Enhancing the organic solvent resistance of ω-amine transaminase for enantioselective synthesis of (R)-(+)-1(1-naphthyl)-ethylamine.

BACKGROUND: Biocatalysis in high-concentration organic solvents has been applied to produce various industrial products with many advantages. However, using enzymes in organic solvents often suffers from inactivation or decreased catalytic activity and stability. An R-selective ω-amine transaminase from Aspergillus terreus (AtATA) exhibited activity toward 1-acetylnaphthalene. However, AtATA displayed unsatisfactory organic solvent resistance, which is required to enhance the solubility of the hydrophobic substrate 1-acetylnaphthalene. So, improving the tolerance of enzymes in organic solvents is essential. MAIN METHODS AND RESULTS: The method of regional random mutation combined with combinatorial mutation was used to improve the resistance of AtATA in organic solvents. Enzyme surface areas are structural elements that undergo reversible conformational transitions, thus affecting the stability of the enzyme in organic solvents. Herein, three surface areas containing three loops were selected as potential mutation regions. And the "best" mutant T23I/T200K/P260S (M3) was acquired. In different concentrations of dimethyl sulfoxide (DMSO), the catalytic efficiency (kcat /Km ) toward 1-acetylnaphthalene and the stability (half-life t1/2 ) were higher than the wild-type (WT) of AtATA. The results of decreased Root Mean Square Fluctuation (RMSF) values via 20-ns molecular dynamics (MD) simulations under 15%, 25%, 35%, and 45% DMSO revealed that mutant M3 had lower flexibility, acquiring a more stable protein structure and contributing to its organic solvents stability than WT. Furthermore, M3 was applied to convert 1-acetylnaphthalene for synthesizing (R)-(+)-1(1-naphthyl)-ethylamine ((R)-NEA), which was an intermediate of Cinacalcet Hydrochloride for the treatment of secondary hyperthyroidism and hypercalcemia. Moreover, in a 20-mL scale-up experiment, 10 mM 1-acetylnaphthalene can be converted to (R)-NEA with 85.2% yield and a strict R-stereoselectivity (enantiomeric excess (e.e.) value >99.5%) within 10 h under 25% DMSO. CONCLUSION: The beneficial mutation sites were identified to tailor AtATA's organic solvents stability via regional random mutation. The "best" mutant T23I/T200K/P260S (M3) holds great potential application for the synthesis of (R)-NEA.

Production of Salvianic Acid A from l-DOPA via Biocatalytic Cascade Reactions.

Salvianic acid A (SAA), as the main bioactive component of the traditional Chinese herb Salvia miltiorrhiza, has important application value in the treatment of cardiovascular diseases. In this study, a two-step bioprocess for the preparation of SAA from l-DOPA was developed. In the first step, l-DOPA was transformed to 3,4-dihydroxyphenylalanine (DHPPA) using engineered Escherichia coli cells expressing membrane-bound L-amino acid deaminase from Proteus vulgaris. After that, the unpurified DHPPA was directly converted into SAA by permeabilized recombinant E. coli cells co-expressing d-lactate dehydrogenase from Pediococcus acidilactici and formate dehydrogenase from Mycobacterium vaccae N10. Under optimized conditions, 48.3 mM of SAA could be prepared from 50 mM of l-DOPA, with a yield of 96.6%. Therefore, the bioprocess developed here was not only environmentally friendly, but also exhibited excellent production efficiency and, thus, is promising for industrial SAA production.

Anatomical Basis for Malar Augmentation Injection With the Zygomatic Ligamentous System.

BACKGROUND: The malar augmentation injection has gained popularity in recent years, but the exact location of each injection site has not been clearly identified. OBJECTIVE: To discover ideal injection sites by comprehensively considering the distributions of ligaments, muscles, and vessels. MATERIALS AND METHODS: Eighteen cadaver heads were dissected to investigate the zygomatic ligamentous system and to measure the position of muscles. Sixty-six cadaver heads were subjected to computed tomographic scanning and three-dimensional vessel reconstruction. Radiological evaluation of the fillers was performed before and after experimental injection in one hemiface and dissected to confirm safe delivery. Five patients were enrolled in a prospective clinical study. 2D and 3D photographs were taken before and after the injections for comparison. RESULTS: Site 1 was defined along the zygomatic arch, except the first 1/4 length and the midline of the arch. Site 2 was on the body of the zygoma, superior to the level of the infraorbital foramen and medial to the jugale. Site 3 was defined in the anteromedial midface approximately 30 mm below the lateral canthus. CONCLUSION: Injections at these 3 sites can be performed within the range of the ligaments to achieve effective lifting effects and minimize potential complications.

Improving the Thermostability and Activity of Transaminase From Aspergillus terreus by Charge-Charge Interaction.

Transaminases that promote the amination of ketones into amines are an emerging class of biocatalysts for preparing a series of drugs and their intermediates. One of the main limitations of (R)-selective amine transaminase from Aspergillus terreus (At-ATA) is its weak thermostability, with a half-life (t 1/2) of only 6.9 min at 40°C. To improve its thermostability, four important residue sites (E133, D224, E253, and E262) located on the surface of At-ATA were identified using the enzyme thermal stability system (ETSS). Subsequently, 13 mutants (E133A, E133H, E133K, E133R, E133Q, D224A, D224H, D224K, D224R, E253A, E253H, E253K, and E262A) were constructed by site-directed mutagenesis according to the principle of turning the residues into opposite charged ones. Among them, three substitutions, E133Q, D224K, and E253A, displayed higher thermal stability than the wild-type enzyme. Molecular dynamics simulations indicated that these three mutations limited the random vibration amplitude in the two α-helix regions of 130-135 and 148-158, thereby increasing the rigidity of the protein. Compared to the wild-type, the best mutant, D224K, showed improved thermostability with a 4.23-fold increase in t 1/2 at 40°C, and 6.08°C increase in T 50 10 . Exploring the three-dimensional structure of D224K at the atomic level, three strong hydrogen bonds were added to form a special "claw structure" of the α-helix 8, and the residues located at 151-156 also stabilized the α-helix 9 by interacting with each other alternately.

Three-Dimensional Computed Tomography Scanning of Temporal Vessels to Assess the Safety of Filler Injections.

BACKGROUND: Temple filler injection is one of the most common minimally invasive cosmetic procedures involving the face; however, vascular complications are not uncommon. OBJECTIVES: This study aimed to investigate the anatomy of the temporal vessels and provide a more accurate protocol for temple filler injection. METHODS: Computed tomography (CT) scans of 56 cadaveric heads injected with lead oxide were obtained. We then used Mimics software to construct 3-dimensional (3D) images of the temporal vessels described by a coordinate system based on the bilateral tragus and right lateral canthus. RESULTS: In the XOY plane, the superficial temporal artery (STA), middle temporal artery (MTA), zygomatico-orbital artery (ZOA), posterior branch of the deep temporal artery (PDTA), and lateral margin of the orbital rim divide the temple into 4 parts (A, B, C, and D). The probabilities of the STA, MTA, ZOA, and PDTA appearing in parts A, B, C, and D were 30.73%, 37.06%, 39.48%, and 77.18%, respectively. In 3D images, these vessels together compose an arterial network that is anastomosed with other vessels, such as the external carotid, facial, and ocular arteries. CONCLUSIONS: 3D CT images can digitally elucidate the exact positions of temporal vessels in a coordinate system, improving the safety of temple filler injections in a clinical setting.

Three-Dimensional Computed Tomographic Study on the Vessels of the Zygomatic Region: Arterial Variations and Clinical Relevance.

BACKGROUND: Injection-based techniques for "cheek augmentation" have gained popularity in recent years. The aim of this study was to perform a topographic analysis of the depth and distribution of the vessels in the zygomatic region to facilitate clinical procedures. METHODS: The external carotid arteries of seven cadaveric heads were infused with lead oxide contrast medium. The facial and superficial temporal arteries of another 12 cadaveric heads were injected sequentially with the same medium. Computed tomographic scanning was then performed, and three-dimensional computed tomographic scans were reconstructed using validated algorithms. RESULTS: The vessels on the zygomatic arch received a double blood supply from across the upper and lower borders of the arch, and the number of the vessels varied from one to four. Ninety percent of the vessels on the zygomatic arch were at a depth of 1 to 2.5 mm, and 75 percent were at a depth of 10 to 30 percent of the soft-tissue thickness. The vessels were concentrated on the midline of the zygomatic arch and the lateral margin of the frontal process. All samples showed a vessel travel along the lateral margin of the frontal process that eventually merged into the superior marginal arcades. CONCLUSIONS: This study reported a topographic analysis of the depth and distribution of the vessels in the zygomatic region based on three-dimensional scanning. The results indicated that injection on the zygomatic arch should be performed deep to the bone, and the vascular zones anterior or posterior to the midline of the zygomatic arch were relatively safe injection areas.

Parallel Strategy Increases the Thermostability and Activity of Glutamate Decarboxylase.

Glutamate decarboxylase (GAD; EC 4.1.1.15) is a unique pyridoxal 5-phosphate (PLP)-dependent enzyme that specifically catalyzes the decarboxylation of L-glutamic acid to produce γ-aminobutyric acid (GABA), which exhibits several well-known physiological functions. However, glutamate decarboxylase from different sources has the common problem of poor thermostability that affects its application in industry. In this study, a parallel strategy comprising sequential analysis and free energy calculation was applied to identify critical amino acid sites affecting thermostability of GAD and select proper mutation contributing to improve structure rigidity of the enzyme. Two mutant enzymes, D203E and S325A, with higher thermostability were obtained, and their semi-inactivation temperature (T5015) values were 2.3 °C and 1.4 °C higher than the corresponding value of the wild-type enzyme (WT), respectively. Moreover, the mutant, S325A, exhibited enhanced activity compared to the wild type, with a 1.67-fold increase. The parallel strategy presented in this work proved to be an efficient tool for the reinforcement of protein thermostability.

A Single Mutation Increases the Thermostability and Activity of Aspergillus terreus Amine Transaminase.

Enhancing the thermostability of (R)-selective amine transaminases (AT-ATA) will expand its application in the asymmetric synthesis of chiral amines. In this study, mutual information and coevolution networks of ATAs were analyzed by the Mutual Information Server to Infer Coevolution (MISTIC). Subsequently, the amino acids most likely to influence the stability and function of the protein were investigated by alanine scanning and saturation mutagenesis. Four stabilized mutants (L118T, L118A, L118I, and L118V) were successfully obtained. The best mutant, L118T, exhibited an improved thermal stability with a 3.7-fold enhancement in its half-life (t1/2) at 40 °C and a 5.3 °C increase in T5010 compared to the values for the wild-type protein. By the differential scanning fluorimetry (DSF) analysis, the best mutant, L118T, showed a melting temperature (Tm) of 46.4 °C, which corresponded to a 5.0 °C increase relative to the wild-type AT-ATA (41.4 °C). Furthermore, the most stable mutant L118T displayed the highest catalytic efficiency among the four stabilized mutants.

Construction of stabilized (R)-selective amine transaminase from Aspergillus terreus by consensus mutagenesis.

Amine transaminases are a class of efficient and industrially-desired biocatalysts for the production of chiral amines. In this study, stabilized variants of the (R)-selective amine transaminase from Aspergillus terreus (AT-ATA) were constructed by consensus mutagenesis. Using Consensus Finder (http://cbs-kazlab.oit.umn.edu/), six positions with the most prevalent amino acid (over 60% threshold) among the homologous family members were identified. Subsequently, these six residues were individually mutated to match the consensus sequence (I77 L, Q97E, H210N, N245D, G292D, and I295 V) using site-directed mutagenesis. Compared to that of the wild-type, the thermostability of all six single variants was improved. The H210N variant displayed the largest shift in thermostability, with a 3.3-fold increase in half-life (t1/2) at 40 °C, and a 4.6 °C increase in T5010 among the single variants. In addition, the double mutant H210N/I77L displayed an even larger shift with 6.1-fold improvement of t1/2 at 40 °C, and a 6.6 °C increase in T5010. Furtherly, the H210N/I77L mutation was introduced into the previously engineered thermostable AT-ATA by the introduction of disulfide bonds, employing B-factor and folding free energy (ΔΔGfold) calculations. Our results showed that the combined variant H210N/I77L/M150C-M280C had the largest shift in thermostability, with a 16.6-fold improvement of t1/2 and a 11.8 °C higher T5010.

Biosynthesis of γ-aminobutyrate by engineered Lactobacillus brevis cells immobilized in gellan gum gel beads.

γ-Aminobutyrate (GABA) is an important chemical in pharmaceutical field. The use of lactic acid bacteria as biocatalysts for the conversion of l-monosodium glutamate (MSG) into GABA opens interesting perspectives for the production of this functional compound. In this work, an engineered GABA high-producing strain Lactobacillus brevis GadAΔC14 was constructed by overexpressing a C-terminally truncated GadA mutant, which is active in expanded pH range. After comparison with agar and κ-carrageenan, gellan gum was selected as the optimal immobilization support, and the properties of L. brevis GadAΔC14 cells encapsulated in this hydrogel were examined. The optimum pH and temperature of immobilized cells were found to be 40°C and pH 4.4, respectively. It was also observed that operational and thermal stabilities of the cells were increased with immobilization. After ten consecutive reaction cycles, the total amounts of GABA produced by the immobilized cells summed up to 87.56 g/L under the optimum experimental conditions. Taken together, the improved stability and good usability make the immobilized L. brevis GadAΔC14 cells more valuable for industrial applications.

Three-Dimensional Computed Tomographic Study on the Periorbital Branches of the Ophthalmic Artery: Arterial Variations and Clinical Relevance.

BACKGROUND: Filler injection is a popular cosmetic procedure, but it can entail vascular complications. Periorbital injections have the highest risk within the entire injection area. OBJECTIVES: The authors sought to systematically screen for periorbital arterial variations prior to treatment. METHODS: The external carotid arteries of 10 cadaveric heads were infused with adequate lead oxide contrast. The facial and superficial temporal arteries of another 11 cadaveric heads were injected with the contrast in sequential order. Computed tomography (CT) scanning was performed after injection of contrast, and 3-dimensional (3D) CT scans were reconstructed using validated algorithms. RESULTS: Three types of periorbital blood vessels were found to derive from the ophthalmic artery, including 30% directly originating from the ophthalmic artery, 65% originating from its trochlear branch, and 5% originating from its supraorbital branch. In the forehead, the ophthalmic artery, originating from the internal carotid arteries, formed anastomoses between the frontal branch of the superficial temporal artery, originating from the external carotid artery, with the deep and superficial branches of the supratrochlear and supraorbital arteries, respectively. The lateral orbit and malar plexus can be classified into 4 types based on the trunk artery: the zygomatic orbital artery (27%), the transverse facial artery (23%), the premasseteric branch of the facial artery (19%), and all 3 contributing equally (31%). CONCLUSIONS: Postmortem 3D CT can map periorbital arterial variations. The branching pattern of the ophthalmic artery, the ophthalmic angiosome in the forehead, and the distribution of the lateral orbit and malar plexus were identified at high resolution to guide clinical practice.

High-Throughput Screening of Full-Face Clinically Relevant Arterial Variations Using Three-Dimensional Postmortem Computed Tomography.

BACKGROUND: Vascular complications resulting from intravascular filler injection and embolism are major safety concerns for facial filler injection. It is essential to systematically screen full-face arterial variations and help design evidence-based safe filler injection protocols. METHODS: The carotid arteries of 22 cadaveric heads were infused with adequate lead oxide contrast. The facial and superficial temporal arteries of another 12 cadaveric heads were injected with the contrast in a sequential order. A computed tomographic scan was acquired after each contrast injection, and each three-dimensional computed tomographic scan was reconstructed using validated algorithms. RESULTS: Three-dimensional computed tomography clearly demonstrated the course, relative depth, and anastomosis of all major arteries in 63 qualified hemifaces. The ophthalmic angiosome consistently deploys two distinctive layers of branch arteries to the forehead. The superficial temporal and superior palpebral arteries run along the preauricular and superior palpebral creases, respectively. The study found that 74.6 percent of the hemifaces had nasolabial trunks coursing along the nasolabial crease, and that 50.8 percent of the hemifaces had infraorbital trunks that ran through the infraorbital region. Fifty percent of the angular arteries were the direct anastomotic channels between the facial and ophthalmic angiosomes, and 29.2 percent of the angular arteries were members of the ophthalmic angiosomes. CONCLUSIONS: Full-face arterial variations were mapped using postmortem three-dimensional computed tomography. Facial creases were in general correlated with underlying deep arteries. Facial and angular artery variations were identified at high resolution, and reclassified into clinically relevant types to guide medical practice.

Lactobacillus brevis CGMCC 1306 glutamate decarboxylase: Crystal structure and functional analysis.

Glutamate decarboxylase (GAD), which is a unique pyridoxal 5-phosphate (PLP)-dependent enzyme, can catalyze α-decarboxylation of l-glutamate (L-Glu) to γ-aminobutyrate (GABA). The crystal structure of GAD in complex with PLP from Lactobacillus brevis CGMCC 1306 was successfully solved by molecular-replacement, and refined at 2.2 Šresolution to an Rwork factor of 18.76% (Rfree = 23.08%). The coenzyme pyridoxal 5-phosphate (PLP) forms a Schiff base with the active-site residue Lys279 by continuous electron density map, which is critical for catalysis by PLP-dependent decarboxylase. Gel filtration showed that the active (pH 4.8) and inactive (pH 7.0) forms of GAD are all dimer. The residues (Ser126, Ser127, Cys168, Ile211, Ser276, His278 and Ser321) play important roles in anchoring PLP cofactor inside the active site and supporting its catalytic reactivity. The mutant T215A around the putative substrate pocket displayed an 1.6-fold improvement in catalytic efficiency (kcat/Km) compared to the wild-type enzyme (1.227 mM-1 S-1 versus 0.777 mM-1 S-1), which was the highest activity among all variants tested. The flexible loop (Tyr308-Glu312), which is positioned near the substrate-binding site, is involved in the catalytic reaction, and the conserved residue Tyr308 plays a vital role in decarboxylation of L-Glu.

Physiology-Oriented Engineering Strategy to Improve Gamma-Aminobutyrate Production in Lactobacillus brevis.

Gamma-aminobutyrate (GABA) is an important chemical in the pharmaceutical field. GABA-producing lactic acid bacteria (LAB) offer the opportunity of developing this health-oriented product. In this study, the gadA, gadB, gadC, gadCB, and gadCA gene segments of Lactobacillus brevis were cloned into pMG36e, and strain Lb. brevis/pMG36e-gadA was selected for thorough characterization in terms of GABA production after analysis of GAD activities. Subsequently, a physiology-oriented engineering strategy was adopted to construct an FoF1-ATPase deficient strain NRA6 with higher GAD activity. As expected, strain NRA6 could produce GABA at a concentration of 43.65 g/L with a 98.42% GABA conversion rate in GYP fermentation medium, which is 1.22-fold higher than that obtained by the wild-type strain in the same condition. This work demonstrates how the acid stress response mechanisms of LAB can be employed to develop cell factories with improved production efficiency and contributes to research into the development of the physiology-oriented engineering.

[Study on the effect of ultrasound on the secondary structure of BSA by FTIR].

Structure changes of bovine serum albumin (BSA) under ultrasound treatment were studied using Fourier transform infrared spectroscopy (FTIR) and fluorescence spectroscopy. The largest emission peak of BSA solution's fluorescence spectra shifted in blue orientation, indicating that the environment of the Trp residues in BSA had altered with ultrasound treatment. The fluorescence intensity of the solution has also decreased with ultrasound, which showed fluorescence quenching effect and the conformation changes of the BSA. The relative contents of a-helix, beta-fold, beta-turn and random coil under different ultrasound treatment power and time were quantitatively determined via analysis of the amide I changes of infrared spectra of BSA using curve fitting method, the secondary structure of BSA had variation trend from alpha-helix to beta-sheet, however, the relative contents random coil had not significant change.