Enzyme-catalyzed Synthesis Research Articles

Optimization of Biocatalytic Rhododendrol Production from Biogenic Rhododendrol Glycosides.

An enzyme-catalyzed synthesis of rhododendrol, an intermediate in the production of raspberry ketone, was investigated. The approach involves the enzymatic hydrolysis of rhododendrol glycosides into rhododendrol and a glycosidic residue. Rhododendrol glycosides, which are naturally derived from the inner bark of birch trees-a renewable resource-vary considerably in composition depending on the origin of the plants. In this study, mixtures of betuloside and apiosylrhododendrin from natural resources were used in different proportions. An in-depth study was conducted to assess the feasibility of the process. A mathematical model was developed based on studies of the kinetics and operational stability of the enzyme. The model for betuloside hydrolysis catalyzed by β-glucosidase was validated in batch, repetitive batch, and ultrafiltration membrane reactors. The highest productivity, ranging from 83.9 to 94.5 g L-1 day-1, was achieved in the latter. After screening nearly 50 enzymes, RAPIDASE emerged as a solution for the hydrolysis of apiosylrhododendrin, and the model was validated in a batch reactor. Model-based optimization enabled the prediction of input parameters for different compositions of biogenic rhododendrol glycosides to obtain consistent process output metrics.

Enzyme-catalyzed transesterification of galactomannan extracted from mesquite seed (Prosopis velutina) with vinyl carboxylate esters

Galactomannans (GM) are hemicellulosic polysaccharides composed of D-mannopyranose chains linked by β (1 → 4) glycosidic linkages with branches of D-galactopyranose linked by α (1 → 6) linkages. This polysaccharide is recognized for its hydrophilic character, as it is rich in hydroxyl groups (-OH). This chemical characteristic, combined with the absence of ionic charges, enables structural modifications such as transesterification of the fatty acid chains (FA), which provides a strategy for obtaining amphiphilic structures. The enzyme-catalyzed syntheses were carried out in DMSO with GM decanoate (GMD) and GM palmitate (GMP) at different molar ratios (0.5 and 1.0) and the resulting structures were evaluated with infrared spectroscopy (FTIR), solid-state nuclear magnetic resonance (CP/MAS 13C NMR) and differential scanning calorimetry (DSC). The FTIR spectrum confirmed the transesterification of GM with the appearance of a CO band (1730-1750 cm−1). These results were confirmed by the signals observed at 177 and 30 ppm in the CP/MAS 13C NMR spectrum, which corresponded to the CO groups of the esters and the terminal –CH3 groups of the FA chains, respectively. Finally, DSC showed glass transition temperatures (Tg) in the range 43–51 °C, while the melting temperatures (Tm) of the GM esters (59 °C) were not affected by different degrees of esterification (DE) for GMD (0.37 and 0.71) and GMP (0.47 and 0.57).

Enzyme-Catalyzed Synthesis in Pharmaceutical Manufacturing

In recent years, with the progress of science and the rise of green chemistry, enzyme catalysis technology has been highly valued as an important branch of green chemistry. Due to the development of biotechnology, enzyme catalysis technology has been increasingly used in organic synthesis, especially asymmetric synthesis, the synthesis of optically active compounds and natural products. It has been applied in medicine, food and textiles industry. Among these application areas, the pharmaceutical field has been a hot topic in enzyme catalysis technology in recent years. The application of enzyme catalysis technology in the pharmaceutical industry is a means to promote sustainable and environmentally friendly practices. It helps to solve continuously upgrading environmental problems and promote the green development of the industry. Therefore, conducting research on enzyme-catalyzed synthesis in pharmaceutical manufacturing is of great significance. In this work, several practical examples of enzyme catalysis techniques used in the pharmaceutical industry are introduced and discussed.

Direct Electrochemical Regeneration of NADH and Its Biomimetics

Enzyme-catalyzed redox transformations are an attractive approach for conversion of unactivated alkanes into value-added products under mild reaction conditions. This is especially true since advancements in the area of enzyme engineering has led to a dramatic expansion in the use of modified oxidoreductases for selective functionalization of commodity petrochemicals in non-aqueous media. Despite their appeal, many redox enzymes rely on stoichiometric quantities of expensive coenzymes, such as nicotinamide adenine dinucleotide (NADH), that are not easily regenerated. The use of direct electrochemistry to catalytically regenerate NADH has long been an aspiration of chemists as a convenient and straightforward method that would enable use of the expensive cofactor in enzyme-catalyzed synthesis on an industrial scale. Unfortunately, direct electrochemical reduction of the oxidized form of nicotinamide adenine dinucleotide (NAD+) results in unstable radical intermediates that rapidly form biologically inactive byproducts. Recent progress has been made towards the development of inexpensive artificial NADH biomimetics as replacements for the natural coenzyme, but the strategies to regenerate these biomimetic coenzymes is equally limiting. A primary focus of our research is to design synthetic analogues of NAD+ and NADH that are both enzymatically active and electrochemically regenerable. We will present our recent progress in understanding the structure-reactivity relationships that lead to dimerization over the preferred formation of 1,4-dihydronicotinamide in NAD+ and its biomimetics, and we will describe strategies to prevent dimerization of electrochemically reduced NAD+.

Enzyme-Catalyzed Synthesis of Glycerol Carbonate in Solventless Liquid One Phase Conditions: Role of Reaction Medium Engineering on Catalytic Performance

Enzyme-Catalyzed Synthesis of Glycerol Carbonate in Solventless Liquid One Phase Conditions: Role of Reaction Medium Engineering on Catalytic Performance

Simple separation operation improved enzyme-catalyzed ε-caprolactone yield

With the widely application of polycaprolactone-based biodegradable materials, the green production of ε-caprolactone (ε-CL) as a major synthetic monomer, had received widespread attention. In this paper, it was first proposed to use a simple separate operation for enzyme-catalyzed synthesis of ε-CL, which enabled the yield of ε-CL to be improved without complex modification of the enzyme, the factors led to enzymes inactivation during the reaction and the structure of the white undissolved filtered particles were analyzed in detail. As a result, compared with the conventional mixed reaction, this reaction method not only improved the effect of enzyme catalysis, and increased the yield of ε-CL to 90%, but also made immobilized enzymes maintain the integrity of the particles during the reaction, and carriers were reused so as to reduce the production cost. At the same time, it was first demonstrated that the main cause of enzyme inactivation was the change in secondary structure content during the reaction, and the white undissolved filtered particles after the reaction was called hydroxyurea.

Insights into the Unusual Activity of a Novel Homospermidine Synthase with a Promising Application to Produce Spermidine.

Spermidine is a naturally occurring polyamine with multiple biological activities and potential food and agricultural applications. However, sustainable and scalable spermidine production has not yet been attained. In this study, a homospermidine synthase (HSS) from Pseudomonas frederiksbergensis (PfHSS) capable of catalyzing the synthesis of spermidine from 1,3-diaminopropane and putrescine was identified based on multiple sequence alignment using Blastochloris viridis HSS (BvHSS) as a template. The optimal reaction pH and temperature for purified PfHSS were determined to be 8.5 and 45 °C, respectively, and K+ was able to promote the enzyme activity. Further analysis of the structural and functional relationships through molecular docking and molecular dynamics simulation indicates that glutamic acid at position 359 is the essential residue for the enzyme-catalyzed synthesis of spermidine. The whole-cell catalytic reaction yielded 1321.4 mg/L spermidine and 678.2 mg/L of homospermidine. This study presents a novel, promising, and sustainable biological method for producing spermidine.

Enzyme-catalyzed synthesis of selenium-doped manganese phosphate for synergistic therapy of drug-resistant colorectal cancer

BackgroundThe development of multidrug resistance (MDR) during postoperative chemotherapy for colorectal cancer substantially reduces therapeutic efficacy. Nanostructured drug delivery systems (NDDSs) with modifiable chemical properties are considered promising candidates as therapies for reversing MDR in colorectal cancer cells. Selenium-doped manganese phosphate (Se-MnP) nanoparticles (NPs) that can reverse drug resistance through sustained release of selenium have the potential to improve the chemotherapy effect of colorectal cancer.ResultsSe-MnP NPs had an organic–inorganic hybrid composition and were assembled from smaller-scale nanoclusters. Se-MnP NPs induced excessive ROS production via Se-mediated activation of the STAT3/JNK pathway and a Fenton-like reaction due to the presence of manganese ions (Mn2+). Moreover, in vitro and in vivo studies demonstrated Se-MnP NPs were effective drug carriers of oxaliplatin (OX) and reversed multidrug resistance and induced caspase-mediated apoptosis in colorectal cancer cells. OX@Se-MnP NPs reversed MDR in colorectal cancer by down-regulating the expression of MDR-related ABC (ATP binding cassette) transporters proteins (e.g., ABCB1, ABCC1 and ABCG2). Finally, in vivo studies demonstrated that OX-loaded Se-MnP NPs significantly inhibited proliferation of OX-resistant HCT116 (HCT116/DR) tumor cells in nude mice.ConclusionsOX@Se-MnP NPs with simple preparation and biomimetic chemical properties represent promising candidates for the treatment of colorectal cancer with MDR.

Enzyme-catalyzed synthesis of 4-methylcatechol oligomer and preliminary evaluations as stabilizing agent in polypropylene

Abstract In the present work, 4-methylcatechol oligomer has been prepared by using enzyme-catalyzed polymerization in water and preliminary evaluations as stabilizing agent in polypropylene (PP) was performed. In comparison with intrinsic PP, the oxidation onset temperature of the 4-methylcatechol oligomer/PP composite increased by 66°C, and the oxidation induction time increased by 40 min. In addition, the mixing of a 4-methylcatechol oligomer with PP (i.e., in the formation of a 4-methylcatechol oligomer/PP composite) did significantly enhance the long-term stability of PP in a thermal oxidative environment. Moreover, the tensile strength of this composite did not significantly decrease after aging for 800 h in an air atmosphere at 120°C. These results show that the addition of a 4-methylcatechol oligomer will markedly delay the aging and degradation of PP materials, even under extreme conditions. Thus, an enzyme-catalyzed polymerization of phenol compounds may provide a new avenue toward the preparation of novel antioxidants.

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.

Protein Hydrolysates from Pleurotus geesteranus Modified by Bacillus amyloliquefaciens γ-Glutamyl Transpeptidase Exhibit a Remarkable Taste-Enhancing Effect

Long-term high salt intake exerts a negative impact on human health. The excessive use of sodium substitutes in the food industry can lead to decreased sensory quality of food. γ-Glutamyl peptides with pronounced taste-enhancing effects can offer an alternative approach to salt reduction. However, the content and yield of γ-glutamyl peptides in natural foods are relatively low. Enzyme-catalyzed synthesis of γ-glutamyl peptides provides a feasible solution. In this study, Pleurotus geesteranus was hydrolyzed by Flavourzyme to generate protein hydrolysates. Subsequently, they were modified by Bacillus amyloliquefaciens γ-glutamyl transpeptidase to generate γ-glutamyl peptides. The reaction conditions were optimized and their taste-enhancing effects were evaluated. Their peptide sequences were identified by parallel reaction monitoring with liquid chromatography-tandem mass spectrometry and analyzed using molecular docking. The optimal conditions for generation of γ-glutamyl peptides were a pH of 10.0, an enzyme condition of 1.2 U/g, and a reaction time of 2 h, which can elicit a strong kokumi taste. Notably, it exhibited a remarkable taste-enhancing effect for umami intensity (76.07%) and saltiness intensity (1.23-fold). Several novel γ-glutamyl peptide sequences were found by liquid chromatography-tandem mass spectrometry, whereas the binding to the calcium-sensing receptor was confirmed by molecular docking analysis. Overall, γ-glutamyl peptides from P. geesteranus could significantly enhance the umami and salt tastes, which can serve as promising taste enhancers.

Exploring the enzyme-catalyzed synthesis of isotope labeled cyclopropanes.

Cyclopropanes are commonly employed structural moieties in drug design since their incorporation is often associated with increased target affinity, improved metabolic stability, and increased rigidity to access bioactive conformations. Robust chemical cyclopropanation procedures have been developed which proceed with high yield and broad substrate scope, and have been applied to labeled substrates. Recently, engineered enzymes have been shown to perform cyclopropanations with remarkable diastereoselectivity and enantioselectivity, but this biocatalytic approach has not been applied to labeled substrates to date. In this study, the use of enzyme catalysis for the synthesis of labeled cyclopropanes was investigated. Two readily available enzymes, a modified CYP450 enzyme and a modified Aeropyrum pernix protoglobin, were investigated for the cyclopropanation of a variety of substituted styrenes. For this biocatalytic transformation, the enzymes required the use of ethyl diazoacetate. Due to the highly energetic nature of this molecule, alternatives were investigated. The final optimized cyclopropanation was successfully demonstrated using n‐hexyl diazoacetate, resulting in moderate to high enantiomeric excess. The optimized procedure was used to generate labeled cyclopropanes from 13C‐glycine, forming all four labeled stereoisomers of phosphodiesterase type‐IV inhibitor, MK0952. These reactions provide a convenient and effective biocatalytic route to stereoselective 13C‐labeled cyclopropanes and serve as a proof‐of‐concept for generating stereoselective labeled cyclopropanes.

Modulation of Enzyme-Catalyzed Synthesis of Prostaglandins by Components Contained in Kidney Microsomal Preparations.

In the kidney, prostaglandins formed by cyclooxygenase 1 and 2 (COX-1 and COX-2) play an important role in regulating renal blood flow. In the present study, we report our observations regarding a unique modulatory effect of renal microsomal preparation on COX-1/2-mediated formation of major prostaglandin (PG) products in vitro. We found that microsomes prepared from pig and rat kidneys had a dual stimulatory–inhibitory effect on the formation of certain PG products catalyzed by COX-1 and COX-2. At lower concentrations, kidney microsomes stimulated the formation of certain PG products, whereas at higher concentrations, their presence inhibited the formation. Presence of kidney microsomes consistently increased the Km values of the COX-1/2-mediated reactions, while the Vmax might be increased or decreased depending on stimulation or inhibition observed. Experimental evidence was presented to show that a protein component present in the pig kidney microsomes was primarily responsible for the activation of the enzyme-catalyzed arachidonic acid metabolism leading to the formation of certain PG products.

Enzyme-catalyzed synthesis of bioactive heterocycles

Abstract Enzymes are proteins that functions as biological catalyst. It is now a known fact that enzyme can catalyze many synthetic operations better than the conventional reagents. Not only in the synthesis of natural products, they can also be applied for construction of varieties of unnatural compounds. In this chapter, Pariyar and Ghosh have discussed in brief synthesis of various biologically active heterocyclic compounds using different enzymes as catalysts. Among various enzymes, laccases, trypsin, α-amylase and Bakers’ yeast are few that are easily available and have been extensively explored for various synthetic strategies. This chapter will definitely serve as valuable source of information to the readers in the field of enzyme-catalyzed reactions.

Enzymatic synthesis of fluorinated compounds.

Fluorinated compounds are widely used in the fields of molecular imaging, pharmaceuticals, and materials. Fluorinated natural products in nature are rare, and the introduction of fluorine atoms into organic compound molecules can give these compounds new functions and make them have better performance. Therefore, the synthesis of fluorides has attracted more and more attention from biologists and chemists. Even so, achieving selective fluorination is still a huge challenge under mild conditions. In this review, the research progress of enzymatic synthesis of fluorinated compounds is summarized since 2015, including cytochrome P450 enzymes, aldolases, fluoroacetyl coenzyme A thioesterases, lipases, transaminases, reductive aminases, purine nucleoside phosphorylases, polyketide synthases, fluoroacetate dehalogenases, tyrosine phenol-lyases, glycosidases, fluorinases, and multienzyme system. Of all enzyme-catalyzed synthesis methods, the direct formation of the C-F bond by fluorinase is the most effective and promising method. The structure and catalytic mechanism of fluorinase are introduced to understand fluorobiochemistry. Furthermore, the distribution, applications, and future development trends of fluorinated compounds are also outlined. Hopefully, this review will help researchers to understand the significance of enzymatic methods for the synthesis of fluorinated compounds and find or create excellent fluoride synthase in future research. Key points • Fluorinated compounds are distributed in plants and microorganisms, and are used in imaging, medicine, materials science.• Enzyme catalysis is essential for the synthesis of fluorinated compounds.• The loop structure of fluorinase is the key to forming the C-F bond.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00253-021-11608-0.

Enzyme-catalyzed synthesis and properties of polyol ester biolubricant produced from Rhodotorula glutinis lipid

Biolubricants do not cause the environmental pollution resulting from the manufacturing and use of mineral-based lubricants, but the commonly adopted chemical method of preparing biolubricants using expensive vegetable oils as raw materials is still environmentally and economically unsatisfactory. In this study, R. glutinis lipid with a high oleic-acid content was esterified with trimethylolpropane (TMP) by using the Candida sp. 99–125 enzyme in a solvent-free system, and the effect of the microbial oil with the high oleic-acid content on the performance of resulting biolubricants was investigated. The purpose of this study was to solve the problems of pollution caused by the chemical synthesis of biolubricants and of the uneconomical use of vegetable oils as raw materials. Further, this study aimed to improve the performance of biolubricants by using the microbial lipid with a high oleic-acid content. As a result of optimization of the reaction conditions, the yield of trimethylolpropane fatty acid triester (TFAT) reached 89.5%. The synthesized biolubricants had excellent low-temperature lubrication performance (pour point=−45 °C), and the physicochemical performance reached the ISOVG-46 grade. The results of the high-frequency reciprocating friction test (HRFT) show that the products have better tribological properties than mineral-based lubricants of the same viscosity grade (friction coefficient=0.085; average wear scar diameter=187 µm).

Accelerating the optimization of enzyme-catalyzed synthesis conditions via machine learning and reactivity descriptors.

Enzyme-catalyzed synthesis reactions are of crucial importance for a wide range of applications. An accurate and rapid selection of optimal synthesis conditions is crucial and challenging for both human knowledge and computer predictions. In this work, a new scenario, which combines a data-driven machine learning (ML) model with reactivity descriptors, is developed to predict the optimal enzyme-catalyzed synthesis conditions and the reaction yield. Fourteen reactivity descriptors in total are constructed to describe 125 reactions (classified into five categories) included in different reaction mechanisms. Nineteen ML models are developed to train the dataset and the Quadratic support vector machine (SVM) model is found to exhibit the best performance. The Quadratic SVM model is then used to predict the optimal reaction conditions, which are subsequently used to obtain the highest yield among 109 200 reaction conditions with different molar ratios of substrates, solvents, water contents, enzyme concentrations and temperatures for each reaction. The proposed protocol should be generally applicable to a diverse range of chemical reactions and provides a black-box evaluation for optimizing the reaction conditions of organic synthesis reactions.

Synthesis of Aryl or Heteroaryl C-Nucleosides by Direct Coupling of a Carbohydrate Moiety with a Preformed Aglycon Unit

C-Nucleosides have similar structure compared to native N-nucleosides, while the carbohydrate unit and the base in a C-nucleoside are connected through a carbon-carbon bond. Because they can also be recognized and utilized by enzymes associated with N-nucleosides in cells, C-nucleosides can inhibit enzyme-catalyzed synthesis or degradation of nucleic acids, thereby inhibiting the proliferation of viruses or cancer cells. C-Nucleoside synthesis has drawn much attention since the clinical application of the C-nucleoside drug remdesivir for the treatment of COVID-19. The direct coupling of a carbohydrate moiety with a preformed aglycon unit is an efficient synthetic approach to construct aryl or heteroaryl C-nucleoside. The synthetic methods of aryl or heteroaryl C-nucleosides in recent years are summarized from three main synthetic strategies: coupling of ribose derivatives with organometallic reagents, transition metal-catalyzed coupling of ribose derivatives with organometallic reagents, and acid-catalyzed Friedel-Crafts reaction of ribose derivatives. Each synthetic strategy is categorized in terms of sugar donor precursors, including hemiacetal riboses, ribonolactones, ribofuranosyl halides, glycal, and others. The mechanism of α or β product formation in these reactions are explained in detail as well. © 2021 Chinese Chemical Society & SIOC, CAS.

Enzyme-catalyzed synthesis of malonate polyesters and their use as metal chelating materials.

Following the environmental problems caused by non-degradable plastics there is a need to synthesise greener and more sustainable polymers. In this work we describe, for the first time, the facile enzyme-catalysed synthesis of linear polyesters using dimethyl malonate as the diester. These polymers, containing a different aliphatic diol component (C4, C6 or C8), were synthesised in solventless conditions using immobilized Candida antarctica lipase B as the biocatalyst. The potential of enzymes for catalysing this reaction is compared with the unsuccessful antimony- and titanium-catalysed synthesis (T > 150 °C). The application of the synthesized polymers as effective metal chelators in biphasic, green solvent systems was also described, together with the characterisation of the synthesised materials.

On the thermodynamics of biocatalytic reactions with application of group-contribution correlation and prediction

A thermodynamic analysis is presented to correlate and predict reaction equilibrium properties for enzyme-catalyzed synthesis of valuable chemical compounds, such as small-molecule pharmaceuticals. Utilization of group-contribution methods is shown for obtaining reference-state ideal gas and standard-state solution Gibbs energies of formation of reactants and products, as well as for estimating solution nonidealities. Several transaminase reactions have been treated. These cases were previously shown not to be accurately predicted by estimates of Gibbs energies of formation from group contributions. This work achieves accurate descriptions of the data by regressing some unknown component values with a method that accounts for uncertainties in property and data uncertainties. The results and methodology can be applied to many biosystems, but having some reaction equilibrium measurements with uncertainties that involve the species of interest are essential for reliable results.