Semirational engineering of Cytophaga hutchinsonii polyphosphate kinase for developing a cost-effective, robust, and efficient adenosine 5'-triphosphate regeneration system.

IMPORTANCE: The adenosine 5'-triphosphate (ATP) regeneration system can significantly reduce the cost of many biocatalytic processes. Numerous studies have endeavored to utilize the ATP regeneration system based on Cytophaga hutchinsonii PPK (ChPPK). However, the wild-type ChPPK enzyme possesses limitations such as low enzymatic activity, poor stability, and limited substrate tolerance, impeding its application in catalytic reactions. To enhance the performance of ChPPK, we employed a semi-rational design approach to obtain the variant ChPPK/A79G/S106C/I108F/L285P. The enzymatic kinetic parameters and the catalytic performance in the synthesis of nicotinamide mononucleotide demonstrated that the variant ChPPK/A79G/S106C/I108F/L285P exhibited superior enzymatic properties than the wild-type enzyme. All data indicated that our engineered ATP regeneration system holds inherent potential for implementation in biocatalytic processes.

Identification of a novel promoter for driving antibiotic-resistant genes to reduce the metabolic burden during protein expression and effectively select multiple integrations in Pichia Pastoris.

Routine approaches for the efficient expression of heterogenous proteins in Pichia pastoris include using the strong methanol-regulated alcohol oxidase (AOX1) promoter and multiple inserts of expression cassettes. To screen the transformants harboring multiple integrations, antibiotic-resistant genes such as the Streptoalloteichus hindustanus bleomycin gene are constructed into expression vectors, given that higher numbers of insertions of antibiotic-resistant genes on the expression vector confer resistance to higher concentrations of the antibiotic for transformants. The antibiotic-resistant genes are normally driven by the strong constitutive translational elongation factor 1a promoter (PTEF1). However, antibiotic-resistant proteins are necessary only for the selection process. Their production during the heterogenous protein expression process may increase the burden in cells, especially for the high-copy strains which harbor multiple copies of the expression cassette of antibiotic-resistant genes. Besides, a high concentration of the expensive antibiotic is required for the selection of multiple inserts because of the effective expression of the antibiotic-resistant gene by the TEF1 promoter. To address these limitations, we replaced the TEF1 promoter with a weaker promoter (PDog2p300) derived from the potential promoter region of 2-deoxyglucose-6-phosphate phosphatase gene for driving the antibiotic-resistant gene expression. Importantly, the PDog2p300 has even lower activity under carbon sources (glycerol and methanol) used for the AOX1 promoter-based production of recombinant proteins compared with glucose that is usually used for the selection process. This strategy has proven to be successful in screening of transformants harboring more than 3 copies of the gene of interest by using plates containing 100 µg/ml of Zeocin. Meanwhile, levels of Zeocin resistance protein were undetectable by immunoblotting in these multiple-copy strains during expression of heterogenous proteins.Key points• PDog2p300 was identified as a novel glucose-regulated promoter.• The expression of antibiotic-resistant gene driven by PDog2p300 was suppressed during the recombinant protein expression, resulting in reducing the metabolic burden.• The transformants harboring multiple integrations were cost-effectively selected by using the PDog2p300 for driving antibiotic-resistant genes.

Engineering a Pichia pastoris nitrilase whole cell catalyst through the increased nitrilase gene copy number and co-expressing of ER oxidoreductin 1.

1-Cyanocyclohexaneacetic acid (1-CHAA) is a critical intermediate for the synthesis of the antiepileptic agent gabapentin. Previously, our group has established a novel manufacturing route for 1-CHAA through bioconversion catalyzed by an Escherichia coli (E. coli) nitrilase whole cell catalyst. However, the nitrilase expressed in E. coli has several drawbacks such as a low level of reusability, which hampered its industrial application. Herein, we investigated the potential of using the methylotrophic yeast Pichia pastoris (P. pastoris) for producing the nitrilase whole cell catalyst. To achieve strains with high catalytic activities, we investigated the effects of the promoter choice, expressing cassette copy number, and co-expression of chaperone on the production of nitrilase. Our results demonstrated that the strain harboring the multicopy integrations of nitrilase gene under the control of the alcohol oxidase 1 (AOX1) promoter and co-expressing of ER oxidoreductin 1 (ERO1) exhibited an 18-fold enhancement in the nitrilase activity compared with the strain containing a single integration of nitrilase gene under the control of glyceraldehyde-3-phosphate (GAP) dehydrogenase promoter. This optimized P. pastoris strain, compared with the E. coli nitrilase whole cell catalyst, shows greatly improved levels of reusability and thermostability while has a similar high-substrate tolerance.

Highly efficient conversion of 1-cyanocycloalkaneacetonitrile using a "super nitrilase mutant".

Nitrilase is the member of carbon-nitrogen hydrogen hydrolase superfamily, which has been widely used for the hydrolysis of nitriles into corresponding carboxylic acids. But most nitrilases are plagued by product inhibition in the industrial application. In this study, a "super nitrilase mutant" of nitrilase with high activity, thermostability and improved product tolerance from Acidovorax facilis ZJB09122 was characterized. Then, an efficient process was developed by employing the whole cell of recombinant E. coli for the conversion of high concentration of 1-cyanocyclohexylacetonitrile-to-1-cyanocyclohexaneacetic acid, an important intermediate of gabapentin. Under the optimized conditions, the higher substrate concentrations such as 1.3 M, 1.5 M and 1.8 M could be hydrolyzed by 13.58 g DCW/L with outstanding productivity (> 740 g/L/day). This study developed a highly efficient bioprocess for the preparation of 1-cyanocyclohexaneacetic acid which has the great potential for industrial application.

Design of a Molecular Hybrid of Dual Peptide Inhibitors Coupled on AuNPs for Enhanced Inhibition of Amyloid ß-Protein Aggregation and Cytotoxicity.

Aggregation of amyloid-ß protein (Aß) is a pathological hallmark of Alzheimer's disease (AD), so the inhibition of Aß aggregation is an important strategy for the prevention and treatment of AD. Herein, we proposed to design molecular hybrids of peptide inhibitors by combining two peptide inhibitors, VVIA and LPFFD, into single sequences and examined their effects on Aß42 aggregation and cytotoxicity. The hybrid peptides exhibit increased but moderate inhibitory activity as compared to their two precursors. By conjugating the peptides onto gold nanoparticles (AuNPs), however, the inhibition activity of the corresponding peptide@AuNPs against Aß42 aggregation and cytotoxicity is greatly improved. Among them, VVIACLPFFD (VCD10)@AuNP is the most effective, which increases cell viability from 48% to 82% at a dosage as low as 0.1 nmol L-1 (NPs) or 40 nmol L-1 (peptide). The superior capacity of VCD10@AuNPs is considered due to its branched dual-inhibitor sequence, and its special surface orientation and conformation. These structural features promote its synergetic interactions with Aß on AuNP surface, leading to strong inhibitions of Aß oligomerization and fibrillation and the cytotoxicity caused by the aggregation species. The findings suggest that potent inhibitors can be derived by hybridization of multiple peptide inhibitors with the hybrid products coupled onto nanoparticles.

Quantitatively Visualizing the Thermal Dehydration Process and Isotope Effect in Single HKUST-1 Metal-Organic Framework Particles.

Quantitatively visualizing the thermal dehydration in metal-organic frameworks (MOFs), especially at the single-particle level, is still challenging, hindering a deeper understanding of the reaction dynamics. Using in situ dark-field microscopy (DFM), we image the thermal dehydration process of single water-containing HKUST-1 (H2O-HKUST-1) metal-organic framework (MOF) particles. DFM maps the color intensity of single H2O-HKUST-1, which is linearly correlated with the water content in the HKUST-1 framework, enabling a direct quantification of several reaction kinetic parameters of single HKUST-1 particles. Interestingly, when H2O-HKUST-1 is transformed into deutoxide (D2O)-containing HKUST-1, the corresponding thermal dehydration reaction displays higher temperature parameters and activation energy but shows a lower rate constant and diffusion coefficient, revealing the isotope effect. The significant variation of the diffusion coefficient is also confirmed by molecular dynamics simulations. The present operando results are anticipated to provide valuable guidelines for the design and development of advanced porous materials.

Immobilization of Escherichia coli cells harboring a nitrilase with improved catalytic properties though polyethylenemine-induced silicification on zeolite.

In the chemical-biological synthesis route of gabapentin, immobilized Escherichia coli cells harboring nitrilase are used to catalyze the biotransformation of intermediate 1-cyanocyclohexaneacetonitile to 1-cyanocyclohexaneacetic acid. Herein, we present a novel cell immobilization method, which is based on cell adsorption using 75 g/L Escherichia coli cells and 6 g/L zeolite, cell crosslinking using 3 g/L polyethylenemine and biomimetic silicification using 18 g/L hydrolyzed tetramethylorthosilicate. The constructed "hybrid biomimetic silica particles (HBSPs)" with core-shell structure showed a specific activity of 147.2 ± 2.3 U/g, 82.6 ± 2.8% recovery of nitrilase activity and a half-life of 19.1 ± 1.9 h at 55 °C. 1-Cyanocyclohexaneacetonitrile (1.0 M) could be completely hydrolyzed by 50 g/L of HBSPs at pH 7.5, 35 °C in 4 h, providing 92.1 ± 3.2% yield of 1-cyanocyclohexaneacetic acid. In batch reactions, the HBSPs could be reused for 13 cycles and maintained 79.9 ± 4.1% residual activity after the 10th batch, providing an average product yield of 92.6% in the first 10 batches with a productivity of 619.3 g/L/day. In addition, multi-layer structures consisting of silica coating and polyethylenemine/glutaraldehyde crosslinking were constructed to enhance the mechanical strength of immobilized cells, and the effects of coating layers on the catalytic properties of immobilized cells was discussed.

Numerical study on the influence of wall temperature gradient on aerodynamic characteristics of low aspect ratio flying wing configuration.

With the aim for a low-aspect-ratio flying wing configuration, this study explores the influence of wall temperature gradient on the laminar and turbulent boundary layers of aircraft surface and determines the effect on the transition Reynolds number and wall friction drag. A four-equation turbulence model with transition mode is used to numerically simulate the flow around the model. The variation of wall friction coefficient, transition Reynolds number, and turbulent boundary layer flow with wall temperature are emphatically investigated. Results show that when the wall temperature increases from 288 to 500 K, the boundary layer transition Reynolds number for the wing section increased by approximately 28% and the surface friction drags decreases by approximately 10.7%. The hot wall enhances the viscous effects of the laminar temperature boundary layer, reduces the Reynolds shear stress and turbulent kinetic energy, and increases the flow stability. However, the velocity gradient and shear stress in the bottom of the turbulent boundary layer decreases, which leads to reduced friction shear stress on the wall surface. Therefore, for the low-aspect-ratio flying wing model, the hot wall can delay the boundary layer transition and reduce the friction drag coefficient in the turbulent region.

Leaching of Valuable Elements from the Waste Chromite Ore Processing Residue: A Kinetic Analysis.

The efficacious treatment and resource utilization of the chromite ore processing residue (COPR) is important for chromate salt production. In this study, the leaching of valuable elements from the waste COPR was investigated. X-ray diffraction (XRD) analysis showed that the COPR mainly contained periclase (MgCr2O4), magnesiochromite ((Fe, Mg) (Cr, Fe)2O4), Fe (Cr, Al)2O4, and MgFeAlO4. The optimum parameters for COPR leaching were as follows: mechanical ball-milling time of 120 min, sulfuric acid concentration (w/w % H2SO4) of 60%, reaction temperature (T) of 403 K, liquid-solid ratio (L/S) of 8 mL/g, and reaction time (t) of 6 h. Under these conditions, the valuable components such as Fe, Al, and Cr were extracted with an ideal leaching efficiency of 94.8, 75.1, and 76%, respectively. The results of the leaching kinetics analysis indicated that the leaching of Fe and Cr from the COPR was controlled by a surface chemical reaction, and the leaching of Al was controlled by diffusion through a product layer. The apparent activation energy of the leaching of Fe, Cr, and Al was calculated to be 23.03, 44.15, and 17.54 kJ/mol, respectively. It is believed that this approach has potential applications for the chromate salt industry because of its advantage of ideal leaching efficiency.

Integrated guidance and control for missile with narrow field-of-view strapdown seeker.

Due to the removal of the mechanically stable platform in the conventional gimbaled seeker, the strapdown seeker's measurement is coupled with the missile body attitude motion, such that the inertial line-of-sight (LOS) angular rate required to implement traditional guidance laws cannot be measured, and the field-of-view (FOV) limit must be considered when designing guidance and control systems for a strapdown homing missile. To address these practical problems, an integrated guidance and control (IGC) scheme with considering the FOV limit is proposed in this paper. A novel IGC model is first derived based on the body-LOS (BLOS) angle that a strapdown seeker can directly measure, and then an IGC controller is designed using the dynamic surface control technique. A great merit of this design is that the inertial LOS angle and its angular rate are not needed, and thus the filters/estimators required to extract this guidance information in previous studies can be canceled. Next, by using the output to input saturation transformation (OIST) technique, the FOV limit, which is always considered as a state/output constraint, is transformed to a time-varying boundary limitation on the control input, and then is handled simultaneously with the actuator saturation constraint. Finally, extensive numerical simulations against both stationary and moving targets are performed to fully demonstrate the efficiency of the proposed IGC law.

Separation and purification of l-methionine from E. coli fermentation broth by macroporous resin chromatography.

Methionine is an essential sulfur-containing amino acid for organisms. The separation and purification are important for the production of l-methionine from fermentation broth. In this work, the adsorption properties for l-methionine separation of ten macroporous resins were firstly evaluated. Macroporous cation resin D72 showed the best adsorption capacity (52.37 mg/g) and desorption rate (99.12%). The adsorption kinetics of l-methionine on D72 resin followed the pseudo second-order model and adsorption isotherm fitted well to the Sips model, the adsorption process mainly followed a physisorption mechanism. D72 resin packed columns were then used in the batch separation of fermentation broth, the operation parameters were optimized during the dynamic adsorption and desorption experiments. Under the optimized conditions: fermentation broth adjusted to pH = 2, loading flow rate at 2 BV/h, loading volume 55 mL, 1 mol/L NH3·H2O as eluent, elution flow rate at 2 BV/h, column height-diameter ratio at 14:1, excellent recovery and purity of l-methionine (82.37% and 85.69%, respectively) could be achieved.

Engineering the residues on "A" surface and C-terminal region to improve thermostability of nitrilase.

Nitrilases can hydrolyze nitriles to corresponding carboxylic acids in one single step, which have great potential as valuable biocatalysts for chemical synthesis. However, the poor thermostability of the nitrilases restrict their applications in industry. In this work, error-prone PCR and site-directed mutagenesis were utilized to improve the thermostability of nitrilases. Several mutants (AcN-Q339K, AcN-Q343K, AcN-T201F, AcN-T201W, AcN-T201L, AcN-T201I) were obtained with dramatically improved thermostability. The best mutant AcN-T201F/Q339K/Q343K exhibited about 14-fold longer half-life at 45 °C. The result of homology modeling suggested that the site 201, which was located on the "A" surface (the dimer interaction), played an important role in the oligomerization of nitrilase and the stabilization of substrate binding pocket. The Phe substitution on site 201 was selected in protein engineering of nitrilase LNIT5, which also demonstrated an improvement of thermostability. In addition, lysine substitution on Q339 and Q343 which brought positive charges to the α helix in the C-terminal region stabilized the surface.

Design of LVFFARK and LVFFARK-functionalized nanoparticles for inhibiting amyloid ß-protein fibrillation and cytotoxicity.

Aggregation of amyloid ß-protein (Aß) into amyloid oligomers and fibrils is pathologically linked to Alzheimer's disease (AD). Hence, the inhibition of Aß aggregation is essential for the prevention and treatment of AD, but the development of potent agents capable of inhibiting Aß fibrillogenesis has posed significant challenges. Herein, we designed Ac-LVFFARK-NH2 (LK7) by incorporating two positively charged residues, R and K, into the central hydrophobic fragment of Aß17-21 (LVFFA) and examined its inhibitory effect on Aß42 aggregation and cytotoxicity by extensive physical, biophysical, and biological analyses. LK7 was observed to inhibit Aß42 fibrillogenesis in a dose-dependent manner, but its strong self-assembly characteristic also resulted in high cytotoxicity. In order to prevent the cytotoxicity that resulted from the self-assembly of LK7, the peptide was then conjugated to the surface of poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) to fabricate a nanosized inhibitor, LK7@PLGA-NPs. It was found that LK7@PLGA-NPs had little cytotoxicity because the self-assembly of the LK7 conjugated on the NPs was completely inhibited. Moreover, the NPs-based inhibitor showed remarkable inhibitory capability against Aß42 aggregation and significantly alleviated its cytotoxicity at a low LK7@PLGA-NPs concentration of 20 µg/mL. At the same peptide concentration, free LK7 showed little inhibitory effect. It is considered that several synergetic effects contributed to the strong inhibitory ability of LK7@PLGA-NPs, including the enhanced interactions between Aß42 and LK7@PLGA-NPs brought on by inhibiting LK7 self-assembly, restricting conformational changes of Aß42, and thus redirecting Aß42 aggregation into unstructured, off-pathway aggregates. The working mechanisms of the inhibitory effects of LK7 and LK7@PLGA-NPs on Aß42 aggregation were proposed based on experimental observations. This work provides new insights into the design and development of potent NPs-based inhibitors against Aß aggregation and cytotoxicity.

A novel nickel-chelated surfactant for affinity-based aqueous two-phase micellar extraction of histidine-rich protein.

Aqueous two-phase micellar systems (ATPMSs) composed of nonionic surfactants are considered promising for the separation and purification of proteins. To improve the specificity of ATPMSs, a novel nickel-chelated surfactant was prepared by successive modifications of Triton X-114 (TX). Characterizations by Fourier transformation infrared spectroscopy demonstrated the successful synthesis of the nickel-chelated surfactant (TX-Ni). The cloud point, critical micelle concentration (CMC), molecular interaction parameter and micelle size were measured for the mixed surfactant system of TX-Ni and TX to achieve a full understanding of their aggregation behaviors. The results showed that mixed micelles were formed, and the cloud point increased with the mole fraction of TX-Ni because TX-Ni had a more hydrophilic head group than TX. Moreover, the reduction of micelle size revealed by light scattering experiments indicated that the insertion of TX-Ni inhibited the micellar growth due to the increased steric and electrostatic repulsion. Finally, the efficiency of TX-Ni as an affinity surfactant was demonstrated by the affinity partitioning of histidine-tagged enhanced green fluorescent protein with an over 20-fold increase of the partition coefficient (from 0.60 to 12.42). This affinity-based ATPMS is thus considered promising for providing a versatile platform for the separation of histidine-rich proteins.