Hyoscyamine 6β-hydroxylase (H6H, EC 1.14.11.11), an α-ketoglutarate dependent dioxygenase catalyz... more Hyoscyamine 6β-hydroxylase (H6H, EC 1.14.11.11), an α-ketoglutarate dependent dioxygenase catalyzes the hydroxylation of (-)-hyoscyamine and the subsequent epoxidation of 6β-hydroxyhyoscyamine to form scopolamine, a valuable natural alkaloid. In this study, random mutagenesis and site-directed saturation mutagenesis were used to enhance the hydroxylation activity of H6H from Anisodus acutangulus (AaH6H). A double mutant, AaH6HM1 (S14P/K97A), showed a 3.4-fold improved hydroxylation activity compared with the wild-type enzyme, and the in vivo epoxidation activity was also improved by 2.3 times. After 34h cultivation of Escherichia coli cells harboring Aah6hm1 in a 5-L bioreactor with a working volume of 3L, scopolamine was produced via a single-enzyme-mediated two-step transformation from 500mgL(-1) (-)-hyoscyamine in 97% conversion, and 1.068g of the product were isolated, corresponding to a space-time yield of 251mgL(-1)d(-1). This study shows that the protein engineering of some k...
To avoid adding NAD(+) and effectively transform ethyl 4-chloro-3-oxobutanoate, the mixture of l-... more To avoid adding NAD(+) and effectively transform ethyl 4-chloro-3-oxobutanoate, the mixture of l-glutamine (200mM) and d-xylose (250mM) was added into in n-butyl acetate-water (10:90, v/v) biphasic system instead of NAD(+) for increasing the biocatalytic efficiency. To further improve the synthesis of optically pure ethyl (R)-4-chloro-3-hydroxybutanoate (>99% ee), β-cyclodextrin was also added into this reaction media, and ethyl (R)-4-chloro-3-hydroxybutanoate (>99% ee) could be effectively synthesized from 800mM ethyl 4-chloro-3-oxobutanoate in the yield of 100% by whole-cells of recombinant E. coli CCZU-A13. Finally, the possible mechanism for improving the reductase activity by supplementation of l-glutamine, d-xylose and β-CD was proposed. In conclusion, this strategy has high potential for the effective biosynthesis of ethyl (R)-4-chloro-3-hydroxybutanoate (>99% ee).
The keto ester reductase from Candida glabrata, designated as CgKR1, is a highly versatile biocat... more The keto ester reductase from Candida glabrata, designated as CgKR1, is a highly versatile biocatalyst with a broad substrate spectrum. Its substrate preference was altered by rational design of the active pocket for bioreduction of aromatic α-keto esters in our previous work. However, its practical application is still hindered by its poor thermostability and high biocatalyst loading. In this work, random mutagenesis followed by a saturation mutagenesis was performed aiming to improve its thermostability. Variants M5 (I99Y/D138N/G174A) and M6 (D138N) exhibited an obvious increase in T50(15) from 41.8°C of wild-type to 53.4°C and 48.8°C, respectively, indicating the important role of residue 138 for its thermostability. The homologous three-dimensional structures of the wild-type and variant M6 (D138N) revealed that the increased thermostability might be attributed to the increased hydrogen bonds that asparagine forms with other polar amino acids around it. Combination of the most t...
ABSTRACT For efficient production of (R)-(−)-mandelic acid, a nitrilase gene from Alcaligenes sp.... more ABSTRACT For efficient production of (R)-(−)-mandelic acid, a nitrilase gene from Alcaligenes sp. ECU0401 was cloned and overexpressed in Escherichia coli. After simple optimization of the culture conditions, the biocatalyst production was greatly increased from 500 to 7000 U/l. The recombinant E. coli whole cells showed strong tolerance against a high substrate concentration of up to 200 mM, and the concentration of (R)-(−)-mandelic acid after only 4 h of transformation reached 197 mM with an enantiomeric excess (eep) of 99%. In a fed-batch reaction with 600 mM mandelonitrile as the substrate, the cumulative production of (R)-(−)-mandelic acid after 17.5 h of conversion reached 520 mM. The recombinant E. coli cells could also be repeatedly used in the biotransformation, retaining 40% of the initial activity after 10 batches of reaction. The highly substrate/product tolerable and enantioselective nature of this recombinant nitrilase suggests that it is of great potential for the practical production of optically pure (R)-(−)-mandelic acid.
Proceedings of the National Academy of Sciences of the United States of America, Jan 4, 2014
Optically pure epoxides are essential chiral precursors for the production of (S)-propranolol, (S... more Optically pure epoxides are essential chiral precursors for the production of (S)-propranolol, (S)-alprenolol, and other β-adrenergic receptor blocking drugs. Although the enzymatic production of these bulky epoxides has proven difficult, here we report a method to effectively improve the activity of BmEH, an epoxide hydrolase from Bacillus megaterium ECU1001 toward α-naphthyl glycidyl ether, the precursor of (S)-propranolol, by eliminating the steric hindrance near the potential product-release site. Using X-ray crystallography, mass spectrum, and molecular dynamics calculations, we have identified an active tunnel for substrate access and product release of this enzyme. The crystal structures revealed that there is an independent product-release site in BmEH that was not included in other reported epoxide hydrolase structures. By alanine scanning, two mutants, F128A and M145A, targeted to expand the potential product-release site displayed 42 and 25 times higher activities toward ...
A new isolate, Rhodotorula sp. AS2.2241, capable of reducing acetophenone and α-bromoacetophenone... more A new isolate, Rhodotorula sp. AS2.2241, capable of reducing acetophenone and α-bromoacetophenone with high stereoselectivity, was further studied to exploit its potential in the asymmetric reduction of silicon-containing ketones to silyl alcohols. After encapsulation, the cells were used in an aqueous or an aqueous/organic solvent biphasic system for the asymmetric reduction of acetyltrimethylsilane (ATMS) to prepare (−)-1-trimethylsilylethanol [(−)-TMSE]. It has
Applied and environmental microbiology, Jan 19, 2014
A novel non-heme chloroperoxidase (RhEst1), with promiscuous esterase activity for enantioselecti... more A novel non-heme chloroperoxidase (RhEst1), with promiscuous esterase activity for enantioselective hydrolysis of ethyl (S)-2,2-dimethylcylopropanecarboxylate, was identified from the shot-gun library of Rhodococcus sp. ECU1013. RhEst1 was overexpressed in Escherichia coli BL21 (DE3), purified to homogeneity and functionally characterized. Fingerprinting analysis revealed that RhEst1 prefers to para-nitrophenyl (pNP) esters of short-chain acyl groups. pNP esters with a cyclic acyl moiety, especially that with a cyclobutanyl group, were also substrates for RhEst1. The Km values for methyl 2,2-dimethylcyclopropanecarboxylate (DmCpCm) and ethyl 2,2-dimethylcyclopropane carboxylate (DmCpCe) were 0.25 and 0.43 mM, respectively. RhEst1 could serve as an efficient hydrolase for the bioproduction of optically pure (S)-2,2-dimethyl cyclopropane carboxylic acid (DmCpCa) which is an important chiral building block for cilastatin. As much as 0.5 M of DmCpCe was enantioselectively hydrolyzed int...
Nanoparticles such as nano-SiO(2) are increasingly used in food, cosmetics, diagnosis, imaging an... more Nanoparticles such as nano-SiO(2) are increasingly used in food, cosmetics, diagnosis, imaging and drug delivery. However, toxicological data of nano-SiO(2) on hepatic cells in vitro and their detailed molecular mechanisms still remain unclear. In order to assess toxicity of nano-SiO(2), L-02 cells were exposed to 0.2, 0.4 and 0.6 mg/ml of SiO(2) colloids (21, 48 and 86 nm) for 12, 24, 36 and 48h. Lactate dehydrogenase released from damaged cells were quantified, cellular ultrastructural organization was observed, and the levels of reactive oxygen species (ROS), lipid peroxidation and glutathione were measured. Apoptosis induced by 21 nm SiO(2) was characterized by annexin V-FITC/PI staining and DNA ladder assay. Furthermore, apoptosis related proteins such as p53, Bax and Bcl-2 were analyzed by using western blot analysis. Our data indicated that nano-SiO(2) caused cytotoxicity in size, dose and time dependent manners. Oxidative stress and apoptosis were induced by exposure to 21 nm SiO(2). Moreover, the expression of p53 and Bax was increased in time and dose dependent patterns, whereas the expression of Bcl-2 was not significantly changed. In conclusion, ROS-mediated oxidative stress, the activation of p53 and up-regulation of Bax/Bcl-2 ratio are involved in mechanistic pathways of 21 nm SiO(2) induced apoptosis in L-02 cells.
A new synthesis of harzialactone A together with its stereoisomers was achieved starting from phe... more A new synthesis of harzialactone A together with its stereoisomers was achieved starting from phenyl acetone. The key step of this new route is the kinetic bioresolution of racemic cis- and trans-isomers by recombinant Escherichia coli JM109 cells expressing Fusarium proliferatum lactonase gene (reFPL). Harzialactone A was isolated in excellent ee (>99%), while moderate to good enantioselectivities (80% to >99%
The reduction of aromatic ketones to optically active alcohols, mediated by dried baker's... more The reduction of aromatic ketones to optically active alcohols, mediated by dried baker's yeast, proceeded in moderate conversion and good enantioselectivity (82 ∼ 91% e.e.) in a number of organic solvents, including petroleum ether, toluene, chloroform, and tetrahydrofuran. A small amount of water (0.4 mL: g yeast) was required for the reaction to proceed. The water:yeast ratio and the solvent
Magnetic Fe 3 O 4 nanoparticles were prepared by chemical coprecipitation method and subsequently... more Magnetic Fe 3 O 4 nanoparticles were prepared by chemical coprecipitation method and subsequently coated with 3-aminopropyltriethoxysilane (APTES) via silanization reaction. The synthesized materials were characterized by transmission electron microscopy (TEM) and Fourier ...
An additive method has been developed to obtain nearly optically pure (S)-ketoprofen [(S)-2-(3-be... more An additive method has been developed to obtain nearly optically pure (S)-ketoprofen [(S)-2-(3-benzoyphenyl) propionic acid,> 96% enantiomeric excess, ee] by using lipase OF (from Candida rugosa) for the first time. The effect of surfactants (selected as additives) on the ...
Hyoscyamine 6β-hydroxylase (H6H, EC 1.14.11.11), an α-ketoglutarate dependent dioxygenase catalyz... more Hyoscyamine 6β-hydroxylase (H6H, EC 1.14.11.11), an α-ketoglutarate dependent dioxygenase catalyzes the hydroxylation of (-)-hyoscyamine and the subsequent epoxidation of 6β-hydroxyhyoscyamine to form scopolamine, a valuable natural alkaloid. In this study, random mutagenesis and site-directed saturation mutagenesis were used to enhance the hydroxylation activity of H6H from Anisodus acutangulus (AaH6H). A double mutant, AaH6HM1 (S14P/K97A), showed a 3.4-fold improved hydroxylation activity compared with the wild-type enzyme, and the in vivo epoxidation activity was also improved by 2.3 times. After 34h cultivation of Escherichia coli cells harboring Aah6hm1 in a 5-L bioreactor with a working volume of 3L, scopolamine was produced via a single-enzyme-mediated two-step transformation from 500mgL(-1) (-)-hyoscyamine in 97% conversion, and 1.068g of the product were isolated, corresponding to a space-time yield of 251mgL(-1)d(-1). This study shows that the protein engineering of some k...
To avoid adding NAD(+) and effectively transform ethyl 4-chloro-3-oxobutanoate, the mixture of l-... more To avoid adding NAD(+) and effectively transform ethyl 4-chloro-3-oxobutanoate, the mixture of l-glutamine (200mM) and d-xylose (250mM) was added into in n-butyl acetate-water (10:90, v/v) biphasic system instead of NAD(+) for increasing the biocatalytic efficiency. To further improve the synthesis of optically pure ethyl (R)-4-chloro-3-hydroxybutanoate (>99% ee), β-cyclodextrin was also added into this reaction media, and ethyl (R)-4-chloro-3-hydroxybutanoate (>99% ee) could be effectively synthesized from 800mM ethyl 4-chloro-3-oxobutanoate in the yield of 100% by whole-cells of recombinant E. coli CCZU-A13. Finally, the possible mechanism for improving the reductase activity by supplementation of l-glutamine, d-xylose and β-CD was proposed. In conclusion, this strategy has high potential for the effective biosynthesis of ethyl (R)-4-chloro-3-hydroxybutanoate (>99% ee).
The keto ester reductase from Candida glabrata, designated as CgKR1, is a highly versatile biocat... more The keto ester reductase from Candida glabrata, designated as CgKR1, is a highly versatile biocatalyst with a broad substrate spectrum. Its substrate preference was altered by rational design of the active pocket for bioreduction of aromatic α-keto esters in our previous work. However, its practical application is still hindered by its poor thermostability and high biocatalyst loading. In this work, random mutagenesis followed by a saturation mutagenesis was performed aiming to improve its thermostability. Variants M5 (I99Y/D138N/G174A) and M6 (D138N) exhibited an obvious increase in T50(15) from 41.8°C of wild-type to 53.4°C and 48.8°C, respectively, indicating the important role of residue 138 for its thermostability. The homologous three-dimensional structures of the wild-type and variant M6 (D138N) revealed that the increased thermostability might be attributed to the increased hydrogen bonds that asparagine forms with other polar amino acids around it. Combination of the most t...
ABSTRACT For efficient production of (R)-(−)-mandelic acid, a nitrilase gene from Alcaligenes sp.... more ABSTRACT For efficient production of (R)-(−)-mandelic acid, a nitrilase gene from Alcaligenes sp. ECU0401 was cloned and overexpressed in Escherichia coli. After simple optimization of the culture conditions, the biocatalyst production was greatly increased from 500 to 7000 U/l. The recombinant E. coli whole cells showed strong tolerance against a high substrate concentration of up to 200 mM, and the concentration of (R)-(−)-mandelic acid after only 4 h of transformation reached 197 mM with an enantiomeric excess (eep) of 99%. In a fed-batch reaction with 600 mM mandelonitrile as the substrate, the cumulative production of (R)-(−)-mandelic acid after 17.5 h of conversion reached 520 mM. The recombinant E. coli cells could also be repeatedly used in the biotransformation, retaining 40% of the initial activity after 10 batches of reaction. The highly substrate/product tolerable and enantioselective nature of this recombinant nitrilase suggests that it is of great potential for the practical production of optically pure (R)-(−)-mandelic acid.
Proceedings of the National Academy of Sciences of the United States of America, Jan 4, 2014
Optically pure epoxides are essential chiral precursors for the production of (S)-propranolol, (S... more Optically pure epoxides are essential chiral precursors for the production of (S)-propranolol, (S)-alprenolol, and other β-adrenergic receptor blocking drugs. Although the enzymatic production of these bulky epoxides has proven difficult, here we report a method to effectively improve the activity of BmEH, an epoxide hydrolase from Bacillus megaterium ECU1001 toward α-naphthyl glycidyl ether, the precursor of (S)-propranolol, by eliminating the steric hindrance near the potential product-release site. Using X-ray crystallography, mass spectrum, and molecular dynamics calculations, we have identified an active tunnel for substrate access and product release of this enzyme. The crystal structures revealed that there is an independent product-release site in BmEH that was not included in other reported epoxide hydrolase structures. By alanine scanning, two mutants, F128A and M145A, targeted to expand the potential product-release site displayed 42 and 25 times higher activities toward ...
A new isolate, Rhodotorula sp. AS2.2241, capable of reducing acetophenone and α-bromoacetophenone... more A new isolate, Rhodotorula sp. AS2.2241, capable of reducing acetophenone and α-bromoacetophenone with high stereoselectivity, was further studied to exploit its potential in the asymmetric reduction of silicon-containing ketones to silyl alcohols. After encapsulation, the cells were used in an aqueous or an aqueous/organic solvent biphasic system for the asymmetric reduction of acetyltrimethylsilane (ATMS) to prepare (−)-1-trimethylsilylethanol [(−)-TMSE]. It has
Applied and environmental microbiology, Jan 19, 2014
A novel non-heme chloroperoxidase (RhEst1), with promiscuous esterase activity for enantioselecti... more A novel non-heme chloroperoxidase (RhEst1), with promiscuous esterase activity for enantioselective hydrolysis of ethyl (S)-2,2-dimethylcylopropanecarboxylate, was identified from the shot-gun library of Rhodococcus sp. ECU1013. RhEst1 was overexpressed in Escherichia coli BL21 (DE3), purified to homogeneity and functionally characterized. Fingerprinting analysis revealed that RhEst1 prefers to para-nitrophenyl (pNP) esters of short-chain acyl groups. pNP esters with a cyclic acyl moiety, especially that with a cyclobutanyl group, were also substrates for RhEst1. The Km values for methyl 2,2-dimethylcyclopropanecarboxylate (DmCpCm) and ethyl 2,2-dimethylcyclopropane carboxylate (DmCpCe) were 0.25 and 0.43 mM, respectively. RhEst1 could serve as an efficient hydrolase for the bioproduction of optically pure (S)-2,2-dimethyl cyclopropane carboxylic acid (DmCpCa) which is an important chiral building block for cilastatin. As much as 0.5 M of DmCpCe was enantioselectively hydrolyzed int...
Nanoparticles such as nano-SiO(2) are increasingly used in food, cosmetics, diagnosis, imaging an... more Nanoparticles such as nano-SiO(2) are increasingly used in food, cosmetics, diagnosis, imaging and drug delivery. However, toxicological data of nano-SiO(2) on hepatic cells in vitro and their detailed molecular mechanisms still remain unclear. In order to assess toxicity of nano-SiO(2), L-02 cells were exposed to 0.2, 0.4 and 0.6 mg/ml of SiO(2) colloids (21, 48 and 86 nm) for 12, 24, 36 and 48h. Lactate dehydrogenase released from damaged cells were quantified, cellular ultrastructural organization was observed, and the levels of reactive oxygen species (ROS), lipid peroxidation and glutathione were measured. Apoptosis induced by 21 nm SiO(2) was characterized by annexin V-FITC/PI staining and DNA ladder assay. Furthermore, apoptosis related proteins such as p53, Bax and Bcl-2 were analyzed by using western blot analysis. Our data indicated that nano-SiO(2) caused cytotoxicity in size, dose and time dependent manners. Oxidative stress and apoptosis were induced by exposure to 21 nm SiO(2). Moreover, the expression of p53 and Bax was increased in time and dose dependent patterns, whereas the expression of Bcl-2 was not significantly changed. In conclusion, ROS-mediated oxidative stress, the activation of p53 and up-regulation of Bax/Bcl-2 ratio are involved in mechanistic pathways of 21 nm SiO(2) induced apoptosis in L-02 cells.
A new synthesis of harzialactone A together with its stereoisomers was achieved starting from phe... more A new synthesis of harzialactone A together with its stereoisomers was achieved starting from phenyl acetone. The key step of this new route is the kinetic bioresolution of racemic cis- and trans-isomers by recombinant Escherichia coli JM109 cells expressing Fusarium proliferatum lactonase gene (reFPL). Harzialactone A was isolated in excellent ee (>99%), while moderate to good enantioselectivities (80% to >99%
The reduction of aromatic ketones to optically active alcohols, mediated by dried baker's... more The reduction of aromatic ketones to optically active alcohols, mediated by dried baker's yeast, proceeded in moderate conversion and good enantioselectivity (82 ∼ 91% e.e.) in a number of organic solvents, including petroleum ether, toluene, chloroform, and tetrahydrofuran. A small amount of water (0.4 mL: g yeast) was required for the reaction to proceed. The water:yeast ratio and the solvent
Magnetic Fe 3 O 4 nanoparticles were prepared by chemical coprecipitation method and subsequently... more Magnetic Fe 3 O 4 nanoparticles were prepared by chemical coprecipitation method and subsequently coated with 3-aminopropyltriethoxysilane (APTES) via silanization reaction. The synthesized materials were characterized by transmission electron microscopy (TEM) and Fourier ...
An additive method has been developed to obtain nearly optically pure (S)-ketoprofen [(S)-2-(3-be... more An additive method has been developed to obtain nearly optically pure (S)-ketoprofen [(S)-2-(3-benzoyphenyl) propionic acid,> 96% enantiomeric excess, ee] by using lipase OF (from Candida rugosa) for the first time. The effect of surfactants (selected as additives) on the ...
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