[Objective] This study aimed to investigate the enzymatic kinetics of polyphenol oxidase in peel of Nephelium lappaceum to explore the environmental factors affecting its catalytic activity. [ Method ] With N. lappace...[Objective] This study aimed to investigate the enzymatic kinetics of polyphenol oxidase in peel of Nephelium lappaceum to explore the environmental factors affecting its catalytic activity. [ Method ] With N. lappaceum peel as the experimental material and catechol as substrate, effects of temperature, pH, four inhibitors (EDTA, Vc, sodium bisulfite and citric acid) and substrate concentration on the activity of polyphenol oxidase were investigated. [ Result] The optimal temperature for polyphenol oxidase in N. lappaceum peel was 40 %, and the optimal pH was 6.8. EDTA, Vc, sodium bisulfite and citric acid all showed ideal in- hibitory effects on the activity of polyphenol oxidase; specifically, EDTA had the strongest inhibitory effect. [ Conclusion] At low temperature, EDTA, Vc, sodi- um bisulflte and citric acid with certain concentrations can inhibit the catalytic activity of polyphenol oxidase in peel of N. lappaceum, extend the storage and trans- portation time of N. lappaceum fruit, and inhibit the browning. This study provides theoretical basis for the development and utilization of polyphenol oxidase in peel of N. lappaceum.展开更多
β-Conglycinin,the main protein of soybean,is a key allergen that causes soybean allergies,and hydrolysis is usually applied to lower its antigenicity.We evaluated the enzymolysis characters ofβ-conglycinin from the ...β-Conglycinin,the main protein of soybean,is a key allergen that causes soybean allergies,and hydrolysis is usually applied to lower its antigenicity.We evaluated the enzymolysis characters ofβ-conglycinin from the perspective of enzymolysis kinetics using alkaline protease from B.subtilis ACCC 01746.A dynamic model describing the hydrolysis ofβ-conglycinin was proposed using the initial substrate concentration,enzyme dosage(enzyme to substrate ratio)and hydrolysis time as variables to illustrate the kinetic behavior of enzymatic hydrolysis.The hydrolysis of soybeanβ-conglycinin was carried out at 60 g/L protein concentration,0.6%enzyme dosage,55℃ and pH 8.5 to observe the peptides with anti-enzymatic activities.The hydrolysates were gradually fractionated by ultrafiltration through cut-off membranes with molecular weights of 40,30,20,and 10 kDa,and their antigenicities were evaluated using indirect competitive enzyme-linked immunosorbent assay.The results showed that the degree of hydrolysis(DH)ofβ-conglycinin decreased as theβ-conglycinin concentration(S0)increased,but increased with enzyme dosage(E0)increasing.Thus,the enzymatic hydrolysis ofβ-conglycinin followed the first-order kinetics model.The hydrolysis rate(V)was(527.89C_(E0)-2.5533C_(S0))exp(-0.022DH),the DH-hydrolysis time was 45.454ln[1+(11.614C_(E0)/C_(S0)-0.0562)t],and the correlated kinetic constants k2 and kd were 527.89 min^(−1)and 8.6126 min^(−1),respectively.The hydrolysis behavior ofβ-conglycinin varied considerably among theα',α,andβsubunits.Faster hydrolysis rates were observed for theα'andαsubunits compared to theβsubunit.The relative molecular weights of the intercepted peptides from the hydrolysates were 14.8-40.1 kDa,and the antigenicity of the peptides with smaller molecular weight was reduced,but not removed completely.However,the alkaline protease from the strain appeared to effectively reduce the allergenicity ofβ-conglycinin.Therefore,it is possible to produce less allergenic soybean proteins using enzymatic hydrolysis.Additionally,the microbial alkaline protease may serve as a potential novel food enzyme and should be evaluated for the development of hypoallergenic foods.展开更多
Enzymatic synthesis of ethyl hexanoate from hexanoic acid and ethanol in a solvent-free system, carried out at optimum conditions, has been modeled. The process follows a Ping-Pong Bi-Bi reaction rate and it is endoth...Enzymatic synthesis of ethyl hexanoate from hexanoic acid and ethanol in a solvent-free system, carried out at optimum conditions, has been modeled. The process follows a Ping-Pong Bi-Bi reaction rate and it is endothermic, so microwave heating is provided to prevent its shutdown. The mathematical model has been numerically solved with the FEM method. Results show that microwave heating can be successfully used to carry out this kind of reaction.展开更多
Abstract: Cis-cinnamic acid (CA) is a naturally occurring compound, presumably converted from trans-CA in higher plants. To investigate the effect of cis-CA on the activity of Arabidopsis phenylalanine ammonia lyase (...Abstract: Cis-cinnamic acid (CA) is a naturally occurring compound, presumably converted from trans-CA in higher plants. To investigate the effect of cis-CA on the activity of Arabidopsis phenylalanine ammonia lyase (PAL), AtPAL1, AtPAL2, and AtPAL4 genes were isolated using reverse transcription poly-merase chain reaction. These genes were fused to a glutathione S-transferase gene and overexpressed in a heterologous prokaryotic system of Escherichia coli. The purified PAL1, PAL2 and PAL4 enzymes were characterized biochemically to determine the effects of cis-CA on the kinetic parameter Km. The results showed that cis-CA is a competitive inhibitor for PAL1, but not PAL2 and PAL4, whereas trans-CA acts as a competitive inhibitor for all three PAL isomers, suggesting that cis- and trans-CA have different effects on the catalytic activity of PAL.展开更多
4-Coumarate : coenzyme A Ilgase (4CL) Is one of the key enzymes In phenylpropanoid metabolism leading to series of phenollcs, Including water-soluble phenolic acids, which are important compounds determining the me...4-Coumarate : coenzyme A Ilgase (4CL) Is one of the key enzymes In phenylpropanoid metabolism leading to series of phenollcs, Including water-soluble phenolic acids, which are important compounds determining the medicinal quality of Danshen (Salvia miltiorrhiza Bunge), a traditional Chinese medicinal herb. To Investigate the function of 4CL in the biosynthesis of water-soluble phenolic acid in Danshen, we have cloned two cDNAs (Sm4CL1 and Sm4CL2) encoding divergent 4CL members by applying nested reverse transcrlptlon-polymerase chain reaction (RT-PCR) with degenerate primers followed by 5′/3′rapid amplification of cDNA ends (RACE) (Note, these sequence data have been submitted to the GenBank database under accession numbers AY237163 and AY237164). Either of the coding regions was inserted into a pRSET vector and a kinetic assay was performed with purified recombinant proteins. The substrate utilization profile of Sm4CL1 was distinct from that of Sm4CL2. The Km values of Sm4CL1 and Sm4CL2 to 4-coumarlc acid were (72.20±4.10) and (6.50±1.45) μmol/L, respectively. These results, In conjunction with Northern blotting and other information, imply that Sm4CL2 may play an Important role in the biosynthesis of watersoluble phenolic compounds, whereas Sm4CL1 may play a minor role in the pathway. Southern blotting analysis suggested that both Sm4CL1 and Sm4CL2 genes are present as a single copy and are located at different sites In the genome.展开更多
基金Supported by Leading Academic Discipline Project of Botany in Guizhou Province ([2011] No.275)College-level Planning Project of Kaili University(Z0804)+1 种基金Leading Academic Discipline Project of Botany from Kaili University (KZD2008002)Construction Project for Basic Course Teaching Team of Biological Science from Kaili University (JXTD201003)
文摘[Objective] This study aimed to investigate the enzymatic kinetics of polyphenol oxidase in peel of Nephelium lappaceum to explore the environmental factors affecting its catalytic activity. [ Method ] With N. lappaceum peel as the experimental material and catechol as substrate, effects of temperature, pH, four inhibitors (EDTA, Vc, sodium bisulfite and citric acid) and substrate concentration on the activity of polyphenol oxidase were investigated. [ Result] The optimal temperature for polyphenol oxidase in N. lappaceum peel was 40 %, and the optimal pH was 6.8. EDTA, Vc, sodium bisulfite and citric acid all showed ideal in- hibitory effects on the activity of polyphenol oxidase; specifically, EDTA had the strongest inhibitory effect. [ Conclusion] At low temperature, EDTA, Vc, sodi- um bisulflte and citric acid with certain concentrations can inhibit the catalytic activity of polyphenol oxidase in peel of N. lappaceum, extend the storage and trans- portation time of N. lappaceum fruit, and inhibit the browning. This study provides theoretical basis for the development and utilization of polyphenol oxidase in peel of N. lappaceum.
基金Authors wish to thank to Grain&Corn Engineering Technology Research Center,State Administration of Grain(GA2017004)for funding support.
文摘β-Conglycinin,the main protein of soybean,is a key allergen that causes soybean allergies,and hydrolysis is usually applied to lower its antigenicity.We evaluated the enzymolysis characters ofβ-conglycinin from the perspective of enzymolysis kinetics using alkaline protease from B.subtilis ACCC 01746.A dynamic model describing the hydrolysis ofβ-conglycinin was proposed using the initial substrate concentration,enzyme dosage(enzyme to substrate ratio)and hydrolysis time as variables to illustrate the kinetic behavior of enzymatic hydrolysis.The hydrolysis of soybeanβ-conglycinin was carried out at 60 g/L protein concentration,0.6%enzyme dosage,55℃ and pH 8.5 to observe the peptides with anti-enzymatic activities.The hydrolysates were gradually fractionated by ultrafiltration through cut-off membranes with molecular weights of 40,30,20,and 10 kDa,and their antigenicities were evaluated using indirect competitive enzyme-linked immunosorbent assay.The results showed that the degree of hydrolysis(DH)ofβ-conglycinin decreased as theβ-conglycinin concentration(S0)increased,but increased with enzyme dosage(E0)increasing.Thus,the enzymatic hydrolysis ofβ-conglycinin followed the first-order kinetics model.The hydrolysis rate(V)was(527.89C_(E0)-2.5533C_(S0))exp(-0.022DH),the DH-hydrolysis time was 45.454ln[1+(11.614C_(E0)/C_(S0)-0.0562)t],and the correlated kinetic constants k2 and kd were 527.89 min^(−1)and 8.6126 min^(−1),respectively.The hydrolysis behavior ofβ-conglycinin varied considerably among theα',α,andβsubunits.Faster hydrolysis rates were observed for theα'andαsubunits compared to theβsubunit.The relative molecular weights of the intercepted peptides from the hydrolysates were 14.8-40.1 kDa,and the antigenicity of the peptides with smaller molecular weight was reduced,but not removed completely.However,the alkaline protease from the strain appeared to effectively reduce the allergenicity ofβ-conglycinin.Therefore,it is possible to produce less allergenic soybean proteins using enzymatic hydrolysis.Additionally,the microbial alkaline protease may serve as a potential novel food enzyme and should be evaluated for the development of hypoallergenic foods.
文摘Enzymatic synthesis of ethyl hexanoate from hexanoic acid and ethanol in a solvent-free system, carried out at optimum conditions, has been modeled. The process follows a Ping-Pong Bi-Bi reaction rate and it is endothermic, so microwave heating is provided to prevent its shutdown. The mathematical model has been numerically solved with the FEM method. Results show that microwave heating can be successfully used to carry out this kind of reaction.
文摘Abstract: Cis-cinnamic acid (CA) is a naturally occurring compound, presumably converted from trans-CA in higher plants. To investigate the effect of cis-CA on the activity of Arabidopsis phenylalanine ammonia lyase (PAL), AtPAL1, AtPAL2, and AtPAL4 genes were isolated using reverse transcription poly-merase chain reaction. These genes were fused to a glutathione S-transferase gene and overexpressed in a heterologous prokaryotic system of Escherichia coli. The purified PAL1, PAL2 and PAL4 enzymes were characterized biochemically to determine the effects of cis-CA on the kinetic parameter Km. The results showed that cis-CA is a competitive inhibitor for PAL1, but not PAL2 and PAL4, whereas trans-CA acts as a competitive inhibitor for all three PAL isomers, suggesting that cis- and trans-CA have different effects on the catalytic activity of PAL.
基金Supported by the National Natural Science Foundation of China (30300447).The authors thank Dr Chen Yongning (China Innovation Centre for Drug Development, HK) for useful suggestions and support. The authors also thank to Dr Fanya Zeng and Miss Charis Chan (Department of Zoology, University of Hong Kong) for technical assistance.
文摘4-Coumarate : coenzyme A Ilgase (4CL) Is one of the key enzymes In phenylpropanoid metabolism leading to series of phenollcs, Including water-soluble phenolic acids, which are important compounds determining the medicinal quality of Danshen (Salvia miltiorrhiza Bunge), a traditional Chinese medicinal herb. To Investigate the function of 4CL in the biosynthesis of water-soluble phenolic acid in Danshen, we have cloned two cDNAs (Sm4CL1 and Sm4CL2) encoding divergent 4CL members by applying nested reverse transcrlptlon-polymerase chain reaction (RT-PCR) with degenerate primers followed by 5′/3′rapid amplification of cDNA ends (RACE) (Note, these sequence data have been submitted to the GenBank database under accession numbers AY237163 and AY237164). Either of the coding regions was inserted into a pRSET vector and a kinetic assay was performed with purified recombinant proteins. The substrate utilization profile of Sm4CL1 was distinct from that of Sm4CL2. The Km values of Sm4CL1 and Sm4CL2 to 4-coumarlc acid were (72.20±4.10) and (6.50±1.45) μmol/L, respectively. These results, In conjunction with Northern blotting and other information, imply that Sm4CL2 may play an Important role in the biosynthesis of watersoluble phenolic compounds, whereas Sm4CL1 may play a minor role in the pathway. Southern blotting analysis suggested that both Sm4CL1 and Sm4CL2 genes are present as a single copy and are located at different sites In the genome.