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Metallurgical slag such as solid waste generated in the steel industry carries environmental pollution risks, but it is rich in nutrients required by microalgae. Metallurgical slag used for carbon capture and biomass energy conversion has multiple benefits: (i) reduction and harmless treatment of metallurgical solid waste, (ii) assisting in carbon neutrality by efficient carbon fixation, and (iii) production of biodiesel from CO2. In this study, AOD, BOF, BFS, HVS, and VTS slag were applied to culture Chlorella pyrenoidosa (C. pyrenoidosa) with the regulation of growth, carbon fixation, and lipid synthesis. An excellent fixed amount of CO2 with 94.59 mg is obtained from C. pyrenoidosa biomass at BOF slag added (mass ratio of CO2 captured/microalgae/slag with 1.99/1.00/10.53) since high Ca/Mg mass ratio of 419 (8.38 mg/L Ca and 0.02 mg/L Mg), no Cr and low concentration of Al (0.04 mg/L) contribute to regulating antioxidant enzyme activity (SOD and POD) to resist ROS and improving PEPC activity to reduce carbon flux toward lipid to promote biomass synthesis. Both metal concentrations from Ca (5.86 mg/L), Mg (0.05 mg/L), Al (0.42 mg/L), and Cr (0.006 mg/L) and suitable pH (10.53) in AOD leaching solution at solid/liquid ratio of 0.5 g/L change carbon flow toward efficient lipid synthesis (47.07 wt%) by continuously providing raw materials and energy by regulating ACC, ME, and PEPC activities. High value‐added biodiesel with high concentrations of C16 and C18 methyl esters from lipid of C. pyrenoidosa is achieved, following other ecological and economic benefits including 197 mg CO2 captured and 2198 mg AOD applied with harmless. In this study, C. pyrenoidosa is cultured with elements from metallurgical slag solid waste, which promotes C. pyrenoidosa efficient carbon fixation to assist in carbon neutrality, and provides guidance for CO2 conversion to high‐value‐added products with low cost. [ABSTRACT FROM AUTHOR]

Carbonyl reductases are useful for producing optically active alcohols from their corresponding prochiral ketones. Herein, we applied a computer‐assisted strategy to increase the thermostability of a previously constructed carbonyl reductase, LsCRM4 (N101D/A117G/F147L/E145A), which showed an outstanding activity in the synthesis of the ticagrelor precursor (1S)‐2‐chloro‐1‐(3,4‐difluorophenyl)ethanol. The stability changes introduced by mutations at the flexible sites were predicted using the computational tools FoldX, I‐Mutant 3.0, and DeepDDG, which demonstrated that 12 virtually screened mutants could be thermally stable; 11 of these mutants exhibited increased thermostability. Then a superior mutant LsCRM4‐V99L/D150F was screened out from the library that was constructed by iteratively combining the beneficial sites, which showed a 78% increase in activity and a 17.4°C increase in melting temperature compared to LsCRM4. Our computer‐assisted design and combinatorial strategy dramatically increased the efficiency of thermostable enzyme production. [ABSTRACT FROM AUTHOR]

Nocardia seriolae is the primary aetiological agent of nocardiosis in fish, which causes mass mortality in freshwater and marine fish. β‐ketoacyl‐ACP synthase (KAS) is one of the essential enzymes in the synthesis of mycolic acids (MASs) in Mycobacterium spp. and has been chosen as the target for therapeutic intervention in mycobacterial diseases. In the present study, a kasB homologue gene (kasB) was identified in the genome of N. seriolae, and the gene‐deficient mutant (ΔkasB) was generated based on a clinical isolate, XSYC‐Ns. Compared to the wild‐type (WT) strain, the ΔkasB showed a measurably growth defect in vitro but retained the acid‐fastness in acid‐fast staining. Observation of the cell ultrastructure showed some alterations in the cell wall of the ΔkasB strain. Compared to its original strain, the cell wall lipid layer seemed sparser, and a wider electron‐transparent zone was observed in the cell wall of ΔkasB strain. Moreover, the ΔkasB strain showed impaired ability of cell invasion as well as intracellular survival in the cell line originating from the head‐kidney of the large yellow croaker (LYC‐hK), compared to its original strain. In addition, the deficiency of ΔkasB significantly attenuated the virulence of N. seriolae in largemouth bass. The present study suggested that the ΔkasB gene might be involved in the synthesis of extracellular cell‐wall lipids in N. seriolae and play a crucial role in its pathogenicity. [ABSTRACT FROM AUTHOR]

Aldo‐keto reductases (AKRs) are important biocatalysts that can be used to synthesize chiral pharmaceutical alcohols. In this study, the catalytic activity and stereoselectivity of a NADPH‐dependent AKR from Kluyveromyces dobzhanskii (KdAKR) toward t‐butyl 6‐chloro (5S)‐hydroxy‐3‐oxohexanoate ((5S)‐CHOH) were improved by mutating its residues in the loop regions around the substrate‐binding pocket. And the thermostability of KdAKR was improved by a consensus sequence method targeted on the flexible regions. The best mutant M6 (Y28A/L58I/I63L/G223P/Y296W/W297H) exhibited a 67‐fold higher catalytic efficiency compared to the wild‐type (WT) KdAKR, and improved R‐selectivity toward (5S)‐CHOH (dep value from 47.6% to >99.5%). Moreover, M6 exhibited a 6.3‐fold increase in half‐life (t1/2) at 40°C compared to WT. Under the optimal conditions, M6 completely converted 200 g/L (5S)‐CHOH to diastereomeric pure t‐butyl 6‐chloro‐(3R, 5S)‐dihydroxyhexanoate ((3R, 5S)‐CDHH) within 8.0 h, with a space‐time yield of 300.7 g/L/day. Our results deepen the understandings of the structure−function relationship of AKRs, providing a certain guidance for the modification of other AKRs. [ABSTRACT FROM AUTHOR]

Structural information can help engineer enzymes. Usually, specific amino acids in particular regions are targeted for functional reconstruction to enhance the catalytic performance, including activity, stereoselectivity, and thermostability. Appropriate selection of target sites is the key to structure‐based design, which requires elucidation of the structure–function relationships. Here, we summarize the mutations of residues in different specific regions, including active center, access tunnels, and flexible loops, on fine‐tuning the catalytic performance of enzymes, and discuss the effects of altering the local structural environment on the functions. In addition, we keep up with the recent progress of structure‐based approaches for enzyme engineering, aiming to provide some guidance on how to take advantage of the structural information. [ABSTRACT FROM AUTHOR]

The emerging outbreak of monkeypox is closely associated with the viral infection and spreading, threatening global public health. Virus‐induced cell migration facilitates viral transmission. However, the mechanism underlying this type of cell migration remains unclear. Here we investigate the motility of cells infected by vaccinia virus (VACV), a close relative of monkeypox, through combining multi‐omics analyses and high‐resolution live‐cell imaging. We find that, upon VACV infection, the epithelial cells undergo epithelial–mesenchymal transition‐like transformation, during which they lose intercellular junctions and acquire the migratory capacity to promote viral spreading. After transformation, VACV‐hijacked RhoA signaling significantly alters cellular morphology and rearranges the actin cytoskeleton involving the depolymerization of robust actin stress fibers, leading‐edge protrusion formation, and the rear‐edge recontraction, which coordinates VACV‐induced cell migration. Our study reveals how poxviruses alter the epithelial phenotype and regulate RhoA signaling to induce fast migration, providing a unique perspective to understand the pathogenesis of poxviruses. [ABSTRACT FROM AUTHOR]

Carbonyl reductase (CR)‐catalyzed bioreduction in the organic phase and the neat substrate reaction system is a lasting challenge, placing higher requirements on the performance of enzymes. Protein engineering is an effective method to enhance the properties of enzymes for industrial applications. In the present work, a single point mutation E145A on our previously constructed CR mutant LsCRM3, coevolved thermostability, and activity. Compared with LsCRM3, the catalytic efficiency kcat/KM of LsCRM3‐E145A (LsCRM4) was increased from 6.6 to 21.9 s−1 mM−1. Moreover, E145A prolonged the half‐life t1/2 at 40°C from 4.1 to 117 h, Tm ${T}_{m}$ was increased by 5°C, T5030 ${T}_{50}^{30}$ was increased by 14.6°C, and Topt was increased by 15°C. Only 1 g/L of lyophilized Escherichia coli cells expressing LsCRM4 completely reduced up to 600 g/L 2‐chloro‐1‐(3,4‐difluorophenyl)ethanone (CFPO) within 13 h at 45°C, yielding the corresponding (1S)‐2‐chloro‐1‐(3,4‐difluorophenyl)ethanol ((S)‐CFPL) in 99.5% eeP, with a space‐time yield of 1.0 kg/L d, the substrate to catalyst ratios (S/C) of 600 g/g. Compared with LsCRM3, the substrate loading was increased by 50%, with the S/C increased by 14 times. Compared with LsCRWT, the substrate loading was increased by 6.5 times. In contrast, LsCRM4 completely converted 600 g/L CFPO within 12 h in the neat substrate bioreaction system. [ABSTRACT FROM AUTHOR]

Enzyme engineering toward catalytic‐tetrad residues usually results in activity loss. Unexpectedly, we found that a directed evolution campaign yielded a beneficial residue A100 in KmCR (a carbonyl reductase from Kluyveromyces marxianus ZJB14056), which is a residue of catalytic tetrad and conserved according to multiple sequence alignment. Inspired by this finding, we performed saturation mutagenesis on all the four residues of catalytic tetrad of KmCR. A number of variants with improved enzymatic activities were obtained. Among them, the variant KmCR_A100S exhibited increased catalytic efficiency (kcat/KM = 47.3 s−1·mM−1), improved stereoselectivity (from moderate selectivity (deP = 66.7%) to strict (S)‐selectivity (deP > 99.5%)), and extended substrate scope, compared to those of KmCR_WT. In silico analysis showed that a relay system was rebuilt in KmCR via the beneficial residue S100. Furthermore, comparison of 11 protein engineering campaigns indicated that the beneficial position is easily overlooked due to the long distance (>10 Å) from ketone substrates. Since CRs share similar catalytic mechanism, the knowledge gained from this study has universal significance to CR engineering. [ABSTRACT FROM AUTHOR]

Enzyme engineering usually generates trade‐offs between activity, stability, and selectivity. Herein, we report semirational engineering of an aldo–keto reductase (AKR) KmAKR for simultaneously enhancing its thermostability and catalytic activity. Previously, we constructed KmAKRM9 (W297H/Y296W/K29H/Y28A/T63M/A30P/T302S/N109K/S196C), which showed outstanding activity towards t‐butyl 6‐chloro‐(3R,5S)‐dihydroxyhexanoate ((3R,5S)‐CDHH), and t‐butyl 6‐cyano‐(3R,5R)‐dihydroxyhexanoate, the key chiral building blocks of rosuvastatin and atorvastatin. Under the guidance of computer‐aided design including consensus residues analysis and molecular dynamics (MD) simulations, K164, S182, S232, and Q266 were dug out for their thermostability conferring roles, generating the "best" mutant KmAKRM13(W297H/Y296W/K29H/Y28A/T63M/A30P/T302S/N109K/S196C/K164E/S232A/S182H/Q266D). The Tm and T5015 values of KmAKRM13 were 10.4 and 6.1°C higher than that of KmAKRM9, respectively. Moreover, it displayed a significantly elevated organic solvent tolerance over KmAKRM9. Structural analysis indicated that stabilization of the α‐helixes mainly contributed to thermostability enhancement. Under the optimized conditions, KmAKRM13 completely asymmetrically reduced 400 g/l t‐butyl 6‐chloro‐(5S)‐hydroxy‐3‐oxohexanoate ((5S)‐CHOH) in 8.0 h at a high substrate to catalyst ratio (S/C) of 106.7 g/g, giving diastereomerically pure (3R,5S)‐CDHH (>99.5% d.e.P) with a space‐time yield (STY) of 449.2 g/l·d. [ABSTRACT FROM AUTHOR]

Disconnected interacting protein 2 (DIP2) is a highly conserved protein family among invertebrates and vertebrates, but its function remains unclear. In this paper, we summarized the conservation of gene sequences and protein domains of DIP2 family members and predicted that they may have a similar functional role in acetyl‐coenzyme A (acetyl‐CoA) synthesis. We then used the most characterized member, disconnected interacting protein 2 homolog A (DIP2A), for further study. DIP2A is a cytoplasmic protein that is preferentially localized to mitochondria, and its acetyl‐CoA synthetase activity has been demonstrated in vitro. Furthermore, the level of acetyl‐CoA in HEK293 cells overexpressing DIP2A was increased, which is consistent with its metabolically related function. Together, these data enrich the evolutionary and functional characterization of dip2 genes and provide significant insights into the identification and application of other homologs of DIP2. [ABSTRACT FROM AUTHOR]

t-Butyl 6-cyano-(3 R,5 R)-dihydroxyhexanoate ((3 R,5 R)- 2) is a key chiral diol precursor of atorvastatin calcium (Lipitor®). We have constructed a Kluyveromyces lactis aldo-keto reductase mutant KlAKR-Y295W/W296L ( KlAKRm) by rational design in previous research, which displayed high activity and excellent diastereoselectivity ( dep > 99.5%) toward t-butyl 6-cyano-(5 R)-hydroxy-3-oxohexanoate ((5 R)- 1). To realize in situ cofactor regeneration, a robust KlAKRm and Exiguobacterium sibiricum glucose dehydrogenase ( EsGDH) co-producer E. coli BL 21(DE3) pETDuet- esgdh (MCS1)/pET-28b (+)- klakrm was constructed in this work. Under the optimized conditions, AKR and GDH activities of E. coli BL 21(DE3) pETDuet- esgdh (MCS1)/pET-28b (+)- klakrm peaked at 249.9 U/g DCW (dry cellular weight) and 29100 U/g DCW, respectively. It completely converted (5 R)- 1 at substrate loading size of up to 60.0 g/L (5 R)- 1 in the absence of exogenous NADH, which was one-fifth higher than that of the separately prepared KlAKRm and EsGDH under the same conditions. In this manner, a biocatalytic process for (3 R,5 R)- 2 with productivity of 243.2 kg/m3 d was developed. Compared with the combination of separate expressed KlAKRm with EsGDH, co-expression of KlAKRm and EsGDH has the advantages of alleviating cell cultivation burden and elevating substrate load. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1235-1242, 2017 [ABSTRACT FROM AUTHOR]

To biosynthesize the (3R,5S)-CDHH in an industrial scale, a newly synthesized stereoselective short chain carbonyl reductase (SCR) was successfully cloned and expressed in Escherichia coli. The fermentation of recombinant E. coli harboring SCR was carried out in 500 L and 5000 L fermenters, with biomass and specific activity of 9.7 g DCW/L, 15749.95 U/g DCW, and 10.97 g DCW/L, 19210.12 U/g DCW, respectively. The recombinant SCR was successfully applied for efficient production of (3R,5S)-CDHH. The scale-up synthesis of (3R,5S)-CDHH was performed in 5000 L bioreactor with 400 g/L of (S)-CHOH at 30°C, resulting in a space-time yield of 13.7 mM/h/g DCW, which was the highest ever reported. After isolation and purification, the yield and d.e. of (3R,5S)-CDHH reached 97.5% and 99.5%, respectively. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:612-620, 2017 [ABSTRACT FROM AUTHOR]

Optically pure t-butyl 6-cyano-( 3R, 5R)-dihydroxyhexanoate (( R)- 1b) is the key precursor for atorvastatin calcium, the most widely used cholesterol-lowering drug. In this work, a strain ZJB-09224 capable of asymmetrically reducing t-butyl 6-cyano-( 5R)-hydroxy-3-oxohexanoate ( 1a) to corresponding optically pure ( R)- 1b was successfully isolated from soil sample, identified belonging to Rhodotorula glutinis based on the morphology, physiological tests, and the 18S rDNA sequence analysis. It was found that heat treatment of cell suspension at 45 °C for 25 Min significantly improved R. glutinis ZJB-09224 stereoselectivity. The asymmetric bioreduction of 1a was most efficient at pH 7.5, 35 °C, 50 mM (15.0 g L−1) substrate concentration, 40.0 g DCW L−1 cell loading size, 0.54 M (60.0 g L−1) sodium lactate acting as co-substrate. Under these optimal conditions, 0.046 M ( R)- 1b was produced with de (diastereomeric excess) value of 99.2% after 40 H conversion. Moreover, R. glutinis ZJB-09224 has a broad substrate spectrum, making it a potential tool for some valuable chiral alcohol pharmaceutical intermediates synthesis. [ABSTRACT FROM AUTHOR]

A water-soluble cationic gallium corrole, 5,10,15-tris( N-methyl-4-pyridyl)corrolatogallium(III) ( 3), was prepared and characterized. The photocytotoxicity of 3 was investigated using Hep G2 cancer cell line. Upon treatment with corrole 3 and irradiation, of tumor cell nuclei was observed, which led to apoptosis. Flow cytometric analysis clearly showed the efficient induction of apoptotic cell death, and corrole 3 exhibited high photocytotoxicity towards Hep G2 cancer cells (IC50 = 60 nM). Furthermore, the binding behavior of corrole 3 with c-MYC G-quadruplex DNA, a potent target for antitumor drugs, was investigated using spectroscopic methods and molecular docking simulation. Copyright © 2015 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

In this work, a separation strategy for fructose purification from high-fructose corn syrup (HFCS) was established, involving two consecutive instances of chromatography with Ca2+-immobilized cation exchanger XA 2004/22. Among the 14 calcium ion form of strong acid cation exchangers tested, cation exchanger XA 2004/22 displayed the best adsorption performances for fructose, with the adsorption ratio of fructose to glucose reaching 2.32 at 303 K. The static equilibrium adsorption data of fructose on Ca2+-immobilized cation exchanger XA 2004/22 closely fitted the linear equation, and the adsorption kinetic followed the pseudo-second order model. Close agreement between the theoretical curves and the experimental breakthrough data was observed using the Thomas and Yoon-Nelson models. Under the optimized conditions, fructose was prepared at a purity of 0.924 g/g (92.4 mass %) and 0.243 g/g (24.3 mass %) yield by two-step packed bed chromatography with Ca2+-immobilized cation exchanger XA 2004/22. Further introducing one step of simple cooling crystallization gave crystalline fructose at 0.99 g/g (99 mass %) purity. [ABSTRACT FROM AUTHOR]

ABSTRACT In this article, we present a process for preparing organovermiculites, which consist of expanded vermiculite (EVMT)-poly(vinyl alcohol) (PVOH) created by the mechanical ball-milling of EVMT in a PVOH-water solution. We then discusses the influence of EVMT-PVOH on the barrier performance, crystallization behavior, thermal stability, and mechanical properties of modified blown polypropylene (PP) films. EVMT was intercalated and exfoliated by PVOH macromolecules to obtain a kind of hybrid EVMT-PVOH. PVOH served as both an intercalating agent into EVMT and a compatibilizer between EVMT and PP. Compared with the original (unmodified) PP, when the EVMT loading ranged from 0.1 to 2.0%, although the crystallinity decreased for most PP films, the thermal stability and mechanical properties all improved. Moreover, EVMT platelike particles with a high aspect ratio (ca. 550) dispersed in the PP matrix also improved the barrier properties of the modified PP films, which was in accordance with the Nielsen model. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016, 132, 42846. [ABSTRACT FROM AUTHOR]

A novel and broadband semidisk microstrip antenna suitable for a portable and compact terminal integrated with the 3G IMT-2000 cellular system, DECT mobile system, and Bluetooth wireless technology is discussed both theoretically and experimentally. Using both bandwidth-enhancement and size-miniaturization methods, a broad impedance bandwidth (return loss ≤-10 dB) of 32.4% from 1.86 to 2.58 GHz is obtained. Typical characteristics of the proposed antenna are addressed and discussed. Good agreements are obtained in the measured and simulated results. © 2002 John Wiley & Sons, Inc. Microwave Opt Technol Lett 32: 204–207, 2002. [ABSTRACT FROM AUTHOR]

t-Butyl 6-cyano-(3R,5R)-dihydroxyhexanoate ((3R,5R)-2) is a key chiral diol precursor of atorvastatin calcium (Lipitor®). We have constructed a Kluyveromyces lactis aldo-keto reductase mutant KlAKR-Y295W/W296L (KlAKRm) by rational design in previous research, which displayed high activity and excellent diastereoselectivity (de p  > 99.5%) toward t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate ((5R)-1). To realize in situ cofactor regeneration, a robust KlAKRm and Exiguobacterium sibiricum glucose dehydrogenase (EsGDH) co-producer E. coli BL 21(DE3) pETDuet-esgdh (MCS1)/pET-28b (+)-klakrm was constructed in this work. Under the optimized conditions, AKR and GDH activities of E. coli BL 21(DE3) pETDuet-esgdh (MCS1)/pET-28b (+)-klakrm peaked at 249.9 U/g DCW (dry cellular weight) and 29100 U/g DCW, respectively. It completely converted (5R)-1 at substrate loading size of up to 60.0 g/L (5R)-1 in the absence of exogenous NADH, which was one-fifth higher than that of the separately prepared KlAKRm and EsGDH under the same conditions. In this manner, a biocatalytic process for (3R,5R)-2 with productivity of 243.2 kg/m 3  d was developed. Compared with the combination of separate expressed KlAKRm with EsGDH, co-expression of KlAKRm and EsGDH has the advantages of alleviating cell cultivation burden and elevating substrate load. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1235-1242, 2017., (© 2017 American Institute of Chemical Engineers.)

The title compound, C(18)H(22)O(6), was obtained by the domino oxa-Michael-aldol (DOMA) reaction and has the cyclo-hexa-none ring in a chair conformation with intra-annular torsion angles in the range 49.9 (2)-58.9 (2)°. The two eth-oxy-carbonyl substituents on the cyclo-hexa-none ring adopt a syn configurations. In the crystal, the mol-ecules self-assemble through duplex inter-molecular hy-droxy-carbonyl O-H⋯O hydrogen bonds, giving centrosymmetric cyclic dimers [graph set R(2) (2)(12)] which inter-associate through weak C-H⋯O hydrogen-bonding inter-actions.