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Overexpression of phytase and functional analysis of disulfide bonds
New Biotechnology · Volume 25S · September 2009
ABSTRACTS
for substrates were accomplished throughout the determination of the activity using different triglycerides (C4 , C8 , C10 , C14 , C18:1 ) as substrate. In the selection stage, from 372 studied wild yeasts only three strains were selected, with potential for lipase production. Concerning the substrate specificity, the enzyme originated from the yeast entitled AY3 presented superior lipolytic activity in tributyrin (C4 ) and tricaprylin (C8 ) when compared with olive oil, demonstrating larger specificity for triglycerides of short and medium chain. Hence, the search of new lipases through programs of selection of microorganisms is of fundamental importance to enlarge the field of application of those enzymes. doi:10.1016/j.nbt.2009.06.352
2.1.106 Overexpression of phytase and functional analysis of disulfide bonds X. Mu ∗ , M. Wang, X. Wang Department of Biochemistry and Molecular Biology, Taian, China
The supplementation of animal feed with phytase increases the bioavailability of phosphorus in monogastric animals besides reducing the phosphorus pollution. Increasing the expression and improving the stability of phytase are of great urgency. As the development of gene engineering, it is to be a promising approach to improve enzyme property via protein engineering. Disulfide bonds are indispensable for the stability and function of enzyme. Phytase contains five pairs of disulfide bonds: Cys12—Cys21, Cys52—Cys395, Cys196—Cys446, Cys245—Cys263, Cys417—Cys425. To provide theoretical basis for improving stability by introducing disulfide bond, we studied the contribution of disulfide bonds to the catalytic activity and conformational stability of phytase. In this study, we cloned the phyA gene from selected A. niger strain which produces extracellular phytase. This gene was transformed into Pichia pastoris and phytase with potential industrial application was overexpressed. We also investigated the effects of the five disulfide bonds on the conformation and thermostability. The main results were as follows: 1. The phyA gene was cloned by PCR with the genomic DNA of A. niger N-J as template. We transformed phyA into P. pastoris and overexpressed phytase with potential industrial application. 2. Five expression plasmids of phytase mutants with deletion of each of the five disulfide bonds, respectively (C12S, C263S, C395S, C417S, C446S) were constructed. All the five mutants were expressed at high level in P. pastoris. 3. Thermostability analysis indicated that the deletion of any disulfide bonds made the conformation of the active-site fragile and easier to be changed. The deletion of disulfide bonds of Cys52—Cys395, Cys245—Cys263 and Cys417—Cys425 extremely decreased the thermostability. 4. Assay of the enzymological characteristics showed that all the five mutants hold high activity in the range of pH 2.5—5.5, which are similar to the stomach and intestines of animal. The deletion of disulfide bonds of Cys12—Cys21 S86
www.elsevier.com/locate/nbt
and Cys196—Cys446 decreased the optimum temperature slightly. doi:10.1016/j.nbt.2009.06.353
2.1.107 Detoxification and decolorization of raw textile effluents by a few marine fungi A. Verma National Institute of Oceanography, Panjim, India
Very few studies address the issue of bioremediation of dyecontaining textile effluents varying in their composition, toxicity, pH and salt content. We have used two marine-derived ascomycetes and two basidiomycetes fungi for treatment of raw dye-containing effluents, varying in their pHs and dye compositions. Textile effluent A (TEA) with pH of 8.9 containing azo dye and textile effluent B (TEB) with pH of 2.5 containing reactive dyes were decolorized 70—90% by these fungi. Effluents at 20, 50 and 90% concentrations were added to the culture medium for decolorization, reduction in COD and phenolics and for toxicity assays. A reduction of 80—90% in COD and total phenolics was brought about by these fungi by day 6. Toxicity assay using Artemia larvae showed two to fivefold reduction in toxicity of both the effluents. Both the effluents supported growth and laccase production in these fungi when grown in seawater medium with 15 ppt salinity. The fungal biomass could be used up to three cycles for decolorization of TEB. It can be concluded from this study that marine-derived ascomycetes fungi are capable of decolorization and detoxification of textile effluents, either by bioadsorption or laccase-mediated or by a concerted effect of these two processes, whereas in basidiomycetes laccase played a major role in these process. Mass spectrometric studies revealed that both the effluents were degraded to low molecular weight compounds. To study the actual pathway of degradation, individual component dyes of the effluents were also treated in the same way at two different concentrations of 300 and 3000 mg l−1 by one of the fungi. Mass spectrometric and NMR analyses were done with these dyes treated with the fungus to follow their degradation/biotransformation pathway. Bioremediation of these two effluents was effective by using immobilized laccase of one of these fungi in a bioreactor. doi:10.1016/j.nbt.2009.06.354
2.1.108 The new strains of Bacillus spp., potential biocontrol agent of mosquito M. Yazici ∗ , F. Sahin, A.U. Dogan, E. Kilic Yeditepe University, Istanbul, Turkey
Mosquitoes are vectors of many diseases such as Mosquito-borne arboviruses, malaria, filariasis and Japan encephalitis. Generally mosquito control was performed based on application of chemical pesticides in the World. All of the chemical pesticides have
ABSTRACTS
for substrates were accomplished throughout the determination of the activity using different triglycerides (C4 , C8 , C10 , C14 , C18:1 ) as substrate. In the selection stage, from 372 studied wild yeasts only three strains were selected, with potential for lipase production. Concerning the substrate specificity, the enzyme originated from the yeast entitled AY3 presented superior lipolytic activity in tributyrin (C4 ) and tricaprylin (C8 ) when compared with olive oil, demonstrating larger specificity for triglycerides of short and medium chain. Hence, the search of new lipases through programs of selection of microorganisms is of fundamental importance to enlarge the field of application of those enzymes. doi:10.1016/j.nbt.2009.06.352
2.1.106 Overexpression of phytase and functional analysis of disulfide bonds X. Mu ∗ , M. Wang, X. Wang Department of Biochemistry and Molecular Biology, Taian, China
The supplementation of animal feed with phytase increases the bioavailability of phosphorus in monogastric animals besides reducing the phosphorus pollution. Increasing the expression and improving the stability of phytase are of great urgency. As the development of gene engineering, it is to be a promising approach to improve enzyme property via protein engineering. Disulfide bonds are indispensable for the stability and function of enzyme. Phytase contains five pairs of disulfide bonds: Cys12—Cys21, Cys52—Cys395, Cys196—Cys446, Cys245—Cys263, Cys417—Cys425. To provide theoretical basis for improving stability by introducing disulfide bond, we studied the contribution of disulfide bonds to the catalytic activity and conformational stability of phytase. In this study, we cloned the phyA gene from selected A. niger strain which produces extracellular phytase. This gene was transformed into Pichia pastoris and phytase with potential industrial application was overexpressed. We also investigated the effects of the five disulfide bonds on the conformation and thermostability. The main results were as follows: 1. The phyA gene was cloned by PCR with the genomic DNA of A. niger N-J as template. We transformed phyA into P. pastoris and overexpressed phytase with potential industrial application. 2. Five expression plasmids of phytase mutants with deletion of each of the five disulfide bonds, respectively (C12S, C263S, C395S, C417S, C446S) were constructed. All the five mutants were expressed at high level in P. pastoris. 3. Thermostability analysis indicated that the deletion of any disulfide bonds made the conformation of the active-site fragile and easier to be changed. The deletion of disulfide bonds of Cys52—Cys395, Cys245—Cys263 and Cys417—Cys425 extremely decreased the thermostability. 4. Assay of the enzymological characteristics showed that all the five mutants hold high activity in the range of pH 2.5—5.5, which are similar to the stomach and intestines of animal. The deletion of disulfide bonds of Cys12—Cys21 S86
www.elsevier.com/locate/nbt
and Cys196—Cys446 decreased the optimum temperature slightly. doi:10.1016/j.nbt.2009.06.353
2.1.107 Detoxification and decolorization of raw textile effluents by a few marine fungi A. Verma National Institute of Oceanography, Panjim, India
Very few studies address the issue of bioremediation of dyecontaining textile effluents varying in their composition, toxicity, pH and salt content. We have used two marine-derived ascomycetes and two basidiomycetes fungi for treatment of raw dye-containing effluents, varying in their pHs and dye compositions. Textile effluent A (TEA) with pH of 8.9 containing azo dye and textile effluent B (TEB) with pH of 2.5 containing reactive dyes were decolorized 70—90% by these fungi. Effluents at 20, 50 and 90% concentrations were added to the culture medium for decolorization, reduction in COD and phenolics and for toxicity assays. A reduction of 80—90% in COD and total phenolics was brought about by these fungi by day 6. Toxicity assay using Artemia larvae showed two to fivefold reduction in toxicity of both the effluents. Both the effluents supported growth and laccase production in these fungi when grown in seawater medium with 15 ppt salinity. The fungal biomass could be used up to three cycles for decolorization of TEB. It can be concluded from this study that marine-derived ascomycetes fungi are capable of decolorization and detoxification of textile effluents, either by bioadsorption or laccase-mediated or by a concerted effect of these two processes, whereas in basidiomycetes laccase played a major role in these process. Mass spectrometric studies revealed that both the effluents were degraded to low molecular weight compounds. To study the actual pathway of degradation, individual component dyes of the effluents were also treated in the same way at two different concentrations of 300 and 3000 mg l−1 by one of the fungi. Mass spectrometric and NMR analyses were done with these dyes treated with the fungus to follow their degradation/biotransformation pathway. Bioremediation of these two effluents was effective by using immobilized laccase of one of these fungi in a bioreactor. doi:10.1016/j.nbt.2009.06.354
2.1.108 The new strains of Bacillus spp., potential biocontrol agent of mosquito M. Yazici ∗ , F. Sahin, A.U. Dogan, E. Kilic Yeditepe University, Istanbul, Turkey
Mosquitoes are vectors of many diseases such as Mosquito-borne arboviruses, malaria, filariasis and Japan encephalitis. Generally mosquito control was performed based on application of chemical pesticides in the World. All of the chemical pesticides have