensis strainAQ5-12 was observed to be between 40 and 60 mM, while forBurkholderiasp. AQ5-13, the optimum molybdate concentration occurred between 40 and 50 mM. Furthermore, 5 mM of phosphate was seen as the optimum concentration supporting molybdenum reduction for both bacteria. The optimum temperature aiding Mo-blue formation ranged from 30 to 40 °C forBurkholderia vietnamiensis strainAQ5-12, whereas forBurkholderiasp. AQ5-13, the range was from 35 to 40 °C. Glucose was the best electron donor for supporting molybdate reduction, followed by sucrose, fructose and galactose for both strains. Ammonium sulphate was the best nitrogen source in supporting molybdenum reduction. Interestingly, increasing the glyphosate concentrations beyond 100 and 300 ppm forBurkholderia vietnamiensis strainAQ5-12 andBurkholderiasp. AQ5-13, respectively, significantly inhibited molybdenum reduction. The ability of these bacteria to reduce molybdenum while degrading glyphosate is a useful process for the bioremediation of both toxicants.
In this study, an alpha-amylase enzyme from a locally isolated Aspergillus flavus NSH9 was purified and characterized. The extracellular α-amylase was purified by ammonium sulfate precipitation and anion-exchange chromatography at a final yield of 2.55-fold and recovery of 11.73%. The molecular mass of the purified α-amylase was estimated to be 54 kDa using SDS-PAGE and the enzyme exhibited optimal catalytic activity at pH 5.0 and temperature of 50 °C. The enzyme was also thermally stable at 50 °C, with 87% residual activity after 60 min. As a metalloenzymes containing calcium, the purified α-amylase showed significantly increased enzyme activity in the presence of Ca2+ ions. Further gene isolation and characterization shows that the α-amylase gene of A. flavus NSH9 contained eight introns and an open reading frame that encodes for 499 amino acids with the first 21 amino acids presumed to be a signal peptide. Analysis of the deduced peptide sequence showed the presence of three conserved catalytic residues of α-amylase, two Ca2+-binding sites, seven conserved peptide sequences, and several other properties that indicates the protein belongs to glycosyl hydrolase family 13 capable of acting on α-1,4-bonds only. Based on sequence similarity, the deduced peptide sequence of A. flavus NSH9 α-amylase was also found to carry two potential surface/secondary-binding site (SBS) residues (Trp 237 and Tyr 409) that might be playing crucial roles in both the enzyme activity and also the binding of starch granules.
In this study, salting-out assisted liquid-liquid extraction (SALLE) as a simple and efficient extraction technique followed by high-performance liquid chromatography (HPLC) was employed for the determination of vitamin D3 in milk samples. The sample treatment is based on the use of water-miscible acetonitrile as the extractant and acetonitrile phase separation under high-salt conditions. Under the optimum conditions, acetonitrile and ammonium sulfate were used as the extraction solvent and salting-out agent, respectively. The vitamin D3 extract was separated using Hypersil ODS (250x i.d 4.6 mm, 5 µm) HPLC column that was coupled with diode array detector. Vitamin D2 was used as internal standard (IS) to offset any variations in chromatographic conditions. The vitamin D3 and the IS were eluted in 18 min. Good linearity (r2 > 0.99) was obtained within the range of 25-600 ng g-1 with the limit of detection of 15 ng g-1 and limit of quantification of 25 ng g-1. The validated method was applied for the determination of vitamin D3 in milk samples. The recoveries for spiked samples were from 94.4 to 113.5%.