Sequence and Structure-Guided Engineering of Urethanase from Agrobacterium tumefaciens d3 for Improved Catalytic Activity.

The amidase from Agrobacterium tumefaciens d3 (AmdA) degrades the carcinogenic ethyl carbamate (EC) in alcoholic beverages. However, its limited catalytic activity hinders practical applications. Here, multiple sequence alignment was first used to predict single variants with improved activity. Afterward, AlphaFold 2 was applied to predict the three-dimensional structure of AmdA and 21 amino acids near the catalytic triad were randomized by saturation mutagenesis. Each of the mutation libraries was then screened, and the improved single variants were combined to obtain the best double variant I97L/G195A that showed a 3.1-fold increase in the urethanase activity and a 1.5-fold increase in ethanol tolerance. MD simulations revealed that the mutations shortened the distance between catalytic residues and the substrate and enhanced the occurrence of a critical hydrogen bond in the catalytic pocket. This study displayed a useful strategy to engineer an amidase for the improvement of urethanase activity, and the variant obtained provided a good candidate for applications in the food industry.

Expression, isolation, and identification of an ethanol-resistant ethyl carbamate-degrading amidase from Agrobacterium tumefaciens d3.

Ethyl carbamate (EC), widely found in alcoholic beverages, has been revealed to be a probable carcinogen in humans. Urethanase (EC 3.5.1.75) is an effective enzyme for the degradation of EC; however, the previously identified urethanases exhibited insufficient acid and alcohol resistance. In this study, an enantioselective amidase (AmdA) screened from Agrobacterium tumefaciens d3 exhibited urethanase activity with excellent alcohol resistance. AmdA was first overexpressed in Escherichia coli; however, the recombinant protein was primarily located in inclusion bodies, and thus, co-expression of molecular chaperones was used. The activity of AmdA increased 3.1 fold to 307 U/L, and the specific activity of urethanase with C-terminal His-tags reached 0.62 U/mg after purification through a Ni-NTA column. Subsequently, the enzymatic properties and kinetic constants of AmdA were investigated. The optimum temperature for AmdA was 55 °C, it showed the highest activity at pH 7.5, and the Km was 0.964 mM. Moreover, after 1 h of heat treatment at 37 °C in a 5-20% (v/v) ethanol solution, the residual urethanase activity was higher than 91%, considerably more than that reported thus far.

Analysis of nicotine-induced metabolic changes in Blakeslea trispora by GC-MS.

Blakeslea trispora is a natural source of carotenoids, including ß-carotene and lycopene, which have industrial applications. Therefore, classical selective breeding techniques have been applied to generate strains with increased productivity, and microencapsulated ß-carotene preparation has been used in food industry (Li et al., 2019). In B. trispora, lycopene is synthesized via the mevalonate pathway (Venkateshwaran et al., 2015). Lycopene cyclase, which is one of the key enzymes in this pathway, is a bifunctional enzyme that can catalyze the cyclization of lycopene to produce ß-carotene and exhibit phytoene synthase activity (He et al., 2017).

Semi-rationally engineered variants of S-adenosylmethionine synthetase from Escherichia coli with reduced product inhibition and improved catalytic activity.

S-adenosylmethionine synthetase (MAT) catalyzes the synthesis of S-adenosylmethionine (SAM) from ATP and L-methionine. SAM is the major methyl donor for more than 100 transmethylation reactions. It is also a common cosubstrate involved in transsulfuration and aminopropylation. However, product inhibition largely restrains the activity of MAT and limits the enzymatic synthesis of SAM. In this research, the product inhibition of MAT from Escherichia coli was reduced via semi-rational modification. A triple variant (Variant III, I303 V/I65 V/L186 V) showed a 42-fold increase in Ki,ATP and a 2.08-fold increase in specific activity when compared to wild-type MAT. Its Ki,ATP was 0.42 mM and specific acitivity was 3.78 ±0.19 U/mg. Increased Ki,ATP means reduced product inhibition which enhances SAM accumulation. The SAM produced by Variant III could reach to 3.27 mM while SAM produced by wild-type MAT was 1.62 mM in the presence of 10 mM substrates. When the residue in 104th of Variant III was further optimized by site-saturated mutagenesis, the specific activity of Variant IV (I303 V/I65 V/L186 V/N104 K) reached to 6.02 ±0.22 U/mg at 37 °C, though the SAM concentration decreased to 2.68 mM with 10 mM substrates. Analysis of protein 3D structure suggests that changes in hydrogen bonds or other ligand interactions around active site may account for the variety of product inhibition and enzyme activity. The Variant III and Variant IV with reduced inhibition and improved enzyme activity in the study would be more suitable candidates for SAM production in the future.

Advancement of multi-target drug discoveries and promising applications in the field of Alzheimer's disease.

Complex diseases (e.g., Alzheimer's disease) or infectious diseases are usually caused by complicated and varied factors, including environmental and genetic factors. Multi-target (polypharmacology) drugs have been suggested and have emerged as powerful and promising alternative paradigms in modern medicinal chemistry for the development of versatile chemotherapeutic agents to solve these medical challenges. The multifunctional agents capable of modulating multiple biological targets simultaneously display great advantages of higher efficacy, improved safety profile, and simpler administration compared to single-targeted agents. Therefore, multifunctional agents would certainly open novel avenues to rationally design the next generation of more effective but less toxic therapeutic agents. Herein, the authors review the recent progress made in the discovery and design processes of selective multi-targeted agents, especially the successful application of multi-target drugs for the treatment of Alzheimer's disease.

Advances in Computational Structure-Based Drug Design and Application in Drug Discovery.

Compared with the increasing and widespread bacterial resistance to clinical medicines and the urgent need for cures of intractable diseases, there is a dramatic decline in the numbers of drugs reaching the market or clinical trials. Accordingly, it has become imperative to discover more rational and efficient strategies to design and develop novel drugs. Structure-based drug design/discovery (SBDD) is one of the computer-aided methods, by which novel drugs are designed or discovered based on the knowledge of 3D structures of the relevant specific targets. During the past few decades, the great potentials and success of SBDD have been seen in the field of drug discovery. In this review, we present an overview of the key mechanisms of SBDD, the frequently used computer programs in SBDD and the reported successful cases. Finally, several typical design processes of lead components from SBDD are also highlighted in detail, such as the discovery of inhibitors of G protein-coupled receptors (GPCRs), antibacterial drugs, and anti-cancer drugs.

Chemical characterization and in vitro antitumor activity of a single-component polysaccharide from Taxus chinensis var. mairei.

An alkali-soluble single-component polysaccharide, named CPTC-2, was isolated and purified from the leaves of Taxus chinensis var. mairei. by ion-exchange and gel-permeation chromatography in series. The weight-average molecular mass (Mw) of CPTC-2 was about 73.53kDa determined by gel permeation chromatography (GPC). The structural characteristics of CPTC-2 were analyzed by gas chromatography (GC), Infrared (IR) spectrum, nuclear magnetic resonance (NMR) spectroscopy, periodate oxidation and Smith degradation studies, as well as methylation analysis. The results showed that CPTC-2 consisted of glucose, mannose, xylose, arabinose, rhamnose, and galactose with a molar ratio of 1.00:0.32:0.27:3.34:1.22:1.84. CPTC-2 was mainly composed of one type of sugar, α-glycosidic linkage. It had a backbone composed of α-(1→3) Araf, α-(1→5) Araf and α-(1→4) Galp with branches composed of α-(1→3,5) Araf and ß-(1→3,6) Manp. In vitro anti-tumor experiments with SGC-7901 cells were performed and assessed by MTS (MPEG-2 Transport Stream) method and flow cytometry. The results showed CPTC-2 could inhibit the growth of SGC-7901 cells in a concentration-dependent manner via increased apoptosis. The relationship between the structure of CPTC-2 and its anti-tumor activity indicated that the α-configuration glycosidic bond residues may be essential for the anti-tumor activity of this polysaccharide.

[The extraction and separation of an active and available component from Taxus mairei].

To study on the 10-deacetyl baccatin III extract from the leaves and branches of Taxus mairei. A new method was proposed to extract and purify 10-DAB III, which was adopted as the parent nucleus of the high efficacious antitumour drug-taxol and docetaxel in the compositive synthesis. Optimization selection was also made in the extraction and purification conditions of taxol. The purity quotient of 10-DAB III obtained via this technics could reach 91%, with the yield above 70%. It has been testified that the technics was feasible in industrial practice.

[Decolorization of dyestuff and dying waste water by laccase solution with self-flocculent mycelial pellets of Coriolus versicolor].

Both laccase production by the white-rot fungus Coriolus versicolor and decolorization of dyestuff and dying waste water with crude solution of laccase were studied in this work. Laccase production meets the definition of secondary metabolism. For laccase production the optimum initial pH is 4.5. Addition of veratryl alcohol or elevated trace metals could both enhance the laccase activity, while Tween80 showed some inhibition. The immobilized mycelia of C. versicolor in polyurethane foam had less laccase production ability than mycelial pellets. A repeated batch cultivation process was found to be a very economical way for laccase harvest. The same pellets could be used for at least 14 times and average laccase activity of each batch could maintain 6.72 IU/mL. This method reduces the enzyme production course, medium consumption and the possibility of contamination, showing high efficient and great economic benefit. Good results were also obtained in decolorization experiments with the crude solution of laccase. With 3.3 IU/mL initial laccase activity, color removal of Acid Orange reached 98.5% after 24 h reaction. Also with 2.6 IU/mL initial laccase activity, color removal of dying waste water reached 93% after 24 h reaction.