The gene encoded for tryptophan decarboxylase (TDC), which is the key enzyme in terpenoil indole alkaloids pathway, was targeted to different subcellular compartments and stably expressed in transgenic tobacco (Nicoti...The gene encoded for tryptophan decarboxylase (TDC), which is the key enzyme in terpenoil indole alkaloids pathway, was targeted to different subcellular compartments and stably expressed in transgenic tobacco (Nicotiana tabacum L.) plants at the levels detected by Western blot and tryptamine accumulation analysis. It was shown that the TDC was located in subcellular compartments, the chloroplasts and cytosol. The recombinant TDC targeted to chloroplasts and cytosol in tobacco plants was effectively expressed as soluble protein by Western blot analysis and enzymatic assay. The level of tryptamine accumulation in chloroplast was higher than that in cytosol and very low in vacuole and endoplasmic reticulum (ER) to be hardly detected by Western blot analysis. It was indicated that the highest amount of tryptamine was in chloroplasts, lower in endoplasmic reticula and the lowest in vacuoles as compared to those in wild type plants. The TDC targeted to different subcellular compartments of tobacco plants and its expression level were studied by different nucleotide sequences coding signal peptides at 5'-end of tdc gene in order to know the effects of the TDC in compartmentation on its functionality.展开更多
Strictosidine synthase (STR) is a key enzyme involved in the biosynthesis of terpenoid indole alkaloids (TIA) by condensing tryptamine and secologanin into strictosidine. The transgenic tobacco plants targeting STR to...Strictosidine synthase (STR) is a key enzyme involved in the biosynthesis of terpenoid indole alkaloids (TIA) by condensing tryptamine and secologanin into strictosidine. The transgenic tobacco plants targeting STR to subcellular compartments were established to express STR in chloroplast, vacuole and endoplasmic reticulum (ER) by the tobacco stable transformation. It was shown that STR was effectively expressed in the above subcellular compartments by Western blot analysis and STR enzymatic assay. In vitro , STR enzymatic assay was measured indirectly by fluorimetrically detecting depletion of tryptamine feeding on secologanin in the reaction mixture. The tryptamine were completely depleted by STR in the crude extract of leaves of transgenic tobacco plants targeting and expressing STR in the chloroplast, vacuole and ER, which ascertained the STR functionally targeted to the three subcellular compartments. To confirm STR correct targeting and expressing in chloroplast, the chloroplasts were isolated and the fractions of purified chloroplasts were analyzed by Western blot. The hypothesis of STR correct targeting to the chloroplast was tested. The results have implications on our understanding of the complex intracellular trafficking in metabolic intermediates of TIA biosynthesis.展开更多
The enzymatic degradation of azo dyes is a promising alternative to ineffective chemical and physical remediation methods.Lignin peroxidase(LiP)from Phanerochaete chrysosporium is a hemecontaining lignin-degrading oxi...The enzymatic degradation of azo dyes is a promising alternative to ineffective chemical and physical remediation methods.Lignin peroxidase(LiP)from Phanerochaete chrysosporium is a hemecontaining lignin-degrading oxidoreductase that catalyzes the peroxide-dependent oxidation of diverse molecules,including industrial dyes.This enzyme is therefore ideal as a starting point for protein engineering.Accordingly,we subjected two positions(165 and 264)in the environment of the catalytic Trp171 residue to saturation mutagenesis,and the resulting library of 104 independent clones was expressed on the surface of yeast cells.This yeast display library was used for the selection of variants with the ability to break down structurally-distinct azo dyes more efficiently.We identified mutants with up to 10-fold greater affinity than wild-type LiP for three diverse azo dyes(Evans blue,amido black 10B and Guinea green)and up to 13-fold higher catalytic activity.Additionally,cell wall fragments displaying mutant LiP enzymes were prepared by toluene-induced cell lysis,achieving significant increases in both enzyme activity and stability compared to a whole-cell biocatalyst.LiPcoated cell wall fragments retained their initial dye degradation activity after 10 reaction cycles each lasting 8 h.The best-performing mutants removed up to 2.5-fold more of each dye than the wild-type LiP in multiple reaction cycles.展开更多
文摘The gene encoded for tryptophan decarboxylase (TDC), which is the key enzyme in terpenoil indole alkaloids pathway, was targeted to different subcellular compartments and stably expressed in transgenic tobacco (Nicotiana tabacum L.) plants at the levels detected by Western blot and tryptamine accumulation analysis. It was shown that the TDC was located in subcellular compartments, the chloroplasts and cytosol. The recombinant TDC targeted to chloroplasts and cytosol in tobacco plants was effectively expressed as soluble protein by Western blot analysis and enzymatic assay. The level of tryptamine accumulation in chloroplast was higher than that in cytosol and very low in vacuole and endoplasmic reticulum (ER) to be hardly detected by Western blot analysis. It was indicated that the highest amount of tryptamine was in chloroplasts, lower in endoplasmic reticula and the lowest in vacuoles as compared to those in wild type plants. The TDC targeted to different subcellular compartments of tobacco plants and its expression level were studied by different nucleotide sequences coding signal peptides at 5'-end of tdc gene in order to know the effects of the TDC in compartmentation on its functionality.
文摘Strictosidine synthase (STR) is a key enzyme involved in the biosynthesis of terpenoid indole alkaloids (TIA) by condensing tryptamine and secologanin into strictosidine. The transgenic tobacco plants targeting STR to subcellular compartments were established to express STR in chloroplast, vacuole and endoplasmic reticulum (ER) by the tobacco stable transformation. It was shown that STR was effectively expressed in the above subcellular compartments by Western blot analysis and STR enzymatic assay. In vitro , STR enzymatic assay was measured indirectly by fluorimetrically detecting depletion of tryptamine feeding on secologanin in the reaction mixture. The tryptamine were completely depleted by STR in the crude extract of leaves of transgenic tobacco plants targeting and expressing STR in the chloroplast, vacuole and ER, which ascertained the STR functionally targeted to the three subcellular compartments. To confirm STR correct targeting and expressing in chloroplast, the chloroplasts were isolated and the fractions of purified chloroplasts were analyzed by Western blot. The hypothesis of STR correct targeting to the chloroplast was tested. The results have implications on our understanding of the complex intracellular trafficking in metabolic intermediates of TIA biosynthesis.
基金supported by funds from the Ministry of Education,Science and Technological Development of the Republic of Serbia via project numbers ON172049,ON173017 and III46010.
文摘The enzymatic degradation of azo dyes is a promising alternative to ineffective chemical and physical remediation methods.Lignin peroxidase(LiP)from Phanerochaete chrysosporium is a hemecontaining lignin-degrading oxidoreductase that catalyzes the peroxide-dependent oxidation of diverse molecules,including industrial dyes.This enzyme is therefore ideal as a starting point for protein engineering.Accordingly,we subjected two positions(165 and 264)in the environment of the catalytic Trp171 residue to saturation mutagenesis,and the resulting library of 104 independent clones was expressed on the surface of yeast cells.This yeast display library was used for the selection of variants with the ability to break down structurally-distinct azo dyes more efficiently.We identified mutants with up to 10-fold greater affinity than wild-type LiP for three diverse azo dyes(Evans blue,amido black 10B and Guinea green)and up to 13-fold higher catalytic activity.Additionally,cell wall fragments displaying mutant LiP enzymes were prepared by toluene-induced cell lysis,achieving significant increases in both enzyme activity and stability compared to a whole-cell biocatalyst.LiPcoated cell wall fragments retained their initial dye degradation activity after 10 reaction cycles each lasting 8 h.The best-performing mutants removed up to 2.5-fold more of each dye than the wild-type LiP in multiple reaction cycles.
