Advances of enzymes in the applications of disease treatment and drug preparation / 生物工程学报

The development of biotechnology and the in-depth research on disease mechanisms have led to increased application of enzymes in the treatment of diseases. In addition, enzymes have shown great potential in drug manufacturing, particularly in production of non-natural organic compounds, due to the advantages of mild reaction conditions, high catalytic efficiency, high specificity, high selectivity and few side reactions. Moreover, the application of genetic engineering, chemical modification of enzymes and immobilization technologies have further improved the function of enzymes. This review summarized the advances of using enzymes as drugs for disease treatment or as catalysts for drug manufacturing, followed by discussing challenges, potential solutions and future perspectives on the application of enzymes in the medical and pharmaceutical field.

Construction and application of recombinant human UDP-glucuronosyltransferases expression systems / 药学学报

In the research and development of new drugs, it is very important to investigate the in vitro metabolism of candidate drugs. Traditional models such as liver microsomes have many limitations, while the in vitro model of recombinant human drug metabolizing enzymes is considered as an important and useful approach because of its convenient access, stable activity and low cost. In this study, six major human UDP-glucuronosyltransferases (UGTs) genes (UGT1A1, 1A3, 1A4, 1A6, 1A9 and 2B7) were cloned from human liver cDNA and heterologously expressed in Saccharomyces cerevisiae and baculovirus-infected insect cell. UGT1A1, 1A3, 1A6 and 1A9 were successfully expressed in yeast and showed glucuronidation activity against a variety of different structural types of substrates, but their activities were low. All six UGTs were successfully expressed and exhibited significantly improved glucuronidation activity when Trichopolusia ni cells BTI-TN5B1-4 (High Five) were used as the host. The recombinant human UGTs expressed in insect cells can catalyze the glucuronidation of their specific substrates, and the glucuronidation products were synthesized at milligram-scale with yields of 13%-66% for the first time, of which the structures were identified via MS, 1H NMR, and 13C NMR spectroscopic analysis. Above all, the recombinant human UGTs yeast and insect cell expression systems constructed in this study can be used for in vitro metabolism evaluation in the early stage of new drugs research and development, and also provide a new tool for the synthesis of glucuronide metabolites.

Characterization of inulosucrase and the enzymatic synthesis of inulin / 生物工程学报

As a type of prebiotics and dietary fiber, inulin performs plenty of significant physiological functions and is applied in food and pharmaceutical fields. Inulosucrase from microorganisms can use sucrose as the substrate to synthesize inulin possessing higher molecular weight than that from plants. In this work, a hypothetical gene coding inulosucrase was selected from the GenBank database. The catalytic domain was remained by N- and C- truncation strategies, constructing the recombinant plasmid. The recombinant plasmid was expressed in E. coli expression system, and after purifying the crude enzyme by Ni²⁺ affinity chromatography, a recombinant enzyme with a molecular weight of approximately 65 kDa was obtained. The optimal pH and temperature of the recombinant enzyme were 5.5 and 45 °C, respectively, when sucrose was used as the sole substrate. The activity of this enzyme was inhibited by various metal ions at different degrees. After purifying the produced polysaccharide, nuclear magnetic resonance analysis was used to determine that the polysaccharide was inulin connected by β-(2,1) linkages. Finally, the conditions for the production of inulin were optimized. The results showed that the inulin production reached the maximum, approximately 287 g/L after 7 h, when sucrose concentration and enzyme dosage were 700 g/L and 4 U/mL, respectively. The conversion rate from sucrose to inulin was approximately 41%.

Promising Novel Applications of Heparin / 中国药学杂志

Heparin has been used as first-line anticoagulant clinically for 80 years. Heparin mainly exerts its anticoagulant activity through interaction with ATIII. It has been found there are more than one hundred heparin-dependent functional proteins which demonstrates promising novel application beyond anticoagulant, such as anti-tumor, anti-malaria anti-inflammation, anti-viral, anti-malaria anti-anemia, etc. Heparin is also hopeful to be used as tool for drug delivery vehicle and functionalized implants material. In addition, heparin oligosaccharides are expected to be produced by chemical/enzymatic and bioengineering synthesis. Above mentioned new developments of heparin are covered in this review.

Enzymatic synthesis of mogroside ⅢE / 中国药科大学学报

The aim was to develop the simple preparation method of mogroside ⅢE,and to lay the foundation for the development of the mogroside sweeteners. In the present study,the glycosidase CPU-GH17,which can regio-selectively biosynthesize mogroside ⅢE from mogroside V,was screened from the established library of glycosi-dases. Then,the soluble expression condition of CPU-GH17 in E. coli was exploited by investigating isopropyl β-D-thiogalactoside (IPTG)concentration,culture temperature and induction time,and 0. 4 mmol/L IPTG,15 °C and 12 h was used as optimal condition. The result showed that mogroside V could be completely converted into mogroside ⅢE under the conditions of pH 6. 0,45 °C,3 U/mL enzyme loading,5 mg/mL substrate concentration for 20 h. In conclusion,a biosynthetic system for the regio-selective preparation of mogroside ⅢE by recombinant CPU-GH17 was successfully established and verified at a preparative scale.

Optimization of enzymatic synthesis of asialoglycoprotein receptor ligand cholesterol-vinyl sebacate-lactitol / 中草药

Objective: To synthesize asialoglycoprotein receptor (ASGPR) ligand cholesterol-vinyl sebacate-lactitol (CH-VS-LA) by using enzymatic reaction in organic phase and to optimize its synthesis process. Methods: MS and 13C-NMR were used to identify the structure of product, enzymatic synthesis conditions were optimized via single-factor test and orthogonal experimental design. Results: The enzymatic optimum conditions were as following: the molar ratio of cholesterol-vinyl sebacate (CH-VS) and lactitol (LA) was 4:1, the amount of lipase Novozym 435 was 25 mg, reactional time was 32 h, and productive rate was 92%. Conclusion: The method is highly effecient, the reaction conditions are reasonable, and the process produces no by-product.

Artificial neural network modeling studies to predict the yield of enzymatic synthesis of betulinic acid ester

3 beta-O-phthalic ester of betulinic acid was synthesized from reaction of betulinic acid and phthalic anhydride using lipase as biocatalyst. This ester has clinical potential as an anticancer agent. In this study, artificial neural network (ANN) analysis of Candida antarctica lipase (Novozym 435) -catalyzed esterification of betulinic acid with phthalic anhydride was carried out. A multilayer feed-forward neural network trained with an error back-propagation algorithm was incorporated for developing a predictive model. The input parameters of the model are reaction time, reaction temperature, enzyme amount and substrate molar ratio while the percentage isolated yield of ester is the output. Four different training algorithms, belonging to two classes, namely gradient descent and Levenberg-Marquardt (LM), were used to train ANN. The paper makes a robust comparison of the performances of the above four algorithms employing standard statistical indices. The results showed that the quick propagation algorithm (QP) with 4-9-1 arrangement gave the best performances. The root mean squared error (RMSE), coefficient of determination (R²) and absolute average deviation (AAD) between the actual and predicted yields were determined as 0.0335, 0.9999 and 0.0647 for training set, 0.6279, 0.9961 and 1.4478 for testing set and 0.6626, 0.9488 and 1.0205 for validation set using quick propagation algorithm (QP).

Peptide synthesis: chemical or enzymatic

Peptides are molecules of paramount importance in the fields of health care and nutrition. Several technologies for their production are now available, among which chemical and enzymatic synthesis are especially relevant. The present review pretends to establish a non-biased appreciation of the advantages, potentials, drawbacks and limitations of both technologies. Chemical synthesis is thoroughly reviewed and their potentials and limitations assessed, focusing on the different strategies and challenges for large-scale synthesis. Then, the enzymatic synthesis of peptides with proteolytic enzymes is reviewed considering medium, biocatalyst and substrate engineering, and recent advances and challenges in the field are analyzed. Even though chemical synthesis is the most mature technology for peptide synthesis, lack of specificity and environmental burden are severe drawbacks that can in principle be successfully overcame by enzyme biocatalysis. However, productivity of enzymatic synthesis is lower, costs of biocatalysts are usually high and no protocols exist for its validation and scale-up, representing challenges that are being actively confronted by intense research and development in this area. The combination of chemical and enzymatic synthesis is probably the way to go, since the good properties of each technology can be synergistically used in the context of one process objective.