Succinimidyl ester surface chemistry: implications of the competition between aminolysis and hydrolysis on covalent protein immobilization.

N-Hydroxysuccinimide (NHS) ester terminal groups are commonly used to covalently couple amine-containing biomolecules (e.g., proteins and peptides) to surfaces via amide linkages. This one-step aminolysis is often performed in buffered aqueous solutions near physiological pH (pH 6 to pH 9). Under these conditions, the hydrolysis of the ester group competes with the amidization process, potentially degrading the efficiency of the coupling chemistry. The work herein examines the efficiency of covalent protein immobilization in borate buffer (50 mM, pH 8.50) using the thiolate monolayer formed by the chemisorption of dithiobis (succinimidyl propionate) (DSP) on gold films. The structure and reactivity of these adlayers are assessed via infrared spectroscopy (IR), X-ray photoelectron spectroscopy (XPS), electrochemical reductive desorption, and contact angle measurements. The hydrolysis of the DSP-based monolayer is proposed to follow a reaction mechanism with an initial nucleation step, in contrast to a simple pseudo first-order reaction rate law for the entire reaction, indicating a strong dependence of the interfacial reaction on the packing and presence of defects in the adlayer. This interpretation is used in the subsequent analysis of IR-ERS kinetic plots which give a heterogeneous aminolysis rate constant, ka, that is over 3 orders of magnitude lower than that of the heterogeneous hydrolysis rate constant, kh. More importantly, a projection of these heterogeneous kinetic rates to protein immobilization suggests that under coupling conditions in which low protein concentrations and buffers of near physiological pH are used, proteins are more likely physically adsorbed rather than covalently linked. This result is paramount for biosensors that use NHS chemistry for protein immobilization due to effects that may arise from noncovalently linked proteins.

Dynamics of Panax ginseng Rhizospheric Soil Microbial Community and Their Metabolic Function.

The bacterial communities of 1- to 6-year ginseng rhizosphere soils were characterized by culture-independent approaches, random amplified polymorphic DNA (RAPD), and amplified ribosomal DNA restriction analysis (ARDRA). Culture-dependent method (Biolog) was used to investigate the metabolic function variance of microbe living in rhizosphere soil. Results showed that significant genetic and metabolic function variance were detected among soils, and, with the increasing of cultivating years, genetic diversity of bacterial communities in ginseng rhizosphere soil tended to be decreased. Also we found that Verrucomicrobia, Acidobacteria, and Proteobacteria were the dominants in rhizosphere soils, but, with the increasing of cultivating years, plant disease prevention or plant growth promoting bacteria, such as Pseudomonas, Burkholderia, and Bacillus, tended to be rare.

[Isolation of endophytic bacteria in roots of Panax ginseng and screening of antagonistic strains against phytopathogens prevalent in P. ginseng].

In this study, endophytic bacteria were isolated from roots of P. ginseng by plate culture method, and as a result, 40 endophytic bacterial strains were isolated, Bacillus spp. and Pseudomonas spp. were predominant. By confront culture method, two antagonistic endophytic bacterial strain, ge15 (Stenotrophomonas maltophilia) and ge25 (Bacillus sp. ) against Cylindrocarpon destructans, Sclerotinia schinseng and Alternaria pana were identified. The inhibition zone of ge15 to them were 5.5, 22.0, 14.8 mm, respectively; and which were 12.7,16.5,9.0 mm for ge25. The Results indicate that endophytic bacteria have biocontrol potential on ginseng pathogens, and which can be used as a bio-control factor on ginseng soilborne diseases control.

[Establishment of ARDRA system for Panax ginseng cultivated soil microbial community study].

In this study, ARDRA system was established for Panax ginseng cultivated soil microbial community analysis. In the process of soil analysis we found that, ARDRA can not only distinguish soil microbial communities, proportion of each microbial type in total microorganisms can be calculated based on profiles of restricted enzyme digested 16S rDNA, also. Results indicated that, ARDRA system established was able to analyze microbial communities of P. ginseng cultivated soil samples.