A double mutantEst1, which is a plastic degrading cutinase-type esterase in Thermobifida alba, has been over-expressed in Escherichia coli. The recombinant protein was purified by a two-step protocol involving immobil...A double mutantEst1, which is a plastic degrading cutinase-type esterase in Thermobifida alba, has been over-expressed in Escherichia coli. The recombinant protein was purified by a two-step protocol involving immobilized metal affinity chromatography and cation-exchange chromatography, yielding 120 mg of protein per liter of bacterial culture. Crystals have been obtained by using the sitting-drop vapor-diffusion technique. Native diffraction data to 1.37 Åresolution were obtained at the BL44XU beam line of SPring-8 from a flash-frozen crystal at 100 K. The crystals belong to space group C2, with unit-cell parameters a = 127.2 Å, b = 42.1 Å, c = 63.2 Å, β = 114.7°, likely containing one Est1 double mutant (296 residues) per asymmetric unit.展开更多
Cutinases are hydrolytic enzymes used by phytopathogenic fungi to gain entry into plants by breaking down the cuticular barrier of higher plants. Cutinase displayed hydrolytic activity not only towards cutin, the main...Cutinases are hydrolytic enzymes used by phytopathogenic fungi to gain entry into plants by breaking down the cuticular barrier of higher plants. Cutinase displayed hydrolytic activity not only towards cutin, the main component of the plant cuticle, but also towards a variety of soluble synthetic esters, insoluble triglycerides and polyesters. Therefore, cutinase was evaluated for use in the chemical, food and cotton bio-scouring industry and for synthetic fibers modification. This research examined the production and purification of extracellular cutinase from the phytopathogenic fungus Fusarium oxysporum. The addition of apple cutin or its hydrolysate to the fungus growth medium resulted in an enhanced secretion of cutinase into the extracellular fluid. Testing 1-hexadecanol as an alternative to natural cutin to induce cutinase production resulted in a high process yield under modified growth conditions. Cutinase enzyme production was followed by an optimized purification method for enzyme preparation using high-performance liquid chromatography and high-specificity 4-nitrophenyl (16-methyl sulfide ester) hexadecanoate (pNMSEH) cutinase substrate. Electrophoresis sodiumdodecyl sulfate-polyacrylamide and isoelectric focusing gels enabled the final separation and identification of the protein. The purified cutinase had an approximate molecular weight of 20 kDa and an isoelectric point of 4.7. The method presented here could be modified and used for cutinase production and purification in other microorganisms that exhibit cutinolytic activity.展开更多
The development of medium for the production of cutinase from Pseudomonas cepacia NRRL B 2320 was carried out using Plackett-Burman experimental design followed by central composite design. The medium components were ...The development of medium for the production of cutinase from Pseudomonas cepacia NRRL B 2320 was carried out using Plackett-Burman experimental design followed by central composite design. The medium components were screened by Plackett-Burman experimental design which suggested that cutin, peptone, KCl and MgSO4·7H2O have influenced the cutinase production significantly with very high confidence levels. The concentration levels of these four components were optimized using 24 full factorial central composite design. An optimum combination of 10.06 g·L-1 of cutin, 17.77 g·L-1 of peptone, 0.635 g·L-1 of KCl and 5.455 g·L-1 of MgSO4·7H2O in the medium gave a maximum cutinase activity of 336 U·mL-1. An overall 2 fold increase in the production of cutinase was observed in the optimized medium. Growth and production of cutinase from P. cepacia NRRL B 2320 have been studied in shake flask and batch bioreactor. Time course of cell growth and enzyme production was fitted to the existing kinetic models reported in the literature to estimate the biokinetic parameters. These models suggested that the production of cutinase is growth associated in shake flask and it is a mixed growth type in a batch bioreactor.展开更多
Thermostability of two homologous cutinases, Cut1 and Cut2 from Thermobifida fusca NRRL B-8184 was inves-tigated at combination of different pH and temperature in the range of pH 6 - 9 and temperature 45℃ - 80℃, re-...Thermostability of two homologous cutinases, Cut1 and Cut2 from Thermobifida fusca NRRL B-8184 was inves-tigated at combination of different pH and temperature in the range of pH 6 - 9 and temperature 45℃ - 80℃, re-spectively. The deactivation rate constants, the half-life and thermodynamic parameters, viz., △H*, △S*, △G* and activation energy kinetics of inactivation of the cutinases were assessed at different combinations of pH and temperature and compared. The optimal pH and temperature for the least degree of deactivation for Cut1 and Cut2 were found to be 8℃ and 45℃, respectively. The deactivation process was found to be faster at pH 6 and 9, with minimum deactivation at pH 8 for both the cutinases. It was found that △S* values are negative for both the enzymes and △H* value of Cut2 was 1.5 fold higher than that of Cut1 in the range of pH studied. Cut2 was found to be thermodynamically more stable with 1.7 fold higher deactivation energy at pH 6 and 7 and 1.4 fold higher deactivation energy at pH 8 and 9 in comparison to Cut1.展开更多
Fusarium verticillioides is an important pathogen of maize and causes serious yield losses and food safety issues worldwide.F.verticillioides produces highly toxic mycotoxin Fumonisin B1(FB1)in infested commodities wh...Fusarium verticillioides is an important pathogen of maize and causes serious yield losses and food safety issues worldwide.F.verticillioides produces highly toxic mycotoxin Fumonisin B1(FB1)in infested commodities which makes these food and feeds unsafe for humans and animals.For pathogenic fungi to successfully penetrate its plant hosts,the pathogen secretes hydrolytic enzymes that can facilitate penetration into the plant cutin layer.However,there is limited information on how cutinases transcriptionally regulated to impact F.verticillioides pathogenicity.In this study,our aim is to functionally characterize cutinase transcription factors that regulate key cutinase activities that are directly associated with F.verticillioides pathogenicity and FB1 biosynthesis.Gene deletion of cutinase transcription factor FvCTF1αdid not affect the growth and morphology of the fungal mycelia on CMII medium,whereas the conidiation,utilization of sodium acetate and sodium oleate,stress tolerance against cell wall interfering agent,and the cutinase and pectinase activities in theΔFvctf1αmutant were negatively impacted.FvCtf1αregulates the expression of induced cutinase genes FvCUT1 and FvCUT4 by binding to their GC-rich promoters.In addition,FvCtf1α,containing a novel function in regulating FB1,interacts with the promoter of FvFUM1 and FvFUM6 to down-regulate the expression of FvFUM1 and FvFUM6,resulting in decreased production of FB1 in theΔFvctf1αstrain.ΔFvctf1αexhibited decreased pathogenicity in maize due to the down-regulation of pathogenicity-related genes as well as key downstream cutinase genes FvCUT3 and FvCUT4 in F.verticillioides.We also demonstrated that FvCtf1αregulated FvCUT3 and FvCUT4 differently;FvCUT4 via direct regulation while FvCUT3 via indirect regulation by interacting with FvFarB,a homologous protein of FvCtf1α.Moreover,RNA-seq analysis showed that FvCtf1αwas associated with many pathways,such as fatty acid metabolism,carbon source utilization,cell wall integrity,oxidative stress,and fumonisin synthesis in F.verticillioides.Our study demonstrated that FvCtf1αwas not only involved in the regulation of cutinases but also a broad spectrum of pathways that ultimately affect F.verticillioides virulence and mycotoxin biosynthesis.展开更多
Although the efficiency of poly(ethylene terephthalate)(PET)degradation has been successfully improved by depolymerase engineering,mostly by using Goodfellow-PET(gf-PET)as a substrate,efforts to degrade unpretreated P...Although the efficiency of poly(ethylene terephthalate)(PET)degradation has been successfully improved by depolymerase engineering,mostly by using Goodfellow-PET(gf-PET)as a substrate,efforts to degrade unpretreated PET materials with high crystallinity remain insufficient.Here,we endeavored to improve the degradation capability of a WCCG mutant of leaf-branch compost cutinase(LCC)on a unpretreated PET substrate(crystallinity>40%)by employing iterative saturation mutagenesis.Using this method,we developed a high-throughput screening strategy appropriate for unpretreated substrates.Through extensive screening of residues around the substrate-binding groove,two variants,WCCG-sup1 and WCCG-sup2,showed good depolymerization capabilities with both high-(42%)and low-crystallinity(9%)substrates.The WCCG-sup1 variant completely depolymerized a commercial unpretreated PET product in 36 h at 72℃.In addition to enzyme thermostability and catalytic efficiency,the adsorption of enzymes onto substrates plays an important role in PET degradation.This study provides valuable insights into the structure-function relationship of LCC.展开更多
文摘A double mutantEst1, which is a plastic degrading cutinase-type esterase in Thermobifida alba, has been over-expressed in Escherichia coli. The recombinant protein was purified by a two-step protocol involving immobilized metal affinity chromatography and cation-exchange chromatography, yielding 120 mg of protein per liter of bacterial culture. Crystals have been obtained by using the sitting-drop vapor-diffusion technique. Native diffraction data to 1.37 Åresolution were obtained at the BL44XU beam line of SPring-8 from a flash-frozen crystal at 100 K. The crystals belong to space group C2, with unit-cell parameters a = 127.2 Å, b = 42.1 Å, c = 63.2 Å, β = 114.7°, likely containing one Est1 double mutant (296 residues) per asymmetric unit.
文摘Cutinases are hydrolytic enzymes used by phytopathogenic fungi to gain entry into plants by breaking down the cuticular barrier of higher plants. Cutinase displayed hydrolytic activity not only towards cutin, the main component of the plant cuticle, but also towards a variety of soluble synthetic esters, insoluble triglycerides and polyesters. Therefore, cutinase was evaluated for use in the chemical, food and cotton bio-scouring industry and for synthetic fibers modification. This research examined the production and purification of extracellular cutinase from the phytopathogenic fungus Fusarium oxysporum. The addition of apple cutin or its hydrolysate to the fungus growth medium resulted in an enhanced secretion of cutinase into the extracellular fluid. Testing 1-hexadecanol as an alternative to natural cutin to induce cutinase production resulted in a high process yield under modified growth conditions. Cutinase enzyme production was followed by an optimized purification method for enzyme preparation using high-performance liquid chromatography and high-specificity 4-nitrophenyl (16-methyl sulfide ester) hexadecanoate (pNMSEH) cutinase substrate. Electrophoresis sodiumdodecyl sulfate-polyacrylamide and isoelectric focusing gels enabled the final separation and identification of the protein. The purified cutinase had an approximate molecular weight of 20 kDa and an isoelectric point of 4.7. The method presented here could be modified and used for cutinase production and purification in other microorganisms that exhibit cutinolytic activity.
文摘The development of medium for the production of cutinase from Pseudomonas cepacia NRRL B 2320 was carried out using Plackett-Burman experimental design followed by central composite design. The medium components were screened by Plackett-Burman experimental design which suggested that cutin, peptone, KCl and MgSO4·7H2O have influenced the cutinase production significantly with very high confidence levels. The concentration levels of these four components were optimized using 24 full factorial central composite design. An optimum combination of 10.06 g·L-1 of cutin, 17.77 g·L-1 of peptone, 0.635 g·L-1 of KCl and 5.455 g·L-1 of MgSO4·7H2O in the medium gave a maximum cutinase activity of 336 U·mL-1. An overall 2 fold increase in the production of cutinase was observed in the optimized medium. Growth and production of cutinase from P. cepacia NRRL B 2320 have been studied in shake flask and batch bioreactor. Time course of cell growth and enzyme production was fitted to the existing kinetic models reported in the literature to estimate the biokinetic parameters. These models suggested that the production of cutinase is growth associated in shake flask and it is a mixed growth type in a batch bioreactor.
文摘Thermostability of two homologous cutinases, Cut1 and Cut2 from Thermobifida fusca NRRL B-8184 was inves-tigated at combination of different pH and temperature in the range of pH 6 - 9 and temperature 45℃ - 80℃, re-spectively. The deactivation rate constants, the half-life and thermodynamic parameters, viz., △H*, △S*, △G* and activation energy kinetics of inactivation of the cutinases were assessed at different combinations of pH and temperature and compared. The optimal pH and temperature for the least degree of deactivation for Cut1 and Cut2 were found to be 8℃ and 45℃, respectively. The deactivation process was found to be faster at pH 6 and 9, with minimum deactivation at pH 8 for both the cutinases. It was found that △S* values are negative for both the enzymes and △H* value of Cut2 was 1.5 fold higher than that of Cut1 in the range of pH studied. Cut2 was found to be thermodynamically more stable with 1.7 fold higher deactivation energy at pH 6 and 7 and 1.4 fold higher deactivation energy at pH 8 and 9 in comparison to Cut1.
基金National Natural Science Foundation of China(32272516)Open project of Fujian Provincial Key Laboratory of Crop Pest Monitoring and Control(MIMCP-202103).
文摘Fusarium verticillioides is an important pathogen of maize and causes serious yield losses and food safety issues worldwide.F.verticillioides produces highly toxic mycotoxin Fumonisin B1(FB1)in infested commodities which makes these food and feeds unsafe for humans and animals.For pathogenic fungi to successfully penetrate its plant hosts,the pathogen secretes hydrolytic enzymes that can facilitate penetration into the plant cutin layer.However,there is limited information on how cutinases transcriptionally regulated to impact F.verticillioides pathogenicity.In this study,our aim is to functionally characterize cutinase transcription factors that regulate key cutinase activities that are directly associated with F.verticillioides pathogenicity and FB1 biosynthesis.Gene deletion of cutinase transcription factor FvCTF1αdid not affect the growth and morphology of the fungal mycelia on CMII medium,whereas the conidiation,utilization of sodium acetate and sodium oleate,stress tolerance against cell wall interfering agent,and the cutinase and pectinase activities in theΔFvctf1αmutant were negatively impacted.FvCtf1αregulates the expression of induced cutinase genes FvCUT1 and FvCUT4 by binding to their GC-rich promoters.In addition,FvCtf1α,containing a novel function in regulating FB1,interacts with the promoter of FvFUM1 and FvFUM6 to down-regulate the expression of FvFUM1 and FvFUM6,resulting in decreased production of FB1 in theΔFvctf1αstrain.ΔFvctf1αexhibited decreased pathogenicity in maize due to the down-regulation of pathogenicity-related genes as well as key downstream cutinase genes FvCUT3 and FvCUT4 in F.verticillioides.We also demonstrated that FvCtf1αregulated FvCUT3 and FvCUT4 differently;FvCUT4 via direct regulation while FvCUT3 via indirect regulation by interacting with FvFarB,a homologous protein of FvCtf1α.Moreover,RNA-seq analysis showed that FvCtf1αwas associated with many pathways,such as fatty acid metabolism,carbon source utilization,cell wall integrity,oxidative stress,and fumonisin synthesis in F.verticillioides.Our study demonstrated that FvCtf1αwas not only involved in the regulation of cutinases but also a broad spectrum of pathways that ultimately affect F.verticillioides virulence and mycotoxin biosynthesis.
文摘Although the efficiency of poly(ethylene terephthalate)(PET)degradation has been successfully improved by depolymerase engineering,mostly by using Goodfellow-PET(gf-PET)as a substrate,efforts to degrade unpretreated PET materials with high crystallinity remain insufficient.Here,we endeavored to improve the degradation capability of a WCCG mutant of leaf-branch compost cutinase(LCC)on a unpretreated PET substrate(crystallinity>40%)by employing iterative saturation mutagenesis.Using this method,we developed a high-throughput screening strategy appropriate for unpretreated substrates.Through extensive screening of residues around the substrate-binding groove,two variants,WCCG-sup1 and WCCG-sup2,showed good depolymerization capabilities with both high-(42%)and low-crystallinity(9%)substrates.The WCCG-sup1 variant completely depolymerized a commercial unpretreated PET product in 36 h at 72℃.In addition to enzyme thermostability and catalytic efficiency,the adsorption of enzymes onto substrates plays an important role in PET degradation.This study provides valuable insights into the structure-function relationship of LCC.
