L-Amino acid deaminase(LAAD) is a key enzyme in the deamination of L-valine(L-val) to produce α-ketoisovalerate(KIV). However, the product inhibition of LAAD is a major hindrance to industrial KIV production.In the p...L-Amino acid deaminase(LAAD) is a key enzyme in the deamination of L-valine(L-val) to produce α-ketoisovalerate(KIV). However, the product inhibition of LAAD is a major hindrance to industrial KIV production.In the present study, a combination strategy of modification of flexible loop regions around the product binding site and the avoidance of dramatic change of main-chain dynamics was reported to reduce the product inhibition.The four mutant PM-LAAD^(M4)(PM-LAAD^(S98A/T105A/S106A/L341A)) achieved a 6.2-fold higher catalytic efficiency and an almost 6.7-fold reduction in product inhibition than the wild-type enzyme. Docking experiments suggested that weakened interactions between the product and enzyme, and the flexibility of the "lid" structure relieved LAAD product inhibition. Finally, the whole-cell biocatalyst PM-LAAD^(M4) has been applied to KIV production,the titer and conversion rate of KIV from L-val were 98.5 g·L^-1 and 99.2% at a 3-L scale, respectively. These results demonstrate that the newly engineered catalyst can significantly reduce the product inhibition, that making KIV a prospective product by bioconversion method, and also provide the understanding of the mechanism of the relieved product inhibition of PM-LAAD.展开更多
A series of novel L-amino acid esters prodrugs of acyclic nucleoside phosphonates was synthesized and their anti-HBV activity was evaluated in HepG2 2.2.15 cells. Compound 1d exhibited more potent anti-HBV activity an...A series of novel L-amino acid esters prodrugs of acyclic nucleoside phosphonates was synthesized and their anti-HBV activity was evaluated in HepG2 2.2.15 cells. Compound 1d exhibited more potent anti-HBV activity and lower cytotoxicity than those of adefovir dipivoxil with EC50 and CC50 values of 0.207 μmol/L and 2530 μmol/L, respectively.展开更多
Monoamine neurotransmitters include catecholamines and indoleamines. The most common catecholamines are do- pamine （DA）, noradrenaline （NA） and adrenaline, which are produced from phenylalanine and tyrosine; where...Monoamine neurotransmitters include catecholamines and indoleamines. The most common catecholamines are do- pamine （DA）, noradrenaline （NA） and adrenaline, which are produced from phenylalanine and tyrosine; whereas the most common indoleamine is serotonin （5-hydroxytrypta mine, 5-HT）, which is produced from 5-hydroxytryptophan （5-HTP）.展开更多
4-Hydroxyphenylpyruvic acid （4-HPPA）, a kind of α-keto acid, is an intermediate in the metabolism of tyrosine and has a wide range of application in food, pharmaceutical and chemical industry. Using amino acids as ...4-Hydroxyphenylpyruvic acid （4-HPPA）, a kind of α-keto acid, is an intermediate in the metabolism of tyrosine and has a wide range of application in food, pharmaceutical and chemical industry. Using amino acids as raw material to prod uce the corresponding α-keto acid is thought to be both economic and efficient. Among the enzymes that convert amino acid to α-keto acid, membrane bound L-amino acid deaminase （mL-AAD）, which is anchored to the outer side of the cytomembrane, becomes an ideal enzyme to prepare α-keto acid since there is no cofactors needed and H2O2 production during the reaction. In this study, the mL-AAD from Proteus vulgaris was used to prepare whole-cell catalysts to produce 4-HPPA from L-tyrosine. The secretory efficiency of mL-AAD conducted by its own twin-arginine signal peptide （twin-arginine translocation pathway, Tat） and integrated pelB （the general secretory pathway, Sec）-Tat signal peptide was determined and compared firstly, using two pET systems （pET28a and pET20b）. It was found that the Tat pathway （pET28a-mlaad） resulted in higher cell-associated mL-AAD activity and cell biomass, and was more beneficial to prepare biocatalyst. In addition, expression hosts BI21 （DE3） and 0.05 mmol. L- 1 IPTG were found to be suitable for mL-AAD expression. The reaction conditions for mL-AAD were optimized and 72.72 mmol,L 1 4-HPPA was obtained from 100 mmol.L 1 tyrosine in 10 h under the optimized conditions. This bioprocess, which is more eco-friendly and economical than the traditional chemical synthesis ways, has great potential for industrial application.展开更多
A new composite chiral liquid crystal containing L-amino acid derivatives was prepared and their liquid crystal properties were investigated. Results show that they could exhibit liquid crystal through hydrogen bonding.
A series of novel mono（2,2,2-trifluoroethyl） esters,mono L-amino acid ester prodrugs of acyclic nucleoside phosphonates was synthesized and their in vitro anti-HBVactivity was evaluated in HepG 2 2.2.15 cells.Compou...A series of novel mono（2,2,2-trifluoroethyl） esters,mono L-amino acid ester prodrugs of acyclic nucleoside phosphonates was synthesized and their in vitro anti-HBVactivity was evaluated in HepG 2 2.2.15 cells.Compound 1d exhibited more potent anti-HBV activity and lower cytotoxicity than those of adefovir dipivoxil and alamifovir（MCC-478） with EC_（50） and CC_（50） values of 0.01μmol/L and 8000μmol/L respectively.展开更多
为挖掘油莎豆匍匐茎形成茎豆过程中的关键代谢物质,调控匍匐茎形成更多的茎豆,以匍匐茎及新茎豆为研究对象,利用高效液相色谱联合三重四极杆质谱仪进行广泛靶向代谢组学分析,采用多元统计学方法挖掘与茎豆形成相关的差异代谢物质,并富...为挖掘油莎豆匍匐茎形成茎豆过程中的关键代谢物质,调控匍匐茎形成更多的茎豆,以匍匐茎及新茎豆为研究对象,利用高效液相色谱联合三重四极杆质谱仪进行广泛靶向代谢组学分析,采用多元统计学方法挖掘与茎豆形成相关的差异代谢物质,并富集差异代谢物相关的KEGG代谢通路。结果表明,未膨大匍匐茎(PFJ1)、膨大匍匐茎(PFJ2)及新茎豆(JD1)3组样本中共检测到1 217个代谢物。按照变量投影重要度(VIP)>1、P<0.05且差异倍数(FC)>2或FC<0.5的条件进行差异代谢物筛选,JD1 vs PFJ1筛选到35个差异代谢物,其中26个上调表达,9个下调表达;JD1 vs PFJ2筛选到57个差异代谢物,其中50个上调表达,7个下调表达;PFJ2 vs PFJ1筛选到39个差异代谢物,其中3个上调表达,36个下调表达。利用数据库对代谢物进行定性分析,差异表达代谢物主要被鉴定为氨基酸及其衍生物、核苷酸及其衍生物、有机氧化合物、黄酮类、苯丙素类、酚类等。差异表达代谢物相对含量分析显示,JD1谷胱甘肽含量是PFJ1的14.021倍、PFJ2的29.195倍;JD1 3-甲基组氨酸含量是PFJ1的10.463倍、PFJ2的129.931倍;JD1山柰酚含量是PFJ1的0.421倍、PFJ2的0.358倍。进一步的KEGG通路分析显示,差异表达代谢物主要富集到氨酰-tRNA生物合成,精氨酸和脯氨酸代谢,丙氨酸、天冬氨酸及谷氨酸代谢,苯丙氨酸代谢,黄酮及黄酮醇生物合成等代谢通路。综上,与油莎豆茎豆形成相关的代谢物质主要为氨基酸类及黄酮类物质;代谢通路主要集中在氨酰-tRNA生物合成及黄酮和黄酮醇生物合成途径中。展开更多
基金financially supported by the national first-class discipline program of Light Industry Technology and Engineering(LITE201820)the Key Technologies Research and Development Program of Jiangsu Province(BE2018623)。
文摘L-Amino acid deaminase(LAAD) is a key enzyme in the deamination of L-valine(L-val) to produce α-ketoisovalerate(KIV). However, the product inhibition of LAAD is a major hindrance to industrial KIV production.In the present study, a combination strategy of modification of flexible loop regions around the product binding site and the avoidance of dramatic change of main-chain dynamics was reported to reduce the product inhibition.The four mutant PM-LAAD^(M4)(PM-LAAD^(S98A/T105A/S106A/L341A)) achieved a 6.2-fold higher catalytic efficiency and an almost 6.7-fold reduction in product inhibition than the wild-type enzyme. Docking experiments suggested that weakened interactions between the product and enzyme, and the flexibility of the "lid" structure relieved LAAD product inhibition. Finally, the whole-cell biocatalyst PM-LAAD^(M4) has been applied to KIV production,the titer and conversion rate of KIV from L-val were 98.5 g·L^-1 and 99.2% at a 3-L scale, respectively. These results demonstrate that the newly engineered catalyst can significantly reduce the product inhibition, that making KIV a prospective product by bioconversion method, and also provide the understanding of the mechanism of the relieved product inhibition of PM-LAAD.
文摘A series of novel L-amino acid esters prodrugs of acyclic nucleoside phosphonates was synthesized and their anti-HBV activity was evaluated in HepG2 2.2.15 cells. Compound 1d exhibited more potent anti-HBV activity and lower cytotoxicity than those of adefovir dipivoxil with EC50 and CC50 values of 0.207 μmol/L and 2530 μmol/L, respectively.
基金supported by the Lundbeck Foundationthe Danish Multiple Sclerosis Foundationthe Danish Medical Research Council
文摘Monoamine neurotransmitters include catecholamines and indoleamines. The most common catecholamines are do- pamine （DA）, noradrenaline （NA） and adrenaline, which are produced from phenylalanine and tyrosine; whereas the most common indoleamine is serotonin （5-hydroxytrypta mine, 5-HT）, which is produced from 5-hydroxytryptophan （5-HTP）.
基金Supported by the National Natural Science Foundation of China(31470793,31670804)China Postdoctoral Science Foundation(2016M592003)+1 种基金the Natural Science Foundation of Zhejiang Province(LZ13B060002)the General Scientific Research Project of Zhejiang Provincial Education Department(Y201432760)
文摘4-Hydroxyphenylpyruvic acid （4-HPPA）, a kind of α-keto acid, is an intermediate in the metabolism of tyrosine and has a wide range of application in food, pharmaceutical and chemical industry. Using amino acids as raw material to prod uce the corresponding α-keto acid is thought to be both economic and efficient. Among the enzymes that convert amino acid to α-keto acid, membrane bound L-amino acid deaminase （mL-AAD）, which is anchored to the outer side of the cytomembrane, becomes an ideal enzyme to prepare α-keto acid since there is no cofactors needed and H2O2 production during the reaction. In this study, the mL-AAD from Proteus vulgaris was used to prepare whole-cell catalysts to produce 4-HPPA from L-tyrosine. The secretory efficiency of mL-AAD conducted by its own twin-arginine signal peptide （twin-arginine translocation pathway, Tat） and integrated pelB （the general secretory pathway, Sec）-Tat signal peptide was determined and compared firstly, using two pET systems （pET28a and pET20b）. It was found that the Tat pathway （pET28a-mlaad） resulted in higher cell-associated mL-AAD activity and cell biomass, and was more beneficial to prepare biocatalyst. In addition, expression hosts BI21 （DE3） and 0.05 mmol. L- 1 IPTG were found to be suitable for mL-AAD expression. The reaction conditions for mL-AAD were optimized and 72.72 mmol,L 1 4-HPPA was obtained from 100 mmol.L 1 tyrosine in 10 h under the optimized conditions. This bioprocess, which is more eco-friendly and economical than the traditional chemical synthesis ways, has great potential for industrial application.
文摘A new composite chiral liquid crystal containing L-amino acid derivatives was prepared and their liquid crystal properties were investigated. Results show that they could exhibit liquid crystal through hydrogen bonding.
基金supported by the grants from the National Natural Science Foundation of China(No.20962004)the Provincial Social Development Foundation of Guizhou,China(No.QKHSYZ[2009]3081)+1 种基金Provincial Special Assistant Foundation for High-level Talents of Guizhou,China(No.TZJF-2009-36)Science and Technology Foundation of Guizhou Province,China(No.QKHJZ[2008]2140)
文摘A series of novel mono（2,2,2-trifluoroethyl） esters,mono L-amino acid ester prodrugs of acyclic nucleoside phosphonates was synthesized and their in vitro anti-HBVactivity was evaluated in HepG 2 2.2.15 cells.Compound 1d exhibited more potent anti-HBV activity and lower cytotoxicity than those of adefovir dipivoxil and alamifovir（MCC-478） with EC_（50） and CC_（50） values of 0.01μmol/L and 8000μmol/L respectively.
文摘为挖掘油莎豆匍匐茎形成茎豆过程中的关键代谢物质,调控匍匐茎形成更多的茎豆,以匍匐茎及新茎豆为研究对象,利用高效液相色谱联合三重四极杆质谱仪进行广泛靶向代谢组学分析,采用多元统计学方法挖掘与茎豆形成相关的差异代谢物质,并富集差异代谢物相关的KEGG代谢通路。结果表明,未膨大匍匐茎(PFJ1)、膨大匍匐茎(PFJ2)及新茎豆(JD1)3组样本中共检测到1 217个代谢物。按照变量投影重要度(VIP)>1、P<0.05且差异倍数(FC)>2或FC<0.5的条件进行差异代谢物筛选,JD1 vs PFJ1筛选到35个差异代谢物,其中26个上调表达,9个下调表达;JD1 vs PFJ2筛选到57个差异代谢物,其中50个上调表达,7个下调表达;PFJ2 vs PFJ1筛选到39个差异代谢物,其中3个上调表达,36个下调表达。利用数据库对代谢物进行定性分析,差异表达代谢物主要被鉴定为氨基酸及其衍生物、核苷酸及其衍生物、有机氧化合物、黄酮类、苯丙素类、酚类等。差异表达代谢物相对含量分析显示,JD1谷胱甘肽含量是PFJ1的14.021倍、PFJ2的29.195倍;JD1 3-甲基组氨酸含量是PFJ1的10.463倍、PFJ2的129.931倍;JD1山柰酚含量是PFJ1的0.421倍、PFJ2的0.358倍。进一步的KEGG通路分析显示,差异表达代谢物主要富集到氨酰-tRNA生物合成,精氨酸和脯氨酸代谢,丙氨酸、天冬氨酸及谷氨酸代谢,苯丙氨酸代谢,黄酮及黄酮醇生物合成等代谢通路。综上,与油莎豆茎豆形成相关的代谢物质主要为氨基酸类及黄酮类物质;代谢通路主要集中在氨酰-tRNA生物合成及黄酮和黄酮醇生物合成途径中。
