ω-Transaminase catalyzes the asymmetric reductive amination of carbonyl compounds, and has great application prospect in the preparation of chiral amines. The application in synthesis of bulky chiral ...amines is limited by the special structure of substrate binding region in the wild-type enzyme. Moreover, there are also some drawbacks in the stereoselectivity and stability of ω-transaminase. So far, -tωransaminase satisfying the industrial requirements is still rare. In this review, we first introduce the structure and catalytic mechanism of ω-transaminase, and then discuss the structural differences between S-selective and R-selective enzymes. Molecular modification of ω-transaminase was introduced in detail, by focusing on the structure and mechanism-based molecular modification, including substrate specificity, stereoselectivity, and stability.
(
R
)-Selective ω-transaminase (ω-TA) is a key enzyme for the asymmetric reductive amination of carbonyl compounds to produce chiral amines which are essential parts of many therapeutic compounds. ...However, its practical industrial applications are hindered by the low catalytic efficiency and poor thermostability of naturally occurring enzymes. In this work, we report the molecular modification of (
R
)-selective ω-TA from
Aspergillus terreus
(
At
TA) to allow asymmetric reductive amination of 4-hydroxy-2-butanone, producing (
R
)-3-amino-1-butanol. Based on substrate docking analysis, 4 residues in the substrate tunnel and binding pocket of
At
TA were selected as mutation hotspots. The screening procedure was facilitated by the construction of a “small-intelligent” library and the use of thin-layer chromatography for preliminary screening. The resulting mutant
At
TA-M5 exhibited a 9.6-fold higher
k
cat
/
K
m
value and 9.4 °C higher
T
1
/
2
10
min
than that of wild-type
At
TA. Furthermore, the conversion of 20 and 50 g L
−1
4-hydroxy-2-butanone by
At
TA-M5 reached 90.8% and 79.1%, suggesting significant potential for production of (
R
)-3-amino-1-butanol. Under the same conditions, wild-type
At
TA achieved less than 5% conversion. Moreover, the key mutation (S215P in
At
TA) was validated in 7 other (
R
)-selective ω-TAs, indicating its general applicability in improving the catalytic efficiency of homologous (
R
)-selective ω-TAs.
This paper proposes a new approach for investigating the mechanism of the formation of the active phase of a hydrodesulfurization (HDS) catalyst via crystalline polyoxometalate (POM) precursors. The ...proposed strategy induces the crystallization of small Ni–Mo–O clusters in an impregnating solution by the coordinate bonding and supramolecular interaction of organic ligands to form POMs. By exploiting the “ligand-induced self-assembly” strategy, two Ni–Mo binary POMs with different frameworks, namely, Mo 2 Ni and PMo 11 Ni, were isolated from the impregnating solution by means of 4,4′-bpy. The sulfidation process of the precursors and the formation mechanism of the NiMoS active phase were fully characterized by a multi-technique approach that comprised, in particular, in situ FT-IR spectroscopy, XRD and Raman spectroscopy for different degrees of sulfidation. The results of the characterization revealed the structure-directing effects (framework effect, promoting effect and ligand effect) of the POM precursors on the structure of the active phase and even its HDS performance. MoS 2 was formed at 200 °C from Mo 2 Ni, and the Ni species interacted with the edges of MoS 2 to form the NiMoS active phase, whereas PMo 11 Ni formed MoS 2 at 300 °C. The structure-directing effects enabled a higher content and better dispersion of the NiMoS active phase, which explains the higher HDS reactivity of sulfided Mo 2 Ni. The bottom-up self-assembly approach not only provides a better understanding of the composition of the impregnating solution and the formation mechanism of the NiMoS active phase but also sheds light on the rational design and controllable preparation of NiMoS catalysts with high performance.
A stepwise control strategy for enhancing glutathione (GSH) synthesis in yeast based on oxidative stress and energy metabolism was investigated. First, molasses and corn steep liquor were selected ...and fed as carbon source mixture at a flow rate of 1.5 g/L/h and 0.4 g/L/h, respectively, for increasing cell density in a 10 L fermenter. When the biomass reached 90 g/L, the KMnO
4
sustained-release particles, composed of 1.5% KMnO
4
, 3% stearic acid, 2% polyethylene glycol and 3% agar powder, were prepared and added to the fermentation broth for maintaining the oxidative stress. The results showed that the maximum GSH accumulation of the group fed KMnO
4
sustained-release particles was 39.0% higher than that of KMnO
4
-fed group. In addition to the improved average GSH productivity and average specific production rate, the activities of GSH peroxidase, γ-glutamylcysteine synthetase and GSH reductase, enzymes taking part in GSH metabolism, were also significantly enhanced by KMnO
4
sustained-release particles feeding. Finally, 6 g/L sodium citrate fed as an energy adjuvant elevated the intracellular ATP level for further enhancing GSH production. Through the above stepwise strategy, the GSH accumulation reached 5.76 g/L, which was 2.84-fold higher than that of the control group. The stepwise control strategy based on oxidative stress and energy metabolism significantly improved GSH accumulation in yeast.
Saccharomyces cerevisiae is often used as a cell factory for the production of S-adenosyl-l-methionine (SAM) for diverse pharmaceutical applications. However, SAM production by S. cerevisiae is ...negatively influenced by glucose repression, which is regulated by a serine/threonine kinase SNF1 complex. Here, a strategy of alleviating glucose repression by deleting REG1 (encodes the regulatory subunit of protein phosphatase 1) and overexpressing SNF1 (encodes the catalytic subunit of the SNF1 complex) was applied to improve SAM production in S. cerevisiae. SAM production, growth conditions, glucose consumption, ethanol accumulation, lifespan, glycolysis and amino acid metabolism were analyzed in the mutant strains. The results showed that the multiple effects of REG1 deletion and/or SNF1 overexpression exhibited a great potential for improving the SAM production in yeast. Enhanced the expression levels of genes involved in glucose transport and glycolysis, which improved the glucose utilization and then elevated the levels of glycolytic intermediates. The expression levels of ACS1 (encoding acetyl-CoA synthase I) and ALD6 (encoding aldehyde dehydrogenase), and the activity of alcohol dehydrogenase II (ADH2) were enhanced especially in the presence of excessive glucose levels, which probably promoted the conversion of ethanol in fermentation broth into acetyl-CoA. The gene expressions involved in sulfur-containing amino acids were also enhanced for the precursor amino acid biosynthesis. In addition, the lifespan of yeast was extended by REG1 deletion and/or SNF1 overexpression. As expected, the final SAM yield of the mutant YREG1DELAPSNF1 reached 8.28 g/L in a 10-L fermenter, which was 51.6% higher than the yield of the parent strain S. cerevisiae CGMCC 2842. This study showed that the multiple effects of REG1 deletion and SNF1 overexpression improved SAM production in S. cerevisiae, providing new insight into the application of the SNF1 complex to abolish glucose repression and redirect carbon flux to nonethanol products in S. cerevisiae.
(R)-Selective omega-transaminase (omega-TA) is a key enzyme for the asymmetric reductive amination of carbonyl compounds to produce chiral amines which are essential parts of many therapeutic ...compounds. However, its practical industrial applications are hindered by the low catalytic efficiency and poor thermostability of naturally occurring enzymes. In this work, we report the molecular modification of (R)-selective omega-TA from Aspergillus terreus (AtTA) to allow asymmetric reductive amination of 4-hydroxy-2-butanone, producing (R)-3-amino-1-butanol. Based on substrate docking analysis, 4 residues in the substrate tunnel and binding pocket of AtTA were selected as mutation hotspots. The screening procedure was facilitated by the construction of a "small-intelligent" library and the use of thin-layer chromatography for preliminary screening. The resulting mutant AtTA-M5 exhibited a 9.6-fold higher k.sub.cat/K.sub.m value and 9.4 °C higher Formula omitted than that of wild-type AtTA. Furthermore, the conversion of 20 and 50 g L.sup.-1 4-hydroxy-2-butanone by AtTA-M5 reached 90.8% and 79.1%, suggesting significant potential for production of (R)-3-amino-1-butanol. Under the same conditions, wild-type AtTA achieved less than 5% conversion. Moreover, the key mutation (S215P in AtTA) was validated in 7 other (R)-selective omega-TAs, indicating its general applicability in improving the catalytic efficiency of homologous (R)-selective omega-TAs.
(R)-Selective omega-transaminase (omega-TA) is a key enzyme for the asymmetric reductive amination of carbonyl compounds to produce chiral amines which are essential parts of many therapeutic ...compounds. However, its practical industrial applications are hindered by the low catalytic efficiency and poor thermostability of naturally occurring enzymes. In this work, we report the molecular modification of (R)-selective omega-TA from Aspergillus terreus (AtTA) to allow asymmetric reductive amination of 4-hydroxy-2-butanone, producing (R)-3-amino-1-butanol. Based on substrate docking analysis, 4 residues in the substrate tunnel and binding pocket of AtTA were selected as mutation hotspots. The screening procedure was facilitated by the construction of a "small-intelligent" library and the use of thin-layer chromatography for preliminary screening. The resulting mutant AtTA-M5 exhibited a 9.6-fold higher k.sub.cat/K.sub.m value and 9.4 #176;C higher Formula omitted than that of wild-type AtTA. Furthermore, the conversion of 20 and 50 g L.sup.-1 4-hydroxy-2-butanone by AtTA-M5 reached 90.8% and 79.1%, suggesting significant potential for production of (R)-3-amino-1-butanol. Under the same conditions, wild-type AtTA achieved less than 5% conversion. Moreover, the key mutation (S215P in AtTA) was validated in 7 other (R)-selective omega-TAs, indicating its general applicability in improving the catalytic efficiency of homologous (R)-selective omega-TAs.
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•Well-defined Strandberg polyoxometalates were used to prepare hydrodesulfurization catalysts.•Highly dispersed and stacked MoS2 was controllably prepared via the polyoxometalate ...route.•Components and structure of polyoxometalates modulated the morphology of the active phase.•Sulfidation behavior of polyoxometalates was tracked by a multitechnique approach.•The new catalyst shows enhanced performance compared with the industrial NiMo catalyst.
Highly active hydrodesulfurization (HDS) catalysts have been prepared successfully through a molecular approach using well-defined Strandberg PMoNi polyoxometalates (POMs) as superior precursors. The hand-picked POMs significantly facilitated not only the formation of highly dispersed NiMo species but also the formation of abundant and accessible NiMoS active sites at lower sulfidation temperatures, thereby resulting in a remarkable activity improvement in comparison with reference catalysts prepared with conventional precursors. Characterization results revealed the multidirecting effects of the POMs component and structure on the morphology and composition of the species in the NiMoS active phase and the evolution of precursors during the catalyst preparation simultaneously. The bottom-up POMs-based preparation methodology provides a better understanding of HDS catalyst structure and performance, thus further shedding light on the rational design and controllable fabrication of efficient HDS catalysts.
This paper proposes a new approach for investigating the mechanism of the formation of the active phase of a hydrodesulfurization (HDS) catalyst
via
crystalline polyoxometalate (POM) precursors. The ...proposed strategy induces the crystallization of small Ni-Mo-O clusters in an impregnating solution by the coordinate bonding and supramolecular interaction of organic ligands to form POMs. By exploiting the "ligand-induced self-assembly" strategy, two Ni-Mo binary POMs with different frameworks, namely, Mo
2
Ni and PMo
11
Ni, were isolated from the impregnating solution by means of 4,4′-bpy. The sulfidation process of the precursors and the formation mechanism of the NiMoS active phase were fully characterized by a multi-technique approach that comprised, in particular,
in situ
FT-IR spectroscopy, XRD and Raman spectroscopy for different degrees of sulfidation. The results of the characterization revealed the structure-directing effects (framework effect, promoting effect and ligand effect) of the POM precursors on the structure of the active phase and even its HDS performance. MoS
2
was formed at 200 °C from Mo
2
Ni, and the Ni species interacted with the edges of MoS
2
to form the NiMoS active phase, whereas PMo
11
Ni formed MoS
2
at 300 °C. The structure-directing effects enabled a higher content and better dispersion of the NiMoS active phase, which explains the higher HDS reactivity of sulfided Mo
2
Ni. The bottom-up self-assembly approach not only provides a better understanding of the composition of the impregnating solution and the formation mechanism of the NiMoS active phase but also sheds light on the rational design and controllable preparation of NiMoS catalysts with high performance.
This paper proposes a new approach for investigating the mechanism of the formation of the active phase of a hydrodesulfurization (HDS) catalyst
via
crystalline polyoxometalate (POM) precursors.
