Enzymatic Synthesis Research Articles

Preparation of Bio‐Orthogonal Self‐Cross‐Linked Arabinose Isomerase for Enzymatic Synthesis of ʟ‐Ribulose

Preparation of Bio‐Orthogonal Self‐Cross‐Linked Arabinose Isomerase for Enzymatic Synthesis of <scp>ʟ</scp>‐Ribulose

Enzymatic synthesis of ω-3 fatty acids-based structured phospholipids: A novel solvent-free preparation approach and its catalytic mechanism

ω-3 fatty acids-based phospholipids (ω-3-PLs) exhibit higher bioavailability within organisms and stability compared to polyunsaturated fatty acids in their neutral forms. In this study, ω-3 fatty acids enriched partial acylglycerols (ω-3-AGs) were produced from hydrolysis of algal oil firstly. The effects of hydrolysis processing parameters such as enzyme type, enzyme loading, oil-water ratio, reaction temperature, and time were evaluated. Subsequently, ω-3-PLs were prepared via interesterification of ω-3-AGs with PLs catalyzed by PLA1. When monoacylglycerols (MAGs) reached 60% in partial acylglycerols, the interesterification reaction showed a marked improvement, leading to the achievement of the optimal final ω-3-PLs content of 38.93%. Using molecular docking techniques to speculate on the catalytic mechanism, the results indicate that MAGs exhibit an affinity of up to −6.33 kcal/mol with PLA1, verifying their critical role in the interesterification process.

Functionalized Metal-Organic Framework for NADH Regeneration by Hydrogen in a Redox Flow Bioreactor.

The electrochemical regeneration of reduced nicotinamide adenine dinucleotide (NADH) using [Rh(Cp*)(bpy)Cl]+ holds significant promise for the industrial synthesis of chiral chemicals. However, challenges persist due to the high consumption of NADH and the limited efficiency of its cyclic regeneration, which currently hinder widespread application. To address these obstacles, based on in-situ growth of 3D ordered metal-organic framework (NU-1000) on the surface of graphite felt, [Rh(Cp*)(bpy)Cl]+ were immobilized on the Zr6 nodes of NU-1000 by solvent-assisted ligand incorporation (SALI), and applied in a flow bioreactor. Moreover, we employ a gas diffusion electrode (GDE) to oxidize H2, providing a clean proton source for the electrochemical regeneration of NADH. Consequently, highly efficient enzymatic electrocatalytic synthesis of L-lactate was achieved when coupled with L-lactate dehydrogenases (LDH) as a model reaction, and the total turnover number (TTN) reached 19600 and 1750 for [Rh(Cp*)(bpy)Cl]+ and NAD+ after 48 h, corresponding to a high turnover frequency (TOF) of 2350 h-1 and 210 h-1 for [Rh(Cp*)(bpy)Cl]+ and NAD+, respectively. This work provides new insights for the construction of efficient enzymatic electrosynthesis systems in industrial production.

Protective effects of cilostazol via the HNF1α/FXR signalling pathway and anti-apoptotic mechanisms in a rat model of estrogen-induced intrahepatic cholestasis

Currently, there is a lack of targeted medications for estrogen-induced intrahepatic cholestasis (EIC) and the primary objective in managing this condition is to safeguard liver function. Consequently, this study was conducted to examine the pharmacological efficacy of cilostazol (CTZ) in the management of EIC and explore its underlying mechanisms through the use of an animal model. Thirty female Sprague-Dawley rats were divided into five groups of six animals each: Normal group, 17-ethinylestradiol (EE)-induced intrahepatic cholestasis group, EE + ursodeoxycholic acid (UDCA)-treated group, EE + CTZ (5 mg/kg)-treated group, and EE + CTZ (10 mg/kg)-treated group. It was found that the therapeutic efficacy of UDCA and low dosage of CTZ (5 mg/kg) was comparable. Nevertheless, when CTZ was administered at a dose of 10 mg/kg, it resulted in the normalization of all liver function parameters, oxidative stress, and pro-inflammatory markers, together with improvement in the histopathological derangements and hepatocytic apoptosis. These effects were mediated through the activation of the hepatocyte nuclear factor-1 alpha (HNF1α)/Farnesoid X receptor (FXR) pathway with subsequent down-regulation of the bile acids (BAs) synthesis enzyme; cholesterol 7α-hydroxylase (CYP7A1), and up-regulation of the BAs-metabolizing enzyme; cytochrome P450 (CYP)3A1 and the bile salt export pump; BSEP. Therefore, the administration of CTZ in a dose-dependent manner can protect against EIC through regulating the HNF1α/FXR pathway and anti-apoptotic mechanisms. This implies that CTZ exhibits considerable promise as a therapeutic agent for the treatment of cholestatic liver disorders.

Ziziphus jujuba (Jujube) in Metabolic Syndrome: From Traditional Medicine to Scientific Validation.

This review evaluates the therapeutic potential of Ziziphus jujuba and its main components in managing complications of metabolic syndrome, including diabetes, dyslipidemia, obesity, and hypertension. The reviewed studies provide evidence supporting the use of Z. jujuba and its main components (lupeol and betulinic acid) as natural treatments for complications of metabolic syndrome. These substances enhance glucose uptake through the activation of signaling pathways such as phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt), reduce hepatic glucose synthesis, and increase glucose uptake by adipocytes and skeletal muscle cells. They also improve insulin sensitivity by modulating AMP-activated protein kinase (AMPK) activity and regulating insulin signaling proteins and glucose transporters. In the field of dyslipidemia, they inhibit triglyceride synthesis, lipid accumulation, and adipogenic enzymes, while influencing key signaling pathways involved in adipogenesis. Z. jujuba and its constituents demonstrate anti-adipogenic effects, inhibiting lipid accumulation and modulating adipogenic enzymes and transcription factors. They also exhibit positive effects on endothelial function and vascular health by enhancing endothelial nitric oxide synthase (eNOS) expression, NO production, and antioxidant enzyme activity. Z. jujuba, lupeol, and betulinic acid hold promise as natural treatments for complications of metabolic syndrome. They improve glucose metabolism, insulin sensitivity, and lipid profiles while exerting anti-adipogenic effects and enhancing endothelial function. However, further research is needed to elucidate the mechanisms and confirm their efficacy in clinical trials. These natural compounds offer potential as alternative therapies for metabolic disorders and contribute to the growing body of evidence supporting the use of natural medicines in their management.

Metabolic adaptations of Escherichia coli to extended zinc exposure: insights into tricarboxylic acid cycle and trehalose synthesis

Balanced bacterial metabolism is essential for cell homeostasis and growth and can be impacted by various stress factors. In particular, bacteria exposed to metals, including the nanoparticle form, can significantly alter their metabolic processes. It is known that the extensive and intensive use of food and feed supplements, including zinc, in human and animal nutrition alters the intestinal microbiota and this may negatively impact the health of the host. This study examines the effects of zinc (zinc oxide and zinc oxide nanoparticles) on key metabolic pathways of Escherichia coli. Transcriptomic and proteomic analyses along with quantification of intermediates of tricarboxylic acid (TCA) were employed to monitor and study the bacterial responses. Multi-omics analysis revealed that extended zinc exposure induced mainly oxidative stress and elevated expression/production of enzymes of carbohydrate metabolism, especially enzymes for synthesis of trehalose. After the zinc withdrawal, E. coli metabolism returned to a baseline state. These findings shed light on the alteration of TCA and on importance of trehalose synthesis in metal-induced stress and its broader implications for bacterial metabolism and defense and consequently for the balance and health of the human and animal microbiome.

Enzymatic synthesis of self-adhesive zein to impart durable hydrophobicity with antioxidant activity to cotton fabrics

In this study, dopamine (DA) was used as the acyl receptor to modify zein through transglutaminase (TGase)-mediated reaction, producing self-adhesive zein (zein-DA complex). The structure of zein-DA complex was characterized by Fourier transform infrared spectrum (FT-IR), 1H nuclear magnetic resonance spectrum (1H NMR), and circular dichroism spectrum (CD), revealing the successful grafting of catechol structure of DA to zein. The results of SDS-PAGE demonstrated that zein-DA complex exhibited great ability of self-polymerization. The zein-DA complex absorbed on the cotton fabric surface imparted hydrophobicity with a water contact angle value (WCA) of 121.34 ± 1.14° to the fabrics. After 5 washing cycles, the WCA still remained 100.28 ± 4.83°, which indicated the fabrics obtained relatively durable hydrophobicity due to the self-adhesion of zein-DA complex. The results of FT-IR, scanning electron microscope (SEM), X-ray photoelectron spectroscopy (XPS), and color reaction tests manifested that much more protein attached on the surface of cotton fabric treated with zein-DA complex after washing compared with that with zein, which verified that the zein-DA complex had strong adhesion properties. Due to the introduction of DA, the relative DPPH free radical scavenging activity of cotton treated with zein-DA complex reached 43.60 ± 0.28 %, which was obviously higher than cotton treated with zein. The functionalized fabrics with zein-DA complex had both hydrophobicity and antioxidant activities.

Modeling enzyme competition in eicosanoid metabolism in macrophage cells using a cybernetic framework

Cellular metabolism is a complex process involving the consumption and production of metabolites, as well as the regulation of enzyme synthesis and activity. Modeling of metabolic processes is important to understand the underlying mechanisms, with a wide range of applications in metabolic engineering and health sciences. Cybernetic modeling is a powerful technique that accounts for unknown intricate regulatory mechanisms in complex cellular processes. It models regulation as goal-oriented, where the levels and activities of enzymes are modulated by the cybernetic control variables to achieve the cybernetic objective. This study used cybernetic model to study the enzyme competition between arachidonic acid (AA) and eicosapentaenoic acid (EPA) metabolism in murine macrophages. AA and EPA compete for the shared enzyme cyclooxygenase. Upon external stimuli, AA produces proinflammatory 2-series prostaglandins and EPA metabolizes to antiinflammatory 3-series prostaglandins, where proinflammatory and antiinflammatory responses are necessary for homeostasis. The cybernetic model adequately captured the experimental data for control and EPA-supplemented conditions. The model is validated by performing an F-test, conducting leave-one-out-metabolite cross-validation, and predicting an unseen experimental condition. The cybernetic variables provide insights into the competition between AA and EPA for the cyclooxygenase enzyme. Predictions from our model suggest that the system undergoes a switch from a predominantly proinflammatory state in the control to an antiinflammatory state with EPA-supplementation. The model can also be used to analytically determine the AA and EPA concentrations required for the switch to occur. The quantitative outcomes enhance understanding of proinflammatory and antiinflammatory metabolism in RAW 264.7 macrophages.

Effect of Partial Knockout of the Plastid Starch Phosphorylase Gene NtPHO1-L1 on the Metabolism of Carbohydrates and Carotenoids in Nicotiana tabacum L. Leaves

Starch metabolism is regulated by a complex catalytic network, one of the key enzymes of which is the plastid starch phosphorylase PHO1. In this study, using the CRISPR-Cas9 system, we obtained tobacco (Nicotiana tabacum L.) plants with a partial knockout of the NtPHO1-L1 gene due to deletion variants of the catalytic domain of the NtPHO1-L1 protein, leading to the formation of nonfunctional forms of the enzyme. The edited lines differed from wild-type plants by increased starch accumulation and decreased content of sugars, chlorophylls, and carotenoids in the leaves. It was shown that, compared to the control, the edited plants were characterized by differential expression of starch (NtPHO1-L1, NtGWD, NtBAM1, NtBAM9, NtAI) and carotenoid (NtPSY2, NtPDS, NtZDS, NtCRTISO, NtVDE) metabolism genes, as well as genes encoding MADS-domain transcription factors (NtFUL1, NtSEP1, NtSEP2, NtSEP3), which are presumably involved in the regulation of transcription of the studied metabolic genes. These data suggest that partial knockout of NtPHO1-L1 alters the functional activity of tobacco starch phosphorylase. This, in turn, may influence the coordinated activity of starch catabolism enzymes, as well as chlorophyll and carotenoid synthesis enzymes, possibly through differential expression of MADS-box genes. The results highlight the critical regulatory role of plastid starch phosphorylase in transient starch metabolism and in stimulating plant photosynthesis.

Enhancement of Bioactive Compounds and Survival of Lactobacillus acidophilus Grown in the Omega-6, -7 Riched Cyanobacteria Spirulina platensis.

Lactobacillus acidophilus is a probiotic commonly used in aquaculture to enhance the growth and immune system of aquatic species through the synthesis of various enzymes, and antimicrobial compounds like lactic acid. Traditional method of growing L. acidophilus involes using the De Man-Rogosa-Sharpe (MRS) medium. However, L. acidophilus belongs to a non-spore forming group, which make it vulnerable to stress conditions, especially during the usage process. Therefore, the present study aimed to improve the survival rate, antibacterial activity, and enrich the polyunsaturated fatty acids (PUFAs) content of L. acidophilus LB when cultured in an algae-supplemented medium, thus increasing its benefits in aquaculture applications. Using different algae biomass species as an alternative to MRS medium for the growth of L. acidophilus LB, the results showed that Spirulina platensis promoted the highest density of L. acidophilus LB. When grown in (S. platensis + glucose) medium, L. acidophilus LB produced the highest lactic acid concentration of 18.24 ± 2.43mg/mL and survived in extreme conditions such as 4% NaCl, pH 1.0-2.0, and 50ºC, and inhibited 99.82 ± 0.24% of Vibrio parahaemolyticus population after 2days of treatment. Additionally, it was observed that the PUFAs content, specifically omega-6, and -7, also increased in the fermentation mixture as compared to the control sample. These findings highlighted the potential of utilizing the cyanobacteria S. platensis as an alternative, eco-friendly growth substance for L. acidophilus LB to enhance its bioactivity and viability under extreme conditions.

Limonene enhances rice plant resistance to a piercing-sucking herbivore and rice pathogens.

Terpene synthases (TPSs) are key enzymes in terpenoids synthesis of plants and play crucial roles in regulating plant defence against pests and diseases. Here, we report the functional characterization of OsTPS19 and OsTPS20, which were upregulated by the attack of brown planthopper (BPH). BPH female adults performed concentration-dependent behavioural responses to (S)-limonene showing preference behaviour at low concentrations and avoidance behaviour at high concentrations. Overexpression lines of OsTPS19 and OsTPS20, which emitted higher amounts of the monoterpene (S)-limonene, decreased the hatching rate of BPH eggs, reduced the lesion length of sheath blight caused by Rhizoctonia solani and bacterial blight caused by Xanthomonas oryzae. While knockout lines of OsTPS19 and OsTPS20, which emitted lower amounts of (S)-limonene, were more susceptible to these pathogens. Overexpression of OsTPS19 and OsTPS20 in rice plants had adverse effects on the incidence of BPH, rice blast, and sheath blight in the field and had no significant impacts on rice yield traits. OsTPS19 and OsTPS20 were found to be involved in fine-tuning the emission of (S)-limonene in rice plants and play an important role in defence against both BPH and rice pathogens.

Based on green synthesis, multisensory evaluations and molecular simulation approaches: Exploring the taste-enhancing characteristics and mechanisms of N-succinyl-L-leucine

N-Succinyl amino acids (N-Suc-AAs) are increasingly recognized for their potential as taste-active compounds. However, research into the green synthesis, taste-enhancing properties, and mechanisms of N-succinyl-L-leucine (N-Suc-Leu) remains limited. This study employed an enzymatic synthesis method, catalyzed by protamex and pancreatin, to produce N-Suc-Leu, with its structure confirmed. Multiple sensory techniques demonstrated that N-Suc-Leu markedly enhanced the umami, saltiness, and kokumi intensity, and prolonged the duration of umami by 25%. Sigmoid curve analysis further revealed the synergistic enhancement of N-Suc-Leu on the perceptions of umami and saltiness. Molecular docking and dynamics simulations revealed that N-Suc-Leu could bind with T1R1, T1R3, TMC4, and CaSR, enhancing the sensations of saltiness, umami, and kokumi, and bound closely to these receptors without altering their overall conformation. These findings offered a systematic explanation of the potential and mechanism of enzymatically synthesized N-Suc-Leu in enhancing taste and provided novel insights into potential strategies for the development and innovation of taste enhancers and food flavors.

Enzymatic synthesis of azide by a promiscuous N-nitrosylase.

Azides are energy-rich compounds with diverse representation in a broad range of scientific disciplines, including material science, synthetic chemistry, pharmaceutical science and chemical biology. Despite ubiquitous usage of the azido group, the underlying biosynthetic pathways for its formation remain largely unknown. Here we report the characterization of an enzymatic route for de novo azide construction. We demonstrate that Tri17, a promiscuous ATP- and nitrite-dependent enzyme, catalyses organic azide synthesis through sequential N-nitrosation and dehydration of aryl hydrazines. Through biochemical, structural and computational analyses, we further propose a plausible molecular mechanism for azide synthesis that sets the stage for future biocatalytic applications and biosynthetic pathway engineering.

Direct asymmetric synthesis of β-branched aromatic α-amino acids using engineered phenylalanine ammonia lyases

β-Branched aromatic α-amino acids are valuable building blocks in natural products and pharmaceutically active compounds. However, their chemical or enzymatic synthesis is challenging due to the presence of two stereocenters. We design phenylalanine ammonia lyases (PAL) variants for the direct asymmetric synthesis of β-branched aromatic α-amino acids. Based on extensive computational analyses, we unravel the enigma behind PAL’s inability to accept β-methyl cinnamic acid (β-MeCA) as substrate and achieve the synthesis of the corresponding amino acids of β-MeCA and analogs using a double (PcPAL-L256V-I460V) and a triple mutant (PcPAL-F137V-L256V-I460V). The reactions are scaled-up using an optimized E. coli based whole-cell biotransformation system to produce ten β-branched phenylalanine analogs with high diastereoselectivity (dr > 20:1) and enantioselectivity (ee > 99.5%) in yields ranging from 41-71%. Moreover, we decipher the mechanism of PcPAL-L256V-I460V for the acceptance of β-MeCA and converting it with excellent stereoselectivity by computational simulations. Thus, this study offers an efficient method for synthesizing β-branched aromatic α-amino acids.

Impact of nitrogen rates on biosynthesis pathways: A comparative study of diterpene synthases in clerodane diterpenoids and enzymes in benzylisoquinoline alkaloids

Tinospora sagittata is rich in secondary metabolites used in traditional medicine. However, environmental factors impact key enzymes in metabolite synthesis, highlighting the need for improved growth conditions. This study employs transcriptomics and metabolomics to assess nitrogen's impact on enzymes in secondary metabolites biosynthesis pathways. The gene expressions of berberine bridge enzymes (BBEs) like TsBBE2 had peak expression in low nitrogen treatments (A0 and A1) but were absent in higher nitrogen treatments (A2 and A3). Similar trends were observed for other enzymes such as (S)-scoulerine 9-O-methyltransferase (TsCMT3), Tetrahydroberberine oxidase (TsSTOX), and Columbamine O-methyltransferase (TsCoCOMT2-4) in response to nitrogen levels. In examining gene families related to diterpene synthases (diTPS), 1-deoxyxylulose 5-phosphate synthase (TsDXR1) expression increased with higher nitrogen fertilizer, while TsDXR2 peaked at maximal nitrogen levels. Geranylgeranyl diphosphate synthase (TsGGPP3 and TsGGPP5) decreased with nitrogen levels. (−)-kolavenyl diphosphate synthase (KPS) genes had higher expression in treatments, while ent-kaurene synthase (KSL) genes, especially TsKSL1 and TsKSL2, showed higher expression in control conditions with lower nitrogen fertilizer. Metabolite analysis confirmed more upregulated compounds in A3 compared to A0. These findings have practical implications for agriculture and pharmaceuticals, highlighting the link between nitrogen fertilization and specialized metabolism in medicinal plants.

Action pattern of Sulfolobus O-α-glycoligase for synthesis of highly water soluble resveratrol 3,4′-α-diglucoside

This study presents the enzymatic synthesis of resveratrol-3,4′-O-α-diglucoside (RDG) using a hyperactive O-α-glycoligase (MalA-D416R/Q450S) and α-glucopyranosyl fluoride as the donor substrate. The transglycosylation rate for resveratrol by MalA-D416R/Q450S was maximized in 100 mM Tris-HCl (pH 9.5) containing 20 % DMSO at 45°C. Because the pKa of the 4′-OH group of resveratrol is lower than that of the 3-OH group, the 4′-OH group is more nucleophilic at the alkaline pH, leading to a preference for glycosylation at the 4′-OH site rather than the 3-OH site. This preference makes resveratrol 3-O-α-glucoside (R3G) as the more efficient acceptor than resveratrol 4′-O-α-glucoside (R4′G), resulting in negligible production of resveratrol 3-O-α-glucoside (R3G) due to its complete consumption in the second transglycosylation reaction when using a 2:1 ratio of donor to acceptor substrates. From a preparative scale reaction, R4′G and RDG were isolated with yields of 41.2 % and 43.3 %, respectively. The water solubility of RDG exceeded 1.67 M, which represents more than a 9,800-fold improvement compared to resveratrol. In a hydrolysis experiment using intestinal α-glycosidase from rat, the α-glucosides of resveratrol (R4′G and RDG) were completely deglycosylated to the aglycone.

GABAergic dysfunction in postmortem dorsolateral prefrontal cortex: implications for cognitive deficits in schizophrenia and affective disorders.

The microcircuitry within superficial layers of the dorsolateral prefrontal cortex (DLPFC), composed of excitatory pyramidal neurons and inhibitory GABAergic interneurons, has been suggested as the neural substrate of working memory performance. In schizophrenia, working memory impairments are thought to result from alterations of microcircuitry within the DLPFC. GABAergic interneurons, in particular, are crucially involved in synchronizing neural activity at gamma frequency, the power of which increases with working memory load. Alterations of GABAergic interneurons, particularly parvalbumin (PV) and somatostatin (SST) subtypes, are frequently observed in schizophrenia. Abnormalities of GABAergic neurotransmission, such as deficiencies in the 67 kDA isoform of GABA synthesis enzyme (GAD67), vesicular GABA transporter (vGAT), and GABA reuptake transporter 1 (GAT1) in presynaptic boutons, as well as postsynaptic alterations in GABA A receptor subunits further contribute to impaired inhibition. This review explores GABAergic abnormalities of the postmortem DLPFC in schizophrenia, with a focus on the roles of interneuron subtypes involved in cognition, and GABAergic neurotransmission within presynaptic boutons and postsynaptic alterations. Where available, comparisons between schizophrenia and affective disorders that share cognitive pathology such as bipolar disorder and major depressive disorder will be made. Challenges in directly measuring GABA levels are addressed, emphasizing the need for innovative techniques. Understanding GABAergic abnormalities and their implications for neural circuit dysfunction in schizophrenia is crucial for developing targeted therapies.

Bioinformatics Identification and Expression Analysis of Acetyl-CoA Carboxylase Reveal Its Role in Isoflavone Accumulation during Soybean Seed Development.

Isoflavones belong to the class of flavonoid compounds, which are important secondary metabolites that play a crucial role in plant development and defense. Acetyl-CoA carboxylase (ACCase) is a biotin-dependent enzyme that catalyzes the conversion of Acetyl-CoA into Malonyl-CoA in plants. It is a key enzyme in fatty acid synthesis and also catalyzes the production of various secondary metabolites. However, information on the ACC gene family in the soybean (Glycine max L. Merr.) genome and the specific members involved in isoflavone biosynthesis is still lacking. In this study, we identified 20 ACC family genes (GmACCs) from the soybean genome and further characterized their evolutionary relationships and expression patterns. Phylogenetic analysis showed that the GmACCs could be divided into five groups, and the gene structures within the same groups were highly conserved, indicating that they had similar functions. The GmACCs were randomly distributed across 12 chromosomes, and collinearity analysis suggested that many GmACCs originated from tandem and segmental duplications, with these genes being under purifying selection. In addition, gene expression pattern analysis indicated that there was functional divergence among GmACCs in different tissues. The GmACCs reached their peak expression levels during the early or middle stages of seed development. Based on the transcriptome and isoflavone content data, a weighted gene co-expression network was constructed, and three candidate genes (Glyma.06G105900, Glyma.13G363500, and Glyma.13G057400) that may positively regulate isoflavone content were identified. These results provide valuable information for the further functional characterization and application of GmACCs in isoflavone biosynthesis in soybean.

Biosynthesis and Extraction of Chlorophyll, Carotenoids, Anthocyanins, and Betalaine In Vivo and In Vitro.

As natural bioactive compounds, plant pigments play crucial roles not only in plant phenotype, growth, development, and adaptation to stress but also hold unique value in biotechnology, healthcare, and industrial applications. There is growing interest in the biosynthesis and acquisition of plant pigments. Thus, this paper explores emerging extraction methods of natural pigments and elucidates the biosynthesis pathways of four key plant pigments, chlorophylls, carotenoids, anthocyanins, and betalaine in vivo and in vitro. We comprehensively discuss the application of solvent, supercritical fluid [extraction], ultrasonic, and microwave-assisted extraction techniques, as well as introducing key enzymes, precursors, and synthetic pathways involved in pigment synthesis. δ-Aminolevulinic acid represents a pivotal initiating enzyme for chlorophyll synthesis, whereas isopentenylpyrophosphate, (IPP) and dimethylallyl pyrophosphate, (DMAPP) are closely associated with carotenoid biosynthesis. Phenylalanine and tyrosine are critical substances for anthocyanin and betalaine synthesis, respectively. Hence, crucial genes such as chlI, crtB, PGT8, CYP76AD1, and BvDODA can be employed for heterologous biosynthesis in vitro to meet the demand for increased plant pigment amount. As a pivotal determinant of plant coloration, an in-depth exploration into the high-quality acquisition of plant pigments can provide a basis for developing superior pigments and offer new insights into increasing pigment yield.

Boosting Fructosyl Transferase's Thermostability and Catalytic Performance for Highly Efficient Fructooligosaccharides (FOS) Production.

Achieving enzymatic food processing at high substrate concentrations can significantly enhance production efficiency; however, related studies are notably insufficient. This study focused on the enzymatic synthesis of fructooligosaccharides (FOS) at high temperature and high substrate concentration. Results revealed that increased viscosity and limited substrate solubility in high-concentration systems could be alleviated by raising the reaction temperature, provided it aligned with the enzyme's thermostability. Further analysis of enzyme thermostability in real sucrose solutions demonstrates that the enzyme's thermostability was remarkedly improved at higher sucrose concentrations, evidenced by a 10.3 °C increase in melting temperature (Tm) in an 800 g/L sucrose solution. Building upon these findings, we developed a novel method for enzymatic FOS synthesis at elevated temperatures and high sucrose concentrations. Compared to existing commercial methods, the initial transglycosylation rate and volumetric productivity for FOS synthesis increased by 155.9% and 113.5%, respectively, at 65 °C in an 800 g/L sucrose solution. This study underscores the pivotal role of substrate concentration, incubation temperature, and the enzyme's actual status in advancing enzyme-catalyzed processes and demonstrates the potential of enzymatic applications in enhancing food processing technologies, providing innovative strategies for the food industry.