Cyclic AMP phosphodiesterase (PDE) partially purified from roots of Vigna mungo exhibited optimum activity at pH 5.5 to 6.0 and maximum enzyme activity at 50 degrees C. Levels of PDE activity in roots remained relatively constant from the first to the eleventh day after germination; on the twelfth day there was a 400% increase in PDE activity. The enzyme was stable for at least 48 hours at 28 degrees C, retaining 92% of its original activity. Plant growth hormones including gibberellic acid, indoleacetic acid and kinetin at 1.0 and 10.0 microM concentrations did not have any significant effect on enzyme activity. Nucleotides tested including cyclic 2'3' AMP, cyclic 2'3' GMP completely abolished enzyme activity at 1.0mM while cyclic 3'5' GMP, cyclic 3'5' GMP, 2'deoxy 5' ATP, 2'deoxy 5'GTP and 5'ADP were also inhibitory to the enzyme. The enzyme was stimulated by Mg2+, Fe2+ and NH4+ while Cu2+ and Fe3+ were inhibitory. Theophylline, caffeine, phosphate, pyrophosphate and EDTA were inhibitory to the enzyme.
Musty odor production by actinomycetes is usually related to the presence of geosmin and 2-methylisoborneol (2-MIB), which are synthesized by enzymes encoded by the geoA and tpc genes, respectively. Streptomyces spp. strain S10, which was isolated from a water reservoir in Malaysia, has the ability to produce geosmin when cultivated in a basal salt (BS) solid medium, but no 2-MIB production occurred during growth in BS medium. Strain S10 could produce higher levels of geosmin when the phosphate concentration was limited to 0.05 mg/L, with a yield of 17.53 ± 3.12 ✕ 105 ng/L, compared with growth in BS medium. Interestingly, 2-MIB production was suddenly detected when the nitrate concentration was limited to 1.0 mg/L, with a yield of 1.4 ± 0.11 ✕ 105 ng/L. Therefore, it was concluded that phosphate- and nitrate-limiting conditions could induce the initial production of geosmin and 2-MIB by strain S10. Furthermore, a positive amplicon of geoA was detected in strain S10, but no tpc amplicon was detected by PCR analysis. Draft genome sequence analysis showed that one open reading frame (ORF) contained a conserved motif of geosmin synthase with 95% identity with geoA in Streptomyces coelicolor A3 (2). In the case of the tpc genes, it was found that one ORF showed 23% identity to the known tpc gene in S. coelicolor A3(2), but strain S10 lacked one motif in the N-terminus.
Linalool and nerolidol are terpene alcohols that occur naturally in many aromatic plants and are commonly used in food and cosmetic industries as flavors and fragrances. In plants, linalool and nerolidol are biosynthesized as a result of respective linalool synthase and nerolidol synthase, or a single linalool/nerolidol synthase. In our previous work, we have isolated a linalool/nerolidol synthase (designated as PamTps1) from a local herbal plant, Plectranthus amboinicus, and successfully demonstrated the production of linalool and nerolidol in an Escherichia coli system. In this work, the biochemical properties of PamTps1 were analyzed, and its 3D homology model with the docking positions of its substrates, geranyl pyrophosphate (C10) and farnesyl pyrophosphate (C15) in the active site were constructed. PamTps1 exhibited the highest enzymatic activity at an optimal pH and temperature of 6.5 and 30 °C, respectively, and in the presence of 20 mM magnesium as a cofactor. The Michaelis-Menten constant (Km) and catalytic efficiency (kcat/Km) values of 16.72 ± 1.32 µM and 9.57 × 10-3 µM-1 s-1, respectively, showed that PamTps1 had a higher binding affinity and specificity for GPP instead of FPP as expected for a monoterpene synthase. The PamTps1 exhibits feature of a class I terpene synthase fold that made up of α-helices architecture with N-terminal domain and catalytic C-terminal domain. Nine aromatic residues (W268, Y272, Y299, F371, Y378, Y379, F447, Y517 and Y523) outlined the hydrophobic walls of the active site cavity, whilst residues from the RRx8W motif, RxR motif, H-α1 and J-K loops formed the active site lid that shielded the highly reactive carbocationic intermediates from the solvents. The dual substrates use by PamTps1 was hypothesized to be possible due to the architecture and residues lining the catalytic site that can accommodate larger substrate (FPP) as demonstrated by the protein modelling and docking analysis. This model serves as a first glimpse into the structural insights of the PamTps1 catalytic active site as a multi-substrate linalool/nerolidol synthase.
The initial rate of NH(3)/NH(4)(+) accumulation in a medium containing L(3) Teladorsagia circumcincta was 0.18-0.6 pmol h(-1) larva(-1), which increased linearly with larval density. However it appeared that the larva-generated external concentration of NH(3)/NH(4)(+) did not exceed about 130 μM. The rate of NH(3)/NH(4)(+) accumulation increased with temperature between 4 °C and 37 °C, declined with increasing pH or increasing external NH(3)/NH(4)(+) concentration and was not significantly affected by the concentration of the phosphate buffer or by exsheathing the larvae. We infer from these data that the efflux of NH(3)/NH(4)(+) is a diffusive process and that the secreted or excreted NH(3)/NH(4)(+) is generated enzymatically rather than dissociating from the surface of the nematode. The enzymatic source of the NH(3)/NH(4)(+) is yet to be identified. Since the concentration of NH(3)/NH(4)(+) in the rumen and abomasum is higher than 130 μM, it is unlikely that T. circumcincta contributes to it, but NH(3)/NH(4)(+) may be accumulated from the rumen fluid by the nematode.
Heat exchanger performance degrades rapidly during operation due to formation of deposits on heat transfer surfaces which ultimately reduces service life of the equipment. Due to scaling, product deteriorates which causes lack of proper heating. Chemistry of milk scaling is qualitatively understood and the mathematical models for fouling at low temperatures have been produced but the behavior of systems at ultra high temperature processing has to be studied further to understand in depth. In diversified field, the effect of whey protein fouling along with pressure drop in heat exchangers were conducted by many researchers. Adding additives, treatment of heat exchanger surfaces and changing of heat exchanger configurations are notable areas of investigation in milk fouling. The present review highlighted information about previous work on fouling, influencing parameters of fouling and its mitigation approach and ends up with recommendations for retardation of milk fouling and necessary measures to perform the task.
Extensive use of metals in various industrial applications has caused substantial environmental pollution. Molybdenum-reducing bacteria isolated from soils can be used to remove molybdenum from contaminated environments. In this work we have isolated a local bacterium with the capability to reduce soluble molybdate to the insoluble molybdenum blue. We studied several factors that would optimize molybdate reduction. Electron donor sources such as glucose, sucrose, lactose, maltose and fructose (in decreasing efficiency) supported molybdate reduction after 24 h of incubation with optimum glucose concentration for molybdate reduction at 1.5% (w/v). The optimum pH, phosphate and molybdate concentrations, and temperature for molybdate reduction were pH 6.5, 5.0, 25 to 50 mM and 37 degrees C, respectively. The Mo-blue produced by cellular reduction exhibited a unique absorption spectrum with a maximum peak at 865 nm and a shoulder at 700 nm. Metal ions such as chromium, cadmium, copper, silver and mercury caused approximately 73, 71, 81, 77 and 78% inhibition of the molybdenum-reducing activity, respectively. All of the respiratory inhibitors tested namely rotenone, azide, cyanide and antimycin A did not show any inhibition to the molybdenum-reducing activity suggesting components of the electron transport system are not responsible for the reducing activity. The isolate was tentatively identified as Enterobacter sp. strain Dr.Y13 based on carbon utilization profiles using Biolog GN plates and partial 16S rDNA molecular phylogeny.
Porous calcium phosphate ceramics have found enormous use in biomedical applications including bone tissue regeneration, cell proliferation, and drug delivery. In bone tissue engineering it has been applied as filling material for bone defects and augmentation, artificial bone graft material, and prosthesis revision surgery. Their high surface area leads to excellent osteoconductivity and resorbability providing fast bone ingrowths. Porous calcium phosphate can be produced by a variety of methods. This paper discusses briefly fundamental aspects of porous calcium phosphate for biomedical applications as well as various techniques used to prepare porous calcium phosphate.
This paper chronicled the development of a locally produced bone graft substitute based on calcium phosphate bioceramics called "GranuMaS--from concepts to clinics, and finally to its successful commercialization all within 5-year duration. It was a Prioritized Research (PR) collaborative project of 5 institutions namely SIRIM, ANM, USM, UKM and IIUM, funded by MOSTI to the amount of approximately RM2.5 millions under RM8. This paper also highlighted the requirements needed in terms of technical expertise/manpower, facilities and infrastructure, and government/institutional supports, as well as the challenge faced in developing and commercializing such product.
The effectiveness of different soil tests in assessing soil phosphorus (P) in soils amended with phosphate rocks (PRs) is uncertain. We evaluated the effects of triple superphosphate (TSP) and PRs on extractable P by conventional soil tests (Mehlich 3 [Meh3] and Bray-1 [B1]) and a nonconventional test (iron oxide-impregnated paper, strip). Extracted amounts of P were in the order: Meh3 >B1 > strip. All the tests were significantly correlated (p = 0.001). Acidic reagents extracted more P from TSP than PRs, while the strip removed equal amounts from the two sources. The P removed by the three tests was related significantly to dry matter yield (DMY), but only in the first harvest, except for B1. Established critical P levels (CPLs) differed for TSP and PRs. In PR-fertilized soils, CPLs were 27, 17, and 12 mg P kg(-1) soil for Meh3, B1, and strip, respectively, and 42, 31, and 12 mg P kg(-1) soil, respectively, in TSP-fertilized soils. Thus, the strip resulted in a common CPL for TSP and PRs (12 mg P kg(-1) soil). This method can be used effectively in soils where integrated nutrient sources have been used, but there is need to establish CPLs for different crops. For cost-effective fertilizer P recommendations based on conventional soil tests, there is a need to conduct separate calibrations for TSP- and PR-fertilized soils.
Biomaterial, an essential component of tissue engineering, serves as a scaffold for cell attachment, proliferation, and differentiation; provides the three dimensional (3D) structure and, in some applications, the mechanical strength required for the engineered tissue. Both synthetic and naturally occurring calcium phosphate based biomaterial have been used as bone fillers or bone extenders in orthopedic and reconstructive surgeries. This study aims to evaluate two popular calcium phosphate based biomaterial i.e., hydroxyapatite (HA) and tricalcium phosphate/hydroxyapatite (TCP/HA) granules as scaffold materials in bone tissue engineering. In our strategy for constructing tissue engineered bone, human osteoprogenitor cells derived from periosteum were incorporated with human plasma-derived fibrin and seeded onto HA or TCP/HA forming 3D tissue constructs and further maintained in osteogenic medium for 4 weeks to induce osteogenic differentiation. Constructs were subsequently implanted intramuscularly in nude mice for 8 weeks after which mice were euthanized and constructs harvested for evaluation. The differential cell response to the biomaterial (HA or TCP/HA) adopted as scaffold was illustrated by the histology of undecalcified constructs and evaluation using SEM and TEM. Both HA and TCP/HA constructs showed evidence of cell proliferation, calcium deposition, and collagen bundle formation albeit lesser in the former. Our findings demonstrated that TCP/HA is superior between the two in early bone formation and hence is the scaffold material of choice in bone tissue engineering.
A recently reported stable and efficient EBPR system at high temperatures around 30 °C has led to characterization of kinetic and stoichiometric parameters of the Activated Sludge Model no. 2d (ASM2d). Firstly, suitable model parameters were selected by identifiability analysis. Next, the model was calibrated and validated. ASM2d was found to represent the processes well at 28 and 32 °C except in polyhyroxyalkanoate (PHA) accumulation of the latter. The values of the kinetic parameters for PHA storage (q PHA), polyphosphate storage (q PP) and growth (μ PAO) of polyphosphate-accumulating organisms (PAOs) at 28 and 32 °C were found to be much higher than those reported by previous studies. Besides, the value of the stoichiometric parameter for the requirement of polyphosphate for PHA storage (Y PO4) was found to decrease as temperature rose from 28 to 32 °C. Values of two other stoichiometric parameters, i.e. the growth yield of heterotrophic organisms (Y H) and PAOs (Y PAO), were high at both temperatures. These calibrated parameters imply that the extremely active PAOs of the study were able to store PHA, store polyphosphate and even utilize PHA for cell growth. Besides, the parameters do not follow the Arrhenius correlation due to the previously reported unique microbial clade at 28 and 32 °C, which actively performs EBPR at high temperatures.
The application of bone substitutes and cements has a long standing history in augmenting fractures as a complement to routine fracture fixation techniques. Nevertheless, such use is almost always in conjunction with definite means of fracture fixation such as intramedullary pins or bone plates. The idea of using biomaterials as the primary fixation bears the possibility of simultaneous fixation and bone enhancement. Intramedullary recruitment of bone cements is suggested in this study to achieve this goal. However, as the method needs primary testings in animal models before human implementation, and since the degree of ambulation is not predictable in animals, this pilot study only evaluates the outcomes regarding the feasibility and safety of this method in the presence of primary bone fixators. A number of two sheep were used in this study. Tibial transverse osteotomies were performed in both animals followed by external skeletal fixation. The medullary canals, which have already been prepared by removing the marrow through proximal and distal drill holes, were then injected with calcium phosphate cement (CPC). The outcomes were evaluated postoperatively by standard survey radiographs, morphology, histology and biomechanical testings. Healing processes appeared uncomplicated until week four where one bone fracture recurred due to external fixator failure. The results showed 56% and 48% cortical thickening, compared to the opposite site, in the fracture site and proximal and distal diaphyses respectively. This bone augmentative effect resulted in 264% increase in bending strength of the fracture site and 148% increase of the same value in the adjacent areas of diaphyses. In conclusion, IMCO, using CPC in tibia of sheep, is safe and biocompatible with bone physiology and healing. It possibly can carry the osteopromotive effect of the CPCs to provide a sustained source of bone augmentation throughout the diaphysis. Although the results must be considered preliminary, this method has possible advantages over conventional methods of bone fixation at least in bones with compromised quality (i.e. osteoporosis and bone cysts), where rigid metal implants may jeopardize eggshell cortices.
With inoculum sludge from a conventional activated sludge wastewater treatment plant, three sequencing batch reactors (SBRs) fed with synthetic wastewater were operated at different high temperatures (30, 40 and 50±1°C) to study the formation of aerobic granular sludge (AGS) for simultaneous organics and nutrients removal with a complete cycle time of 3h. The AGS were successfully cultivated with influent loading rate of 1.6CODg(Ld)(-1). The COD/N ratio of the influent wastewater was 8. The results revealed that granules developed at 50°C have the highest average diameter, (3.36mm) with 98.17%, 94.45% and 72.46% removal efficiency observed in the system for COD, ammonia and phosphate, respectively. This study also demonstrated the capabilities of AGS formation at high temperatures which is suitable to be applied for hot climate conditions.
Phosphate solubilizing bacteria (PSB) can convert insoluble form of phosphorous to an available form. Applications of PSB as inoculants increase the phosphorus uptake by plant in the field. In this study, isolation and precise identification of PSB were carried out in Malaysian (Serdang) oil palm field (University Putra Malaysia). Identification and phylogenetic analysis of 8 better isolates were carried out by 16S rRNA gene sequencing in which as a result five isolates belong to the Beta subdivision of Proteobacteria, one isolate was related to the Gama subdivision of Proteobacteria, and two isolates were related to the Firmicutes. Bacterial isolates of 6upmr, 2upmr, 19upmnr, 10upmr, and 24upmr were identified as Alcaligenes faecalis. Also, bacterial isolates of 20upmnr and 17upmnr were identified as Bacillus cereus and Vagococcus carniphilus, respectively, and bacterial isolates of 31upmr were identified as Serratia plymuthica. Molecular identification and characterization of oil palm strains as the specific phosphate solubilizer can reduce the time and cost of producing effective inoculate (biofertilizer) in an oil palm field.
This study focuses on the evaluation of the performance of Chlorella sp. in removing nutrient in aquaculture wastewater and its correlation with the kinetic growth of Chlorella sp. The treatment was applied with various Chlorella sp. inoculation dosage ranging from 0% to 60% (v/v) of wastewater. The optimum inoculation dosage was recorded at 30% (v/v) with effluent concentration of ammonia and orthophosphate recording at 0.012mgL(-1) and 0.647mgL(-1), respectively on Day 11. The optimum dosage for bio-flocculation process was obtained at 30mgL(-1) of Aspergillus niger with a harvesting efficiency of 97%. This type of development of phytoremediation with continuous bio-harvesting could promote the use of sustainable green technology for effective wastewater treatment.
Primers corresponding to conserved bacterial repetitive of BOX elements were used to show that BOX-DNA sequences are widely distributed in phosphate solubilizing Pseudomonas strains. Phosphate solubilizing Pseudomonas was isolated from oil palm fields (tropical soil) in Malaysia. BOX elements were used to generate genomic fingerprints of a variety of Pseudomonas isolates to identify strains that were not distinguishable by other classification methods. BOX-PCR, that derived genomic fingerprints, was generated from whole purified genomic DNA by liquid culture of phosphate solubilizing Pseudomonas. BOX-PCR generated the phosphate solubilizing Pseudomonas specific fingerprints to identify the relationship between these strains. This suggests that distribution of BOX elements' sequences in phosphate solubilizing Pseudomonas strains is the mirror image of their genomic structure. Therefore, this method appears to be a rapid, simple, and reproducible method to identify and classify phosphate solubilizing Pseudomonas strains and it may be useful tool for fast identification of potential biofertilizer strains.
Molybdenum is very toxic to agricultural animals. Mo-reducing bacterium can be used to immobilize soluble molybdenum to insoluble forms, reducing its toxicity in the process. In this work the isolation of a novel molybdate-reducing Gram positive bacterium tentatively identified as Bacillus sp. strain A.rzi from a metal-contaminated soil is reported. The cellular reduction of molybdate to molybdenum blue occurred optimally at 4 mM phosphate, using 1% (w/v) glucose, 50 mM molybdate, between 28 and 30 °C and at pH 7.3. The spectrum of the Mo-blue product showed a maximum peak at 865 nm and a shoulder at 700 nm. Inhibitors of bacterial electron transport system (ETS) such as rotenone, sodium azide, antimycin A, and potassium cyanide could not inhibit the molybdenum-reducing activity. At 0.1 mM, mercury, copper, cadmium, arsenic, lead, chromium, cobalt, and zinc showed strong inhibition on molybdate reduction by crude enzyme. The best model that fitted the experimental data well was Luong followed by Haldane and Monod. The calculated value for Luong's constants p max, K(s), S(m), and n was 5.88 μmole Mo-blue hr(-1), 70.36 mM, 108.22 mM, and 0.74, respectively. The characteristics of this bacterium make it an ideal tool for bioremediation of molybdenum pollution.