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

The redox biosynthesis system has important applications in green biomanufacturing of chiral compounds. Formate dehydrogenase (FDH) catalyzes the oxidation of formate into carbon dioxide, which is associated with the reduction of NAD(P)+ into NAD(P)H. Due to this property, FDH is used as a crucial enzyme in the redox biosynthesis system for cofactor regeneration. Nevertheless, the application of natural FDH in industrial production is hampered by low catalytic efficiency, poor stability, and inefficient coenzyme utilization. This review summarized the structural characteristics and catalytic mechanism of FDH, as well as the advances in protein engineering of FDHs toward improved enzyme activity, catalytic efficiency, stability and coenzyme preference. The applications of using FDH as a coenzyme regeneration system for green biomanufacturing of chiral compounds were summarized.

[Formate dehydrogenase and its application in biomanufacturing of chiral chemicals].

A Light-controlled Biocatalytic System for Precise Regulation of Enzymatic Decarboxylation

R-2-(4-hydroxyphenoxy)propionicacid (HPOPA) is a valuable intermediate for the synthesis of enantiomerically pure aryloxyphenoxypropionic acid herbicides. In this work, to improve the HPOPA biosynthesis by Beauveria bassiana ZJB16002 from the substrate R-2-phenoxypropionic acid (POPA), the original HPOPA producer B. bassiana ZJB16002 was subjected to physical mutagenesis with 137 Cs-γ irradiation and chemical mutagen N-methyl-N'-nitro-N-nitrasoguanidine (NTG) induced mutagenesis. The effects of different treatment doses of the mutagens on the lethal rate and positive mutation rate were investigated, and the results showed that the optimal 137 Cs-γirradiation dose and NTG concentration was 850 Gy and 500 µg/mL, respectively. Under these conditions, a mutant strain CCN-7 with the highest HPOPA production capacity was obtained through two rounds of 137 Cs-γ irradiation treatment followed by one round of NTG mutagenesis. At the substrate (POPA) concentration of 50 g/L, HPOPA titer of CCN-7 reached 36.88 g/L, which was 9.73-fold higher than the parental strain. The morphology of the wild-type and mutant strain was compared and the results might provide helpful information in exploration of the correlation of morphology and biochemical features of B. bassiana.

Improvement of R-2-(4-hydroxyphenoxy) propionic acid biosynthesis of Beauveria bassiana by combined mutagenesis.

Efficient biosynthesis of 1-cyanocyclohexaneacetic acid using a highly soluble nitrilase by N-terminus modification of novel peptide tags

In the synthesis of hypoglycemic agent miglitol, the quantitative analysis of two key intermediates, N-2-hydroxyethyl glucamine and 6-(N-hydroxyethyl)-amino-6-deoxy-alpha-L-sorbofuranose, has long been a challenge In this study, derivatization reagents were screened, and then a novel HPLC method coupled with 9-fluorenylmethyl chloroformate derivatization was developed for the quantitative analysis of the two miglitol intermediates The derivatization was performed by reacting Fmoc-Cl and the intermediates at a molar ratio of 16:1 and sodium borate at a final concentration of 02 mol/L at pH 80, 35 °C for 40 min, followed by the addition of 15 times the volume of acetic acid (01%, w/v) The analysis was achieved on Hypersil ODS2 (46 mm × 250 mm) column applying an isocratic mobile phase containing acetonitrile and 01% acetic acid aqueous solution (45:55, v/v) at 08 mL/min flow rate and an injection volume of 10 μL The analyte was detected at 254 nm The method showed good linearity, precision, and accuracy It was capable of detecting both intermediates at low concentrations Interestingly, we found that a small amount (> 5%) of N-2-hydroxyethyl glucamine was not converted to 6-(N-hydroxyethyl)-amino-6-deoxy-alpha-L-sorbofuranose in the biotransformation This method was reliable and efficient for the analysis of miglitol intermediates

Ya-Ping Xue

Papers

[Formate dehydrogenase and its application in biomanufacturing of chiral chemicals].

A Light-controlled Biocatalytic System for Precise Regulation of Enzymatic Decarboxylation

Improvement of R-2-(4-hydroxyphenoxy) propionic acid biosynthesis of Beauveria bassiana by combined mutagenesis.

Efficient biosynthesis of 1-cyanocyclohexaneacetic acid using a highly soluble nitrilase by N-terminus modification of novel peptide tags

Development of a Simple and Sensitive Pre-column Derivatization HPLC Method for the Quantitative Analysis of Miglitol Intermediates