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Amino acids are the most important components of black tea and are associated with the physiological and biochemical changes that occur during tea processing. Herein, we review the enzymatic and non-enzymatic reactions of the main proteinaceous and non-proteinaceous amino acids that occur during the manufacturing process of black tea from the postharvest processing steps of tea leaves to the final product. Enzymatic reactions are responsible for the amino acid variation in the withering, rolling, and fermentation stages. The significant increase in the total amino acid content during the withering stage is mainly derived from proteolysis. Furthermore, the transformation reactions in the rolling and fermentation stages decrease the amino acid levels slightly, whereas the non-enzymatic reactions, such as the Maillard and Strecker reactions, contribute to the significant decrease in the amino acid content of the drying stage. This study provides deep insights into the mechanisms underlying amino acid variation during black tea processing. [ABSTRACT FROM AUTHOR]

Herein, the effect of immobilizing lipase onto layered α-zirconium phosphate ceramic nanosheets (α-ZrP NS) on the enzymatic transesterification of allylic alcohols was investigated in organic solvents. α-ZrP NS modified with quaternary ammonium ions show a excellent dispersion stability in n-hexane. Lipase immobilized on α-ZrP NS enables the improved synthesis of chiral allylic alcohols and esters, with significantly reduced reaction times compared to reactions performed in the absence of α-ZrP NS. The coexistence of α-ZrP NS also results in more efficient steric inversion of the resulting allyl ester, suggesting the utility of α-ZrP NS as a support for lipase-induced stereospecific reactions. The present manuscript discusses on mechanisms of high dispersion stability of α-ZrP NS and their preferable immobilizing effect for the lipase inducing the steric inversion in detail. [ABSTRACT FROM AUTHOR]

SNAP-tag® is a powerful technology for the labelling of protein/enzymes by using benzyl-guanine (BG) derivatives as substrates. Although commercially available or ad hoc produced, their synthesis and purification are necessary, increasing time and costs. To address this limitation, here we suggest a revision of this methodology, by performing a chemo-enzymatic approach, by using a BG-substrate containing an azide group appropriately distanced by a spacer from the benzyl ring. The SNAP-tag® and its relative thermostable version (SsOGT-H5) proved to be very active on this substrate. The stability of these tags upon enzymatic reaction makes possible the exposition to the solvent of the azide-moiety linked to the catalytic cysteine, compatible for the subsequent conjugation with DBCO-derivatives by azide-alkyne Huisgen cycloaddition. Our studies propose a strengthening and an improvement in terms of biotechnological applications for this self-labelling protein-tag. [ABSTRACT FROM AUTHOR]

The formation of aromatic esters in intermittently dried noni fruit by endogenous enzymatic activity was investigated. Gas chromatography mass spectrometry was used to determine the volatile compounds of Morinda citrifolia (noni). Large amounts of free fatty acids, especially hexanoic and octanoic acids, were found in fully matured noni. Significant amounts of esters derived from hexanoic and octanoic acids were detected in dried noni, especially in the product that intermittently dried. We postulate that autolysis occurred during ripening and that ester formation was catalyzed by endogenous lipase under low water activity (0.6 ± 0.1) during the intermittent drying stage. By controlling the water activities of noni fruit, biochemical reactions leading to the transformation of the sharp odor into an aromatic smell in dried noni products were investigated. [ABSTRACT FROM AUTHOR]

The production of biodiesel from transesterification of oils is the currently available alternative to consumable fuels. The study focuses on the performance assessment of a microreactor platform for biodiesel production. Since a microreactor provides better mass transfer and a higher surface-to-volume ratio, this significantly contributes to augmenting the reaction rate of biodiesel production at reduced cost. In this study, firstly the T-shaped microfluidic reactor was fabricated on a PMMA (poly methyl methacrylate) sheet. Then, the kinetics of enzymatic transesterification reaction for biodiesel synthesis in this reactor was investigated. A combination of sunflower oil and methanol was used as feedstock in the presence of lipase from Thermomyces lanuginous as a catalyst and tert-butanol as a solvent. The reaction was carried out under ambient conditions (1 atm and 30 °C) where 285 v% of tert-butanol was loaded to oil with 4 wt% lipase, and the molar ratio of oil to methanol was 1:2.5. Gas Chromatography was used to measure the concentration of biodiesel. The kinetic parameters such as maximum reaction rate (Vmax) and Michaelis –Menten constant (KM) were found to be 0.725 (mol/L.min) and 0.092 mol/L, respectively. High Vmax and low KM values indicated the better performance of the reactor. In addition, a Lineweaver–Burk plot with excellent coefficient of determination (R2) of 99.5% supported the high performance of the microreactor. [ABSTRACT FROM AUTHOR]

The biosynthetic pathways of phytosterols and steroidal saponins are located in two adjacent branches which share cycloartenol as substrate. The rate-limiting enzyme S-adenosyl-L-methionine-sterol-C24-methyltransferase 1 (SMT1) facilitates the metabolic flux toward phytosterols. It catalyzes the methylation of the cycloartenol in the side chain of the C24-alkyl group, to generate 24(28)-methylene cycloartenol. In this study, we obtained two full-length sequences of SMT1 genes from Pari polyphylla, designated PpSMT1-1 and PpSMT1-2. The full-length cDNA of PpSMT1-1 was 1369 bp long with an open reading frame (ORF) of 1038 bp, while the PpSMT1-2 had a length of 1222 bp, with a 1005 bp ORF. Bioinformatics analysis confirmed that the two cloned SMTs belong to the SMT1 family. The predicted function was further validated by performing in vitro enzymatic reactions, and the results showed that PpSMT1-1 encodes a cycloartenol-C24-methyltransferase, which catalyzes the conversion of cycloartenol to 24-methylene cycloartenol, whereas PpSMT1-2 lacked this catalytic activity. The tissue expression patterns of the two SMTs revealed differential expression in different organs of Paris polyphylla plants of different developmental stage and age. These results lay the foundation for detailed genetic studies of the biosynthetic pathways of steroid compounds, which constitute the main class of active substances found in P. polyphylla. [ABSTRACT FROM AUTHOR]

Phytate content, bioavailability and myo-inositol phosphates profile were analysed in pseudocereals (i.e., white and coloured quinoa, buckwheat) subjected to thermal processing and solid-state fermentation withRhizopus oligosporus. Fair buckwheat groats were characterised by the highest level of phytate (18.3 µmol/g), with 54% share of InsP6fraction. Processing (cooking, roasting, tempe-type fermentation) resulted in the significant decrease in phytate content, its bioavailability and the share of higher phosphorylated forms of myo-inositol (InsP6-5) in the profile of all the samples. Fermentation of cereals resulted in generation of high amounts of inositol triphosphates, particularly in the case of buckwheat (32% share in the whole profile). The path of dephosphorylation, identified after fermentation of quinoa seeds including intermediate products – Ins(1,2,4,5,6)P5and Ins(1,2,6)P3– confirmed that the dominant phytase activity could be attributed to the presence of microbial 3-phytases fromR. oligosporous. [ABSTRACT FROM AUTHOR]

We report a method for preparation of mammalian cell-enclosing hydrogel particles through horseradish peroxidase (HRP)-catalysed hydrogelation by dropping cell-suspending aqueous solution into an aqueous coagulation solution. An aqueous solution of 10% (w/v) gelatin derivative possessing phenolic hydroxyl (Ph) moieties (Gelatin-Ph), HepG2 cells and 10 U/mL HRP was dropped into an aqueous coagulation solution containing 1 mM H2O2. The resultant hydrogel formed through the HRP-catalysed reaction consuming H2O2 had a spherical shape. The sphericity decreased with decreasing concentrations of Gelatin-Ph, HRP and H2O2. The thickness of the hydrogel membrane layer of the hydrogel particles could be controlled by altering incubation time in the H2O2 solution. The cells encapsulated in the particles with a thinner hydrogel membrane grew faster. These results demonstrate that we successfully established the method of cell-encapsulation in hydrogel particles based on dropping aqueous polymer solution into aqueous coagulation solution through HRP-catalysed reaction. [ABSTRACT FROM AUTHOR]

We prepared phospholipid polymer nanoparticles immobilized with luciferase, and the nanoparticles were applied as photochemical sensing nanoparticles. An amphiphilic water-soluble polymer having a phosphorylcholine group was used as an emulsifier and a surface modifier to prepare the nanoparticles. The polymer was composed of three kinds of monomer units, that is, 2-methacryloyloxyethyl phosphorylcholine (MPC) as a hydrophilic and bioinert unit, n-butyl methacrylate as a hydrophobic unit and p-nitrophenyl ester having methacrylate as an enzyme-immobilizing unit. The p-nitrophenyl ester groups to immobilize the proteins were located on the surface of the nanoparticles. Luciferase was immobilized by the reaction between the p-nitrophenyl ester groups and the amino group. The enzymatic reaction on the nanoparticles was followed using a microdialysis system with an optical fiber having a 800 μm diameter in the probe. The nanoparticles conjugated with luciferase reacted with ATP, luciferin and oxygen. It is concluded that the nanoparticles are a promising tool for a photochemical sensing microdiagnostic system. [ABSTRACT FROM AUTHOR]

The bovine trypsin-catalyzed synthesis of N-α-benzoyl-DL-arginine esters from N-benzoyl-DL-arginine were studied in various organic solvents. Trypsin was immobilized to polyvinyl alcohol(PVA) by adsorption from its aqueous solutions. Immobilized enzyme showed higher catalytic activities than free enzyme for amino acid esterification in ethanol. The yield of ester is strongly dependent upon the PVA/trypsin ratio and water content in the reaction medium. The rate and equilibrium constant of the ester formation reaction are also dependent on water content. [ABSTRACT FROM AUTHOR]

An automatic flow procedure for cholesterol determination in animal blood serum using enzymatic reaction and detection by chemiluminescence is described. Cholesterol esterase and cholesterol oxidase were immobilized on glass beads packed into two mini-columns (15 mm × 5 mm), which were coupled to flow system. Produced H2O2 reacted with luminol produced chemiluminescence that was detected using a solid-state detector. The flow system comprised a set of three-way solenoid valves assembled to implement multicommutation. A microcomputer with an electronic interface controlled the solenoid valves and performed data acquisition. Profitable features such as a sampling throughput of 40 determinations per hour, linear response ranging from 25 to 125 mg L−1 cholesterol (r = 0.998), detection limit of 3.7 mg L−1 cholesterol, relative standard deviation of 2.3% (n = 20), and low reagents consumption 0.22 mg luminol and 2.7 mg hexacyanoferrate(III) per determination were achieved. Comparing the results with those obtained using the conventional spectrophotometric method no significant difference at the 95% confidence level was observed. [ABSTRACT FROM AUTHOR]

Enzymatic synthesis of the aspartame precursor, N -(benzyloxycarbonyl)- l -aspartyl- l -phenylalanine methyl ester (Z-AspPheOMe) was performed with highly concentrated molten substrates. A mixture composed of molten N -(benzyloxycarbonyl)- l -aspartic acid (Z-Asp) and l -phenylalanine methyl ester (PheOMe) mixtures of 20 M could be prepared at 50°C. This Z-Asp/PheOMe mixture was applied to the enzymatic synthesis of Z-AspPheOMe using free thermolysin. Synthesis of Z-AspPheOMe was observed in the range of 100-150 μl of NaOH solution (12.5 M) addition to a reaction mixture consisting of 1.0 mmol Z-Asp and 1.0 mmol PheOMe at 50°C. The enzymatic activity increased with increasing water addition, and reached a maximum at 100 μl in addition to the reaction mixture of 1.0 mmol Z-Asp, 1.0 mmol PheOMe and 125 μl of the NaOH solution. In this reaction system, the conversion at the reaction equilibrium was about 60%, the initial reaction rate calculated on the basis of the enzyme weight was 2.2 μmol/g s, and the productivity calculated on the basis of the reaction mixture volume was 300 mol/m 3 h. [ABSTRACT FROM AUTHOR]