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
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
Soil salinity badly affects agriculture productivity through accumulation of salts in upper layers of soils. The harmful effects of salts in arable lands have influenced modern as well as ancient civilizations. A pot ...Soil salinity badly affects agriculture productivity through accumulation of salts in upper layers of soils. The harmful effects of salts in arable lands have influenced modern as well as ancient civilizations. A pot study was carried out to test the performance of two PGPR isolates (KS 8, KS 28) on sunflower (SMH-0917) under different salinity levels (8, 10 and 12 dS·m-1). These salinity levels were developed by adding calculated amount of salts (NaCl, Na2SO4, CaCl2 and MgSO4) with ratio of 3:4:2:1. The bacterial strains KS 8 and KS 28 were applied separately in two treatments while third treatment was co-inoculation (KS mix). Completely randomized experimental design (CRD) was used and data were collected at flowering stage about pre-decided plant growth parameters (plant height, shoot dry weight and root dry weight). The bacterial isolate KS 8 showed an increase of 26, 102% and 83% in plant height, shoot dry weight and root dry weight at EC 8 dS·m-1, while this improvement was 67%, 163% and 296% at EC 10 dS·m-1, however an increase of 100%, 74% and 382% was recorded over control respectively at EC 12 dS·m-1. Similarly isolate KS 28 exhibited an increase of 14%, 69% and 54% in plant height;shoot dry weight and root dry weight at EC 8 dS·m-1, whereas this improvement was 56%, 163% and 188% at EC 10 dS·m-1, while an increase of 60%, 41% and 282% was registered respectively over control at EC 12 dS·m-1. The increase due to mixture treatments was 4%, 41% and 16% in plant height, shoot dry weight and root dry weight at EC 8 dS·m-1, while an increase of 33%, 57% and 100% at EC 10 dS·m-1, whereas an improvement of 53%, 33% and 164% respectively was noted at EC 12 dS·m-1 over un-inoculated. The isolate KS 8 performed better than KS 28 and mixture treatment. These two PGPR strains could be used to mitigate the adverse impact caused by salinity stress on sunflower.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
Plant-associated bacteria that inhabit the rhizosphere may influence the plant growth by their contribution to the endogenous pool of phytohormones and by the activity of ACC deaminase to decrease the ethylene concent...Plant-associated bacteria that inhabit the rhizosphere may influence the plant growth by their contribution to the endogenous pool of phytohormones and by the activity of ACC deaminase to decrease the ethylene concentration. The aim of this study was to analyse the root length growth by the promoting effect of indole acetic acid producers phytobacteria with ACC deaminase activity, on inoculated seeds of Lens esculenta as synergistic effect on root elongation. In this study, although the roots of L. esculenta seedlings do not show a significant promotion, these phytobacteria could be recommended to treat plants analyzing their added inoculum to increase plant biomass and retard the effect of ethylene on cultures supplied with Tryptophan and ACC.展开更多
We searched for a superior melamine-degrading bacterium for the bioremediation of melamine. Cyanuric acid, which is a by-product produced during the biodegradation of melamine, shows strong nephrotoxicity. Therefore, ...We searched for a superior melamine-degrading bacterium for the bioremediation of melamine. Cyanuric acid, which is a by-product produced during the biodegradation of melamine, shows strong nephrotoxicity. Therefore, the melamine-degrading bacterium is also required to show a high ability to degrade cyanuric acid. We selected a melamine-degrading strain (MEL1) among ten cyanuric acid-degrading bacteria isolated from the soil. The species of MEL1 strain was Microbacterium esteramaticum or was extremely similar to that species, and the enzymatic activity of the melamine deaminase in the MEL1 strain was similar to that in the NRRLB-12227 strain. The ability of the MEL1 strain to degrade cyanuric acid was higher than its ability to degrade melamine, and therefore, the accumulation of by-products (ammeline, ammelide and cyanuric acid) during the degradation of melamine was minimal. These results suggest that the MEL1 strain is useful for the bioremediation of melamine.展开更多
目的:探究血清ADA、LDH及EBV-DNA水平对传染性单核细胞增多症肝损害患儿病情及预后评估的临床价值。方法:选取109例传染性单核细胞增多症(IM)患儿为研究对象,按是否出现肝损害将患儿分为肝损害组(n=62)与非肝损害组(n=47)。检测两组患...目的:探究血清ADA、LDH及EBV-DNA水平对传染性单核细胞增多症肝损害患儿病情及预后评估的临床价值。方法:选取109例传染性单核细胞增多症(IM)患儿为研究对象,按是否出现肝损害将患儿分为肝损害组(n=62)与非肝损害组(n=47)。检测两组患儿血清腺苷脱氨酶(ADA)、乳酸脱氢酶(LDH)水平及EB病毒脱氧核糖核酸(EBV-DNA)载量,分析不同肝损害程度上述指标表达水平及其与传染性单核细胞增多症并发肝损害的相关性,绘制ROC曲线分析上述指标的诊断效能。结果:肝损害组患儿发生肝肿大率、ALT、AST、TBil水平均高于非肝损害组(P<0.05);肝损害组患儿血清ADA、LDH及EBV-DNA水平高于非肝损害组(P<0.05),且均随肝损害程度升高而升高(P<0.05);传染性单核细胞增多症患儿血清ADA、LDH及EBV-DNA水平与ALT、AST均呈正相关(P<0.05);ROC曲线显示,血清ADA、LDH及EBV-DNA的曲线下面积(AUC)分别为0.831、0.861和0.934,截断值分别为30.60 U/L、544.63 U/L和2.35 lg copies/mL,联合诊断的AUC为0.957。结论:血清ADA、LDH及EBV-DNA水平与IM患儿肝损害相关,对于评估IM合并肝损害诊断以及预后评估方面具有一定参考价值,值得临床广泛应用。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.
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Soil salinity badly affects agriculture productivity through accumulation of salts in upper layers of soils. The harmful effects of salts in arable lands have influenced modern as well as ancient civilizations. A pot study was carried out to test the performance of two PGPR isolates (KS 8, KS 28) on sunflower (SMH-0917) under different salinity levels (8, 10 and 12 dS·m-1). These salinity levels were developed by adding calculated amount of salts (NaCl, Na2SO4, CaCl2 and MgSO4) with ratio of 3:4:2:1. The bacterial strains KS 8 and KS 28 were applied separately in two treatments while third treatment was co-inoculation (KS mix). Completely randomized experimental design (CRD) was used and data were collected at flowering stage about pre-decided plant growth parameters (plant height, shoot dry weight and root dry weight). The bacterial isolate KS 8 showed an increase of 26, 102% and 83% in plant height, shoot dry weight and root dry weight at EC 8 dS·m-1, while this improvement was 67%, 163% and 296% at EC 10 dS·m-1, however an increase of 100%, 74% and 382% was recorded over control respectively at EC 12 dS·m-1. Similarly isolate KS 28 exhibited an increase of 14%, 69% and 54% in plant height;shoot dry weight and root dry weight at EC 8 dS·m-1, whereas this improvement was 56%, 163% and 188% at EC 10 dS·m-1, while an increase of 60%, 41% and 282% was registered respectively over control at EC 12 dS·m-1. The increase due to mixture treatments was 4%, 41% and 16% in plant height, shoot dry weight and root dry weight at EC 8 dS·m-1, while an increase of 33%, 57% and 100% at EC 10 dS·m-1, whereas an improvement of 53%, 33% and 164% respectively was noted at EC 12 dS·m-1 over un-inoculated. The isolate KS 8 performed better than KS 28 and mixture treatment. These two PGPR strains could be used to mitigate the adverse impact caused by salinity stress on sunflower.
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Authors are grateful to the Research Projects:SIP:20131494 of the Secretaría de Investigación y Posgrado del I.P.N.ISITDF/325/11 AREAS PRIORITARIAS-IPN and COFAA-IPN,EDI-IPN,SNI-CONACYT fel-lowships
文摘Plant-associated bacteria that inhabit the rhizosphere may influence the plant growth by their contribution to the endogenous pool of phytohormones and by the activity of ACC deaminase to decrease the ethylene concentration. The aim of this study was to analyse the root length growth by the promoting effect of indole acetic acid producers phytobacteria with ACC deaminase activity, on inoculated seeds of Lens esculenta as synergistic effect on root elongation. In this study, although the roots of L. esculenta seedlings do not show a significant promotion, these phytobacteria could be recommended to treat plants analyzing their added inoculum to increase plant biomass and retard the effect of ethylene on cultures supplied with Tryptophan and ACC.
文摘We searched for a superior melamine-degrading bacterium for the bioremediation of melamine. Cyanuric acid, which is a by-product produced during the biodegradation of melamine, shows strong nephrotoxicity. Therefore, the melamine-degrading bacterium is also required to show a high ability to degrade cyanuric acid. We selected a melamine-degrading strain (MEL1) among ten cyanuric acid-degrading bacteria isolated from the soil. The species of MEL1 strain was Microbacterium esteramaticum or was extremely similar to that species, and the enzymatic activity of the melamine deaminase in the MEL1 strain was similar to that in the NRRLB-12227 strain. The ability of the MEL1 strain to degrade cyanuric acid was higher than its ability to degrade melamine, and therefore, the accumulation of by-products (ammeline, ammelide and cyanuric acid) during the degradation of melamine was minimal. These results suggest that the MEL1 strain is useful for the bioremediation of melamine.
文摘目的:探究血清ADA、LDH及EBV-DNA水平对传染性单核细胞增多症肝损害患儿病情及预后评估的临床价值。方法:选取109例传染性单核细胞增多症(IM)患儿为研究对象,按是否出现肝损害将患儿分为肝损害组(n=62)与非肝损害组(n=47)。检测两组患儿血清腺苷脱氨酶(ADA)、乳酸脱氢酶(LDH)水平及EB病毒脱氧核糖核酸(EBV-DNA)载量,分析不同肝损害程度上述指标表达水平及其与传染性单核细胞增多症并发肝损害的相关性,绘制ROC曲线分析上述指标的诊断效能。结果:肝损害组患儿发生肝肿大率、ALT、AST、TBil水平均高于非肝损害组(P<0.05);肝损害组患儿血清ADA、LDH及EBV-DNA水平高于非肝损害组(P<0.05),且均随肝损害程度升高而升高(P<0.05);传染性单核细胞增多症患儿血清ADA、LDH及EBV-DNA水平与ALT、AST均呈正相关(P<0.05);ROC曲线显示,血清ADA、LDH及EBV-DNA的曲线下面积(AUC)分别为0.831、0.861和0.934,截断值分别为30.60 U/L、544.63 U/L和2.35 lg copies/mL,联合诊断的AUC为0.957。结论:血清ADA、LDH及EBV-DNA水平与IM患儿肝损害相关,对于评估IM合并肝损害诊断以及预后评估方面具有一定参考价值,值得临床广泛应用。
