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

Hyperthermophilic pretreatment significantly accelerates thermophilic composting humification through improving bacterial communities and promoting microbial cooperation.

Hydrogenation involved in the chemical–biological synthesis of miglitol: effect of biological impurities on catalytic activity and catalyst reuse

R-2-(4-Hydroxyphenoxy)propionic acid (R-HPPA) is a pivotal intermediate for the synthesis of aryloxyphenoxypropionate (APP) herbicide. To rapidly screen microbial isolates with the capacity of hydroxylating R-2-phenoxypropionic acid to R-HPPA from various environmental samples, a convenient and safe 96-well microplate assay method with sodium nitrite (NaNO2) as chromogenic reagent was proposed and optimized. The optimum assay conditions were as follows: the detection wavelength was 420 nm, the concentration of NaNO2 solution was 6.0 g/L, color reaction temperature was 60 °C, the pH of the NaNO2 solution was 2.4, and the reaction time was 40 min. With the aid of this method, screening for microorganisms with C-4-specific hydroxylation activity of R-PPA was conducted. As a result, 23 strains among 3744 single colonies isolated from various samples exhibited the hydroxylation activity. Among these strains, the highest bioconversion rate was achieved by Penicillium oxalicum A5 and Aspergillus versicolor A12, respectively. After 72-h cultivation in shake flask, their conversion rates of R-HPPA from 10 g/L R-PPA reached 21.18% and 40.24%, respectively. The established method was effective in rapid screening of microbes capable of biosynthesizing R-HPPA through hydroxylation of R-PPA, and the obtained two fungi species could be potentially used for R-HPPA production.

Screening of Fungi Isolates for C-4 Hydroxylation of R -2-Phenoxypropionic Acid Based on a Novel 96-Well Microplate Assay Method

BACKGROUND
Biocatalytic production of L-phosphinothricin (L-PPT) is currently the most promising method. In this work, we use an Escherichia coli strain coexpressing of D-amino acid oxidase and catalase (E. coli DAAO-CAT) to oxidation biocatalytic D-PPT to PPO, then use the second Escherichia coli strain coexpressing glutamate dehydrogenase and formate dehydrogenase (E. coli GluDH-FDH) to reduce biocatalytic PPO to L-PPT.


MAIN METHODS AND MAJOR RESULTS
We compared the effects of different concentrations of IPTG or lactose on protein expression and enzyme activity in 5 L fermenter. The best induction conditions for E. coli DAAO-CAT were 0.05 mM IPTG, induction for 18 h at 28°C. The specific enzyme activities of DAAO and CAT were 153.20 U/g and 896.23 U/g, respectively. The optimal induction conditions for E. coli GluDH-FDH were 0.2 mM IPTG, induction for 19 h at 28°C. The specific enzyme activities of GluDH and FDH were 41.72 U/g and 109.70 U/g, respectively. The 200 mM D-PPT was bio catalyzed by E. coli DAAO-CAT for 4 h with space-time yield of 9.0 g·L-1 ·h-1 and conversion rate of over 99.0%. Then 220 mM PPO was converted to L-PPT by E. coli GluDH-FDH for 3 h with space-time yield of 14.5 g·L-1 ·h-1 and conversion rate of over 99.0%. To our knowledge, this is the most efficient biocatalytic reaction for L-PPT production.


CONCLUSIONS AND IMPLICATIONS
We found that IPTG has advantages compared with lactose in the enzyme activity and biomass of E. coli DAAO-CAT and E. coli GluDH-FDH, and IPTG is more environmentally friendly. Our data implicated that IPTG can replace lactose in terms of economic feasibility and effectiveness for scaled-up industrial fermentations. This article is protected by copyright. All rights reserved.

IPTG-induced high protein expression for whole-cell biosynthesis of L-phosphinothricin.

The present invention discloses a nitrilase mutant and application thereof. The mutant is obtained by mutating the amino acid at position 201 or replacing one or more amino acids at region 324-381 of the amino acid sequence shown in SEQ ID No. 2. In the present invention, by the protein molecular modification, thermostability of the purified nitrilase LNIT5 is increased by up to 4.5 folds; and by utilizing recombinant E. coli containing the nitrilase mutant to hydrolyze 1-cyanocyclohexylacetonitrile at a high temperature (45° C.), product tolerance is increased, activity of NIT5-L201F is increased by 20%, and the mutant NITLNIT5-AcN can completely hydrolyze 750 mM 1-cyanocyclohexylacetonitrile within 8 hours and achieve an doubled conversion rate. Therefore, the mutants obtained by the present invention have a good application prospect in efficiently catalyzing 1-cyanocyclohexylacetonitrile to synthesize gabapentin intermediate, 1-cyanocyclohexyl acetic acid. In the present invention, by protein molecular modification, thermal stability of pure nitrilase LNIT5 at 45° C. is increased up to 4.5 times, and while 1-cyanocyclohexylacetonitrile is hydrolyzed using recombinant Escherichia coli containing nitrilase mutant at high temperature (45° C.), the product yield is increased. Therefore, the mutants obtained in the present invention have a good application prospect in highly efficiently catalyzing 1-cyanocyclohexylacetonitrile to 1-cyanocyclohexyl acetic acid, the gabapentin intermediate.

Ya-Ping Xue

Papers

Hyperthermophilic pretreatment significantly accelerates thermophilic composting humification through improving bacterial communities and promoting microbial cooperation.

Hydrogenation involved in the chemical–biological synthesis of miglitol: effect of biological impurities on catalytic activity and catalyst reuse

Screening of Fungi Isolates for C-4 Hydroxylation of R -2-Phenoxypropionic Acid Based on a Novel 96-Well Microplate Assay Method

IPTG-induced high protein expression for whole-cell biosynthesis of L-phosphinothricin.

Nitrilase mutants and application thereof