Short protocols in molecular biology

Über die Adsorption in Lösungen

Signal transduction by the Toll-like receptors (TLRs) is central to host defence against many pathogenic microorganisms and also underlies a large burden of human disease. Thus, the mechanisms and regulation of signalling by TLRs are of considerable interest. In this Review, we discuss the molecular basis for the recognition of pathogen-associated molecular patterns, the nature of the protein complexes that mediate signalling, and the way in which signals are regulated and integrated at the level of allosteric assembly, post-translational modification and subcellular trafficking of the components of the signalling complexes. These fundamental molecular mechanisms determine whether the signalling output leads to a protective immune response or to serious pathologies such as sepsis. A detailed understanding of these processes at the molecular level provides a rational framework for the development of new drugs that can specifically target pathological rather than protective signalling in inflammatory and autoimmune disease.

Assembly and localization of Toll-like receptor signalling complexes

The genus Stenotrophomonas comprises at least eight species. These bacteria are found throughout the environment, particularly in close association with plants. Strains of the most predominant species, Stenotrophomonas maltophilia, have an extraordinary range of activities that include beneficial effects for plant growth and health, the breakdown of natural and man-made pollutants that are central to bioremediation and phytoremediation strategies and the production of biomolecules of economic value, as well as detrimental effects, such as multidrug resistance, in human pathogenic strains. Here, we discuss the versatility of the bacteria in the genus Stenotrophomonas and the insight that comparative genomic analysis of clinical and endophytic isolates of S. maltophilia has brought to our understanding of the adaptation of this genus to various niches.

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The versatility and adaptation of bacteria from the genus Stenotrophomonas

Biocatalysis has found numerous applications in various fields as an alternative to chemical catalysis. The use of enzymes in organic synthesis, especially to make chiral compounds for pharmaceuticals as well for the flavors and fragrance industry, are the most prominent examples. In addition, biocatalysts are used on a large scale to make specialty and even bulk chemicals. This review intends to give illustrative examples in this field with a special focus on scalable chemical production using enzymes. It also discusses the opportunities and limitations of enzymatic syntheses using distinct examples and provides an outlook on emerging enzyme classes.

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Biocatalysis: Enzymatic Synthesis for Industrial Applications

Commercial production of acarbose is exclusively via done microbial fermentation with strains from the genera of Actinoplanes. The addition of C7N-aminocyclitols for enhanced production of acarbose and concurrently reduced formation of impurity C by cultivation of A. utahensis ZJB-08196 in 500-mL shake flasks was investigated, and validamine was found to be the most effective strategy. Under the optimal conditions of validamine addition, acarbose titer was increased from 3560 ± 128 mg/L to 4950 ± 156 mg/L, and impurity C concentration was concurrently decreased from 289 ± 24 mg/L to 107 ± 29 mg/L in batch fermentation after 168 h of cultivation. A further fed-batch experiment coupled with the addition of validamine (20 mg/L) in the fermentation medium prior to inoculation was designed to enhance the production of acarbose. When twice feedings of a mixture of 6 g/L glucose, 14 g/L maltose, and 9 g/L soybean flour were performed at 72 h and 96 h, acarbose titer reached 6606 ± 103 mg/L and impurity C concentration was only 212 ± 12 mg/L at 168 h of cultivation. Acarbose titer and proportion of acarbose/impurity C increased by 85.6% and 152.9% when compared with control experiments. This work demonstrates for the first time that validamine addition is a simple and effective strategy for increasing acarbose production and reducing impurity C formation.

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Enhanced Production of Acarbose and Concurrently Reduced Formation of Impurity C by Addition of Validamine in Fermentation of Actinoplanes utahensis ZJB-08196

Validoxylamine A is structurally similar to trehalose and acts a potent competitive inhibitor of trehalase. It has recently been receiving increased attention as a potential material for the development of new insecticides or drugs. In this study, beta-glucosidase extracted from honeybees (Apis cerana Fabr.) was used as a catalyst to produce validoxylamine A through enzymatic hydrolysis of validamycin A. Beta-glucosidase was separated and purified from honeybees, and its characteristics were examined. The results showed that beta-glucosidase was stable across a range of temperatures from 30 to 40 degrees C and across a relatively wide range of pH values from 5.0 to 7.5. Investigation of the biocatalyzed hydrolysis process from validamycin A to validoxylamine A with beta-glucosidase revealed that both the substrate (validamycin A) and the product (validoxylamine A) inhibited beta-glucosidase activity. The inhibition constant of the substrate Kis value was 5.01 mM, and that of the product Kip value was 1.32 mM. This product inhibition was competitive.

Preparation of trehalase inhibitor validoxylamine A by biocatalyzed hydrolysis of validamycin A with honeybee (Apis cerana Fabr.) beta-glucosidase.

Efficient chemoenzymatic synthesis of gabapentin by control of immobilized biocatalyst activity in a stirred bioreactor

A single-transaminase-catalyzed biocatalytic cascade was developed by employing the desired biocatalyst, ATA-117-Rd11, that showed high activity toward 2-oxo-4-[(hydroxy)(methyl)phosphinoyl] butyric acid (PPO) and α-ketoglutarate, and low activity against pyruvate The cascade successfully promotes a highly asymmetric amination reaction for the synthesis of l-phosphinothricin (l-PPT) with high conversion (>95 %) and>99 % ee In a scale-up experiment, using 10 kg pre-frozen E coli cells harboring ATA-117-Rd11 as catalyst, 80 kg PPO was converted to ≈70 kg l-PPT after 24 hours with a high ee value (>99 %)

A Single-Transaminase-Catalyzed Biocatalytic Cascade for Efficient Asymmetric Synthesis of l-Phosphinothricin.

The traditional strategy to improve the efficiency of an entire coupled enzyme system relies on separate direction of the evolution of enzymes involved in their respective enzymatic reactions. This strategy can lead to enhanced single-enzyme catalytic efficiency but may also lead to loss of coordination among enzymes. This study aimed to overcome such shortcomings by executing a directed evolution strategy on multiple enzymes in one combined group that catalyzes the asymmetric biosynthesis of l-phosphinothricin. The genes of a glutamate dehydrogenase from Pseudomonas moorei (PmGluDH) and a glucose dehydrogenase from Exiguobacterium sibiricum (EsGDH), along with other gene parts (promoters, ribosomal binding sites (RBSs), and terminators) were simultaneously evolved. The catalytic efficiency of PmGluDH was boosted by introducing the beneficial mutation A164G (from 1.29 s-1mM-1 to 183.52 s-1mM-1), and the EsGDH expression level was improved by optimizing the linker length between the RBS and the start codon of gdh. The total turnover numbers of the bioreaction increased from 115 (GluDH WTNADPH) to 5846 (A164GNADPH coupled with low expression of EsGDH), and to 33950 (A164GNADPH coupled with high expression of EsGDH). The coupling efficiency was increased from ∼30% (GluDH_WT with low expression of GDH) to 83.3% (GluDH_A164G with high expression of GDH). In the batch production of l-phosphinothricin utilizing whole-cell catalysis, the strongest biocatalytic reaction exhibited a high space-time yield (6410 g·L-1·d-1) with strict stereoselectivity (>99% enantiomeric excess).Importance: The traditional strategy to improve multienzyme-catalyzed reaction efficiency may lead to enhanced single-enzyme catalytic efficiency but may also result in loss of coordination among enzymes. We describe a directed evolution strategy of an entire coupled enzyme system to simultaneously enhance enzyme coordination and catalytic efficiency. The simultaneous evolution strategy was applied to a multienzyme-catalyzed reaction for the asymmetric synthesis of l-phosphinothricin, which not only enhanced the catalytic efficiency of GluDH but also improved the coordination between GluDH and GDH. Since this strategy is enzyme-independent, it may be applicable to other coupled enzyme systems for chiral chemical synthesis.

Ya-Ping Xue

Papers

Enhanced Production of Acarbose and Concurrently Reduced Formation of Impurity C by Addition of Validamine in Fermentation of Actinoplanes utahensis ZJB-08196

Preparation of trehalase inhibitor validoxylamine A by biocatalyzed hydrolysis of validamycin A with honeybee (Apis cerana Fabr.) beta-glucosidase.

Efficient chemoenzymatic synthesis of gabapentin by control of immobilized biocatalyst activity in a stirred bioreactor

A Single-Transaminase-Catalyzed Biocatalytic Cascade for Efficient Asymmetric Synthesis of l-Phosphinothricin.

Simultaneous directed evolution of coupled enzymes for efficient asymmetric synthesis of l-phosphinothricin.