A Novel Method for γ-Aminobutyric Acid Biosynthesis Using Glutamate Decarboxylase Entrapped in Polyvinyl Alcohol-Sodium Alginate Capsules.

γ-aminobutyric acid (GABA) has essential physiological functions in the human body. A novel method using glutamate decarboxylase (GAD) entrapped in polyvinyl alcohol (PVA)-sodium alginate (SA) capsules provides a green biological strategy for GABA synthesis. In this investigation, the stability range of immobilized GAD was effectively broadened, and immobilized GAD could be repeatedly used as a batch and fixed-bed column catalyst. The immobilized enzymes were stable and retained 89% of their activity in a pH range of 4.0-5.6, while there was an approximately 50% decrease in free GAD activity in the pH range of 4.8 ± 0.4. The immobilized GAD affinity to the substrate improved, and this was evidenced by the apparent decrease in Km to 13.3 mmol/L from the 30.9 mmol/L for free GAD. The immobilized GAD retained >90.6% activity after eight cycles and a near-100% enzyme activity retention after 120 h of a continuous fixed-bed column catalyst operation. This study has thus presented an effective PVA-SA-GAD immobilization method that could be used to continuously scale-up GABA biosynthesis.

Advances in 4-Hydroxyphenylacetate-3-hydroxylase Monooxygenase.

Catechols have important applications in the pharmaceutical, food, cosmetic, and functional material industries. 4-hydroxyphenylacetate-3-hydroxylase (4HPA3H), a two-component enzyme system comprising HpaB (monooxygenase) and HpaC (FAD oxidoreductase), demonstrates significant potential for catechol production because it can be easily expressed, is highly active, and exhibits ortho-hydroxylation activity toward a broad spectrum of phenol substrates. HpaB determines the ortho-hydroxylation efficiency and substrate spectrum of the enzyme; therefore, studying its structure-activity relationship, improving its properties, and developing a robust HpaB-conducting system are of significance and value; indeed, considerable efforts have been made in these areas in recent decades. Here, we review the classification, molecular structure, catalytic mechanism, primary efforts in protein engineering, and industrial applications of HpaB in catechol synthesis. Current trends in the further investigation of HpaB are also discussed.

Modification of the 4-Hydroxyphenylacetate-3-hydroxylase Substrate Pocket to Increase Activity towards Resveratrol.

4-Hydroxyphenylacetate-3-hydroxylase (4HPA3H; EC 1.14.14.9) is a heterodimeric flavin-dependent monooxygenase complex that catalyzes the ortho-hydroxylation of resveratrol to produce piceatannol. Piceatannol has various health benefits and valuable applications in food, medicine, and cosmetics. Enhancing the catalytic activity of 4HPA3H toward resveratrol has the potential to benefit piceatannol production. In this study, the critical amino acid residues in the substrate pocket of 4HPA3H that affect its activity toward resveratrol were identified using semi-rational engineering. Two key amino acid sites (I157 and A211) were discovered and the simultaneous "best" mutant I157L/A211D enabled catalytic efficiency (Kcat/Km-resveratrol) to increase by a factor of 4.7-fold. Molecular dynamics simulations indicated that the increased flexibility of the 4HPA3H substrate pocket has the potential to improve the catalytic activity of the enzyme toward resveratrol. On this basis, we produced 3.78 mM piceatannol by using the mutant I157L/A211D whole cells. In this study, we successfully developed a highly active 4HPA3H variant for the hydroxylation of resveratrol to piceatannol.

[Semi-rational evolution of ω-transaminase from Aspergillus terreus for enhancing the thermostability].

ω-transaminase (ω-TA) is a natural biocatalyst that has good application potential in the synthesis of chiral amines. However, the poor stability and low activity of ω-TA in the process of catalyzing unnatural substrates greatly hampers its application. To overcome these shortcomings, the thermostability of (R)-ω-TA (AtTA) from Aspergillus terreus was engineered by combining molecular dynamics simulation assisted computer-aided design with random and combinatorial mutation. An optimal mutant AtTA-E104D/A246V/R266Q (M3) with synchronously enhanced thermostability and activity was obtained. Compared with the wild- type (WT) enzyme, the half-life t1/2 (35 ℃) of M3 was prolonged by 4.8-time (from 17.8 min to 102.7 min), and the half deactivation temperature (T1050) was increased from 38.1 ℃ to 40.3 ℃. The catalytic efficiencies toward pyruvate and 1-(R)-phenylethylamine of M3 were 1.59- and 1.56-fold that of WT. Molecular dynamics simulation and molecular docking showed that the reinforced stability of α-helix caused by the increase of hydrogen bond and hydrophobic interaction in molecules was the main reason for the improvement of enzyme thermostability. The enhanced hydrogen bond of substrate with surrounding amino acid residues and the enlarged substrate binding pocket contributed to the increased catalytic efficiency of M3. Substrate spectrum analysis revealed that the catalytic performance of M3 on 11 aromatic ketones were higher than that of WT, which further showed the application potential of M3 in the synthesis of chiral amines.

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.

End-to-end real-time holographic display based on real-time capture of real scenes.

Holographic display is considered as a promising three-dimensional (3D) display technology and has been widely studied. However, to date, the real-time holographic display for real scenes is still far from being incorporated in our life. The speed and quality of information extraction and holographic computing need to be further improved. In this paper, we propose an end-to-end real-time holographic display based on real-time capture of real scenes, where the parallax images are collected from the scene and a convolutional neural network (CNN) builds the mapping from the parallax images to the hologram. Parallax images are acquired in real time by a binocular camera, and contain depth information and amplitude information needed for 3D hologram calculation. The CNN, which can transform parallax images into 3D holograms, is trained by datasets consisting of parallax images and high-quality 3D holograms. The static colorful reconstruction and speckle-free real-time holographic display based on real-time capture of real scenes have been verified by the optical experiments. With simple system composition and affordable hardware requirements, the proposed technique will break the dilemma of the existing real-scene holographic display, and open up a new direction for the application of real-scene holographic 3D display such as holographic live video and solving vergence-accommodation conflict (VAC) problems for head-mounted display devices.

Complex amplitude modulated holographic display system based on polarization grating.

We propose a holographic display system for complex amplitude modulation (CAM) using a phase-only spatial light modulator (SLM) and two polarization gratings (PG). The two sub-holograms of the complex-amplitude computed generated hologram (CGH) are loaded in different regions of SLM. Two diffractive components couple in space after longitudinal migration from the double PGs, and finally interfered through the line polarizer. The influence of the system error on the reconstructed image quality is analyzed, which provides a theoretical assessment for adding pre-compensation to CGH to compensate the system error. Moreover, on the base of the proposed system, a large depth of field and enlarged display area display is realized and the real-time display can be achieved because of the analytical complex-amplitude computed generated hologram. The optical experimental results show that the proposed system has high energy efficiency, and can provide high-quality holographic display with a large depth of field and enlarged display area.

Turning thermostability of Aspergillus terreus (R)-selective transaminase At-ATA by synthetic shuffling.

As versatile and green biocatalysts for the asymmetric amination of ketones, the insufficient thermostability of transaminases always limits its broad application in the pharmaceutical and fine chemical industries. Here, synthetic shuffling technology was used to enhance stability of (R)-selective transaminase from Aspergillus terreus. The results showed that 30 out of 5000 mutants had improved thermostability by color-based screening method, among which mutants with residual enzyme activity higher than 50% at 45 °C for 10 min were selected for further analysis. Especially, the half-inactivation temperature (T5010), half-life (t1/2), and melting temperature (Tm) of the best mutant M14 (M280C-H210N-M150C-F115L) were 13.7 °C, 165.8 min, and 13.9 °C higher than that of the wild type (WT), respectively. M14 also exhibited a significant biocatalytic efficiency toward acetophenone and 1-acetylnaphthalene, the yield of which were 265.6% and 117.5% higher than WT, respectively. Based on molecular dynamics simulation, improved catalytic efficiency of M14 could be attributed to its increased hydrogen bonds interaction around the mutation sites. Additionally, the introduction of disulfide bond combined with above mutations has a synergistic effect on the improved protein thermostability.

Optimized computer-generated hologram for enhancing depth cue based on complex amplitude modulation.

In this Letter, we introduce a computer-generated hologram (CGH) optimization method to enhance the depth cue based on complex amplitude modulation (CAM). An iterative algorithm is designed to generate the optimized random phase (ORAP) according to the size of the target image and the bandwidth limitation condition. The ORAP with limited bandwidth is used as the initial phase of the target image and the hologram is encoded based on the analytical formula. Our proposal can maintain the advantages of CAM and achieve holographic three-dimensional (3D) display with an enhanced depth cue. It is expected that the proposed method could be widely used in holographic field in the future.

Superpixel-based sub-hologram method for real-time color three-dimensional holographic display with large size.

One of the biggest challenges for large size three-dimensional (3D) holographic display based on the computer-generated hologram (CGH) is the trade-off between computation time and reconstruction quality, which has limited real-time synthesis of high-quality holographic image. In this paper, we propose a superpixel-based sub-hologram (SBS) method to reduce the computation time without sacrificing the quality of the reconstructed image. The superpixel-based sub-hologram method divides the target scene into a collection of superpixels. The superpixels are composed of adjacent object points. The region of the superpixel-based sub-hologram corresponding to each superpixel is determined by an approximation method. Since the size and the complexity of the diffraction regions are reduced, the hologram generation time is decreased significantly. The computation time has found to be reduced by 94.89% compared with the conventional sub-hologram method. It is shown that the proposed method implemented on the graphics processing unit (GPU) framework can achieve real-time (> 24 fps) color three-dimensional holographic display with a display size of 155.52 mm × 276.48 mm.

Color dynamic holographic display based on complex amplitude modulation with bandwidth constraint strategy.

In this Letter, we introduce a multiplexing encoding method with a bandwidth constraint strategy to realize a color dynamic holographic display based on complex amplitude modulation (CAM). The method first uses the angular spectrum method (ASM) with a bandwidth constraint strategy to calculate the diffracted wavefronts of red, green, and blue channels. Then the diffracted wavefronts of three channels are synthesized into a color-multiplexed hologram (CMH) based on the double-phase method after they interfere with off-axis reference lights. The color 3D objects can be reconstructed when the combination of red, green, and blue lights is used to illuminate the double-phase CMH, and a 4f system with a slit filter is introduced to extract the desired spectrums. Numerical simulations and optical experiments are performed to verify the effectiveness of the proposed method and the results show that it can achieve a color holographic display with high quality. Our proposal is simple and fast, and the display system is compact. It is expected that our proposed method could in future be widely used in the holographic field.

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.

Reconstruction of the glutamate decarboxylase system in Lactococcus lactis for biosynthesis of food-grade γ-aminobutyric acid.

Gamma-aminobutyric acid (GABA), an important bioactive compound, is synthesized through the decarboxylation of L-glutamate (L-Glu) by glutamate decarboxylase (GAD). The use of lactic acid bacteria (LAB) as catalysts opens interesting avenues for the biosynthesis of food-grade GABA. However, a key obstacle involved in the improvement of GABA production is how to resolve the discrepancy of optimal pH between the intracellular GAD activity and cell growth. In this work, a potential GAD candidate (LpGadB) from Lactobacillus plantarum was heterologously expressed in Escherichia coli. Recombinant LpGadB existed as a homodimer under the native conditions with a molecular mass of 109.6 kDa and exhibited maximal activity at 40°C and pH 5.0. The Km value and catalytic efficiency (kcat/Km) of LpGadB for L-Glu was 21.33 mM and 1.19 mM-1s-1, respectively, with the specific activity of 26.67 µM/min/mg protein. Subsequently, four C-terminally truncated LpGadB mutants (GadBΔC10, GadBΔC11, GadBΔC12, GadBΔC13) were constructed based on homology modeling. Among them, the mutant GadBΔC11 with highest catalytic activity at near-neutral pH values was selected. In further, the GadBΔC11 and Glu/GABA antiporter (GadC) of Lactococcus lactis were co-overexpressed in the host L. lactis NZ3900. Finally, after 48 h of batch fermentation, the engineered strain L. lactis NZ3900/pNZ8149-gadBΔC11C yielded GABA concentration up to 33.52 g/L by applying a two-stage pH control strategy. Remarkably, this is the highest yield obtained to date for GABA from fermentation with L. lactis as a microbial cell factory.Key points• The GadB from L. plantarum was heterologously expressed in E. coli and biochemically characterized.• Deletion of the C-plug in GadB shifted its pH-dependent activity toward a higher pH.• Reconstructing the GAD system of L. lactis is an effective approach for improving its GABA production.

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.

Improving the Thermostability of Glutamate Decarboxylase from Lactobacillus brevis by Consensus Mutagenesis.

γ-Aminobutyrate (GABA) is an important bioactive compound synthesized through decarboxylation of L-glutamate by the glutamate decarboxylase (GAD). In this study, stabilized variants of the GAD from Lactobacillus brevis were constructed by consensus mutagenesis. Using Consensus Finder ( http://cbs-kazlab.oit.umn.edu/ ), eight positions with the most prevalent amino acid (over 60% threshold) among the homologous family members were identified. Subsequently, these eight residues were individually mutated to match the consensus sequence using site-directed mutagenesis. Compared to the wild-type, T383K variant displayed the largest shift in thermostability among the single variants, with a 3.0 °C increase in semi-inactivation temperature (T5015), a 1.7-fold improvement of half-life (t1/2) at 55 °C, and a 1.2-fold improvement of t1/2 at 37 °C, respectively, while its catalytic efficiency (kcat/Km) was reduced. To obtain the mutant with improvement in both thermostability and catalytic activity, we performed a site-saturation mutation at T383. Notably, mutants T383V and T383G exhibited an increasement in thermostability and kcat/Km than that of wild-type. This study not only emphasizes the value of consensus mutagenesis for improving the thermostability of GAD but also sheds a powerful guidance to study the thermal stability of other enzymes.

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.

[Increasing the thermostability of glutamate decarboxylase from Lactobacillus brevis by introducing proline].

Glutamate decarboxylase, a unique pyridoxal 5'-phosphate-dependent enzyme, catalyzes α-decarboxylation of L-glutamate to γ-aminobutyrate. However, glutamate decarboxylase from different sources has the common problem of poor thermostability that affects its application in industry. In this study, proline was introduced at 13 different positions in glutamate decarboxylase by using the design strategy of homologous sequence alignment between Thermococcus kodakarensis and Lactobacillus brevis CGMCC No.1306. A mutant enzyme G364P with higher thermostability was obtained. Compared to the wild type, thermostability of the mutant G364P was significantly improved, the half-life time (t1/2) at 55 °C and the semi-inactivation temperature (T50 ¹5) of the mutant G364P increased 19.4 min and 5.3 °C, respectively, while kcat/Km of the mutant enzyme remained nearly unchanged. Further analysis of their thermostability by molecular dynamics simulations were performed. The root mean square deviation of G364P and root mean square fluctuation in the loop region including G364 were lower than the wild type at 313 K for 10 ns, and G364P increased one hydrophobic interaction in the loop region. It proves that mutation of flexible 364-Gly to rigid proline endows glutamate decarboxylase with enhanced thermostability.