[Research progress on application of multi-enzyme-catalyzed cascade reactions in enzymatic synthesis of natural products].

As a biocatalyst, enzyme has the advantages of high catalytic efficiency, strong reaction selectivity, specific target products, mild reaction conditions, and environmental friendliness, and serves as an important tool for the synthesis of complex organic molecules. With the continuous development of gene sequencing technology, molecular biology, genetic manipulation, and other technologies, the diversity of enzymes increases steadily and the reactions that can be catalyzed are also gradually diversified. In the process of enzyme-catalyzed synthesis, the majority of common enzymatic reactions can be achieved by single enzyme catalysis, while many complex reactions often require the participation of two or more enzymes. Therefore, the combination of multiple enzymes together to construct the multi-enzyme cascade reactions has become a research hotspot in the field of biochemistry. Nowadays, the biosynthetic pathways of more natural products with complex structures have been clarified, and secondary metabolic enzymes with novel catalytic activities have been identified, discovered, and combined in enzymatic synthesis of natural/unnatural molecules with diverse structures. This study summarized a series of examples of multi-enzyme-catalyzed cascades and highlighted the application of cascade catalysis methods in the synthesis of carbohydrates, nucleosides, flavonoids, terpenes, alkaloids, and chiral molecules. Furthermore, the existing problems and solutions of multi-enzyme-catalyzed cascade method were discussed, and the future development direction was prospected.

Structure-guided preparation of fuctional oil rich in 1,3-diacylglycerols and linoleic acid from Camellia oil by combi-lipase.

BACKGROUND: Diacylglycerol (DAG)-enriched oil has been attracting attention because of its nutritional benefits and biological functions, although the composition of its various free fatty acids (FFAs) and an unclear relationship between substrate and yield make it difficult to be identified and qualified with respect to its production. In the present study, linoleic acid-enriched diacylglycerol (LA-DAG) was synthesized and enriched from Camellia oil by the esterification process using the combi-lipase Lipozyme TL IM/RM IM system. RESULTS: The relationship between FFA composition and DAG species productivity was revealed. The results showed that heterogeneous FFA with a major constituent (more than 50%) exhibited higher DAG productivity and inhibited triacylglycerol productivity compared to homogeneous constituents. Joint characterization by high-performance liquid chromatography-evaporative light scattering detection, gas chromatography-mass spectrometry and ultra-performance liquid chromatography-heated electrospray ionization-tandem mass spectrometry identified that DAG components contained dilinoleic acid acyl glyceride, linoleyl-oleyl glyceride and dioleic acid acyl glyceride in esterification products. Under the optimum conditions, 60.4% 1,3-DAG and 61.3% LA-DAG in the crude product at 1 h reaction were obtained, and further purified to 81.7% LA-DAG and 94.7% DAG via silica column chromatography. CONCLUSION: The present study provides a guideline for the identification of DAG species, as well as a structure-guided preparation method of DAG-enriched oils via the cost-effective combi-lipase. © 2022 Society of Chemical Industry.

Biosynthesis Investigations of Terpenoid, Alkaloid, and Flavonoid Antimicrobial Agents Derived from Medicinal Plants.

The overuse of antibiotics in the past decades has led to the emergence of a large number of drug-resistant microorganisms. In recent years, the infection rate caused by multidrug-resistant microorganisms has been increasing, which has become one of the most challenging problems in modern medicine. Plant-derived secondary metabolites and their derivatives have been identified to display significant antimicrobial abilities with good tolerance and less adverse side effects, potentially having different action mechanisms with antibiotics of microbial origin. Thus, these phyto-antimicrobials have a good prospect in the treatment of multidrug-resistant microorganisms. Terpenoids, alkaloids, and flavonoids made up the predominant part of the currently reported phytochemicals with antimicrobial activities. Synthetic biology research around these compounds is one of the hotspot fields in recent years, which not only has illuminated the biosynthesis pathways of these phyto-antimicrobials but has also offered new methods for their production. In this review, we discuss the biosynthesis investigations of terpenoid, alkaloid, and flavonoid antimicrobial agents-using artemisinin and oleanolic acid (terpenoids), berberine and colchicine (alkaloids), and baicalin (flavonoids) as examples-around their antimicrobial action mechanisms, biosynthesis pathway elucidation, key enzyme identification, and heterologous production, in order to provide useful hints for plant-derived antimicrobial agent discovery and development.

Fabrication, characterization, and purification of nutraceutical diacylglycerol components from Camellia oil.

Converting triacylgycerols (TAGs) from edible oils and fats into structured diacylglycerols (DAGs) is meaningful for reducing obesity. Camellia oil, rich in linoleic acid, has the potential to form structured linoleic acid-1,3-diacylglycerol (LA-1,3-DAG) nutrients in the industry. In this research, the physicochemical properties of modified Camellia oil (MCO) by enzymatic esterification were analyzed by Gas Chromatography-Mass Spectrometry (GC-MS), Differential Scanning Calorimetry (DSC), High Performance Liquid Chromatography-Evaporative Light Scattering Detection (HPLC-ELSD), and Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS). The relationship between reaction conditions and the DAG compositions is disclosed using multiple factors. It is found that high constituents of DAG increase the melting and crystallization temperature of MCO, lipase Novozym 435 gives the best yield of targeted nutrients (DAG, 1,3-DAG, LA-DAG), and the mixture of lipases, Lipozyme TL IM and Lipozyme RM IM, shows a synergistic effect in the synthetic process of DAG. Subsequently, MCO containing 65.4% DAG, 54.7% LA-DAG, and 47.6% 1,3-DAG content at optimal conditions (2% enzyme dosage, 4 h reaction time, 2.4:1 substrate molar ratio, 25.8% t-butanol as solvent, 60°C temperature) has been obtained and purified using silica column to obtain the final DAG oil containing 96.1% DAG, 64.7% 1,3-DAG, and 78.4% LA-DAG. High constituents of structured DAG oil rich in LA-1,3-DAG can be obtained by enzymatic esterification for industrial production.

Robustness and accuracy evaluation of moisture prediction model for black tea withering process using hyperspectral imaging.

The rapid and non-destructive detection of moisture in withering leaves is an unsolved problem because the leaves are stacked together and have random orientation. To address this issue, this study aimed to establish more robust and accurate models. The performance of front side, back side and multi-region models were compared, and the front side model showed the worst transferability. Therefore, five effective wavelength (EW) selection algorithms were combined with a successive projection algorithm (SPA) to select EWs. It was found that the shuffled frog leaping algorithm (SFLA) combined with SPA was the best method for the front side model for moisture analyses. Based on the selected EWs, the extreme learning machine (ELM) became the model with the best self-verification result. Subsequently, moisture distribution maps of withering leaves were successfully generated. Considering the processing demand of withering leaves, local region models developed based on partial least squares and the SFLA-SPA method were applied to predict the moisture of withering leaves in the local and stacked region. The results showed that the RPD, Rcv and Rp values were above 1.6, 0.870 and 0.897, respectively. These results provide a useful reference for the non-destructive detection of moisture in withering leaves.

Molecular cloning and biochemical characterization of a new coumarin glycosyltransferase CtUGT1 from Cistanche tubulosa.

UDP-glycosyltransferases (UGTs) are an important and functionally diverse family of enzymes involved in secondary metabolite biosynthesis. Coumarin is one of the most common skeletons of natural products with candidate pharmacological activities. However, to date, many reported GTs from plants mainly recognized flavonoids as sugar acceptors. Only limited GTs could catalyze the glycosylation of coumarins. In this study, a new UGT was cloned from Cistanche tubulosa, a valuable traditional tonic Chinese herb, which is abundant with diverse glycosides such as phenylethanoid glycosides, lignan glycosides, and iridoid glycosides. Sequence alignment and phylogenetic analysis showed that CtUGT1 is phylogenetically distant from most of the reported flavonoid UGTs and adjacent to phenylpropanoid UGTs. Extensive in vitro enzyme assays found that although CtUGT1 was not involved in the biosynthesis of bioactive glycosides in C. tubulosa, it could catalyze the glucosylation of coumarins umbelliferone 1, esculetine 2, and hymecromone 3 in considerable yield. The glycosylated products were identified by comparison with the reference standards or NMR spectroscopy, and the results indicated that CtUGT1 can regiospecifically catalyze the glucosylation of hydroxyl coumarins at the C7-OH position. The key residues that determined CtUGT1's activity were further discussed based on homology modeling and molecular docking analyses. Combined with site-directed mutagenesis results, it was found that H19 played an irreplaceable role as the crucial catalysis basis. CtUGT1 could be used in the enzymatic preparation of bioactive coumarin glycosides.

Green synthesis of garlic oil nanoemulsion using ultrasonication technique and its mechanism of antifungal action against Penicillium italicum.

Penicillium italicum (P. italicum) can cause significant economic loss of fruits and vegetables. Although garlic oil (GO) is an effective antimicrobial agent, the unstability and hydrophobicity limit its use as an environmentally friendly alternative to the conventional antibiotics against P. italicum. In this study, we focused on the fabrication and characterization of a functional GO nanoemulsion (NE) using ultrasonic technique and revealed the antifungal mechanism of the GO NE on P. italicum based on morphological, structural and molecular analyses. The optimal hydrophilic lipophilic balance (HLB) value determined for GO was 14 through the combination of Tween 80 and Span 80. Then the Box-Benhnken Design (BBD) was applied to produce the GO NE and the effects of different fabrication parameters on the particle size were evaluated. The optimal GO NE was selected with the GO concentration of 5.5%, the Smix concentration of 10%, the ultrasonic time of 5 min and the power of 50%. This GO NE had the smallest particle size of 52.27 nm, the best antifungal effect and the most stability. Furthermore, the antifungal mechanism of the GO NE on P. italicum was evaluated by extracellular conductivity, micro-Raman spectra, fluorescence imaging and scanning electron microscopy (SEM) imaging. The results presented that the GO NE retained the antifungal active ingredients. The fungal cell structure and morphology were malformed after treated with the GO NE and the lipids, nucleic acids and protein of P. italicum were destructed. Finally, the optimal GO NE was applied in vivo and P. italicum in citrus was successfully inhibited. It indicated that the optimal GO NE had the better antifungal activity against P. italicum than the pure GO. Besides, the minimum inhibitory concentration (MIC) of GO after preparing into the NE was changed from 3.7% to 0.01265% with about 300 times improvement of bioavailability. Therefore, the synthetic GO NE which promoted the bioavailability of GO was recommended as a promising alternative to inhibit P. italicum in vegetables and fruits.

Optimization of informative spectral variables for the quantification of EGCG in green tea using Fourier transform near-infrared (FT-NIR) spectroscopy and multivariate calibration.

Epigallocatechin-3-gallate (EGCG) is credited with the majority of the health benefits associated with green tea consumption. It has a high economic and medicinal value. The feasibility of using different variable selection approaches in Fourier transform near-infrared (FT-NIR) spectroscopy for a rapid and conclusive quantitative determination of EGCG in green tea was investigated. Graphically oriented multivariate calibration modeling procedures such as interval partial least squares (iPLS), synergy interval partial least squares (siPLS), and genetic algorithm optimization combined with siPLS (siPLS-GA) were applied to select the most efficient spectral variables that provided the lowest prediction error. The performance of the final model was evaluated according to the root mean square error of prediction (RMSEP) and coefficient of determination (R(2)) for the prediction set. Experimental results showed that the siPLS-GA model obtained the best results in comparison to other models. The optimal models were achieved with R(2)(p) = 0.97 and RMSEP = 0.32. The model can be obtained with only 36 variables retained and it provides a robust model with good estimation accuracy. This demonstrates the potential of NIR spectroscopy with multivariate calibration methods to quickly detect the bioactive component in green tea.

[Determination of three organic acids in Schisandrae Chinensis Fructus by HPLC].

OBJECTIVE: To establish an HPLC method for determination of three organic acids in Schisandrae Chinensis Fructus. METHOD: The separation was performed on a C18 column with acetonitrile-15 mmol x L(-1) KH2 PO4 (1:99) and methanol-0.5% acetic glacial as the mobile phrase to determine the content of kinic acid, citric acid, and protocatechuic acid. RESULT: Three kinds of organic acid can be detective in different samples, and the average content of the three organic acids were about 18%, among which the citric acid was the highest (18%). CONCLUSION: Since organic acids is an important parts of the material basis in Schisandrae Chinensis Fructus, it is necessary to carry out the pharmacological research associated with the function of Schisandrae Chinensis Fructus. Further, it will provide scientific basis for the drug relevance research between material basis and efficiency, as well as the foundation for the relevance research on the properties and Five Tastes of traditional Chinese medicine.

Two new anthraquinone glycosides from the roots of Rheum palmatum.

Two new anthraquinone glycosides, named 1-methyl-8-hydroxyl-9,10-anthraquinone-3-O-ß-D-(6'-O-cinnamoyl)glucopyranoside (1) and rhein-8-O-ß-D-[6'-O-(3''-methoxyl malonyl)]glucopyranoside (2), have been isolated from the roots of Rheum palmatum, together with seven known compounds, rhein-8-O-ß-D-glucopyranoside (3), physcion-8-O-ß-D-glucopyranoside (4), chrysophanol-8-O-ß-D-glucopyranoside (5), aleo-emodin-8-O-ß-D-glucopyranoside (6), emodin-8-O-ß-D-glucopyranoside (7), aleo-emodin-ω-O-ß-D-glucopyranoside (8), and emodin-1-O-ß-D-glucopyranoside (9). Their structures were elucidated on the basis of chemical and spectral analysis.