Synthesis Optimization of L-Aspartic acid β-hydroxamate by a novel Enzyme, β-Aspartyl-γ-glutamyl transferase

L-Aspartic acid β-hydroxamate or L-β-Aspartyl hydroxamate (BAH), water soluble- chemical compound currently obtains popularity due to its role in several important biochemical processes and to its bioactivities. The information regarding synthesis process of BAH is not available yet. Novel enzyme, β-aspartyl-γ-glutamyl transferase from Pseudomonas syringae can catalyze the transfer reaction of β-aspartyl moieties from β-aspartyl compounds to water or to hydroxylamine. In this study we describe the synthesis optimization of BAH using this novel enzyme. We prepared the L-β-aspartyl hydroxamate using L- asparagine as a donor substrate and hydroxylammonium chloride as an acceptor substrate. The effects of temperature, pH, concentrations of substrate donor and acceptor were investigated. Spectrophotometry and HPLC analyses were performed to determine the reaction products. The optimum synthesis reaction was observed in 60˚C. BAH synthesis was optimum at pH 6. The concentrations of donor and acceptor substrates affected the BAH production and the best concentrations of both substrates were 80 mM and 40 mM, respectively. The BAH production of 0.106 mM has been obtained under the optimized condition and it is approximately two-times higher than 0.047 mM produced under in standard reaction. In conclusion, biosynthesis of L-β-aspartyl hydroxamate using a novel enzyme, β- aspartyl-γ-glutamyl transferase from Pseudomonas syringae was successfully performed for the first time. Under the optimized conditions, two times higher L-β-aspartyl hydroxamate production was obtained

Characterization of catalytic α-1,3-glucanase isozymes from Paenibacillus glycanilyticus FH11 by using Brevibacillus system; Essential for suppression of Streptococcus mutans biofilms

S. mutans has been implicated in the etiology of dental caries by facilizing the colonization of tooth surfaces and playing a key role in the development of the virulent dental plaque. α-1,3-Glucan, which is a key structural constituent of the biofilm matrix (dental plaque), synthesized by glucosyltransferase type B (gtfB) in the presence of ingested sucrose. α-1,3-Glucanases also called mutanases, which hydrolyze α-1,3-glucan, are classified into two families of glycoside hydrolases, fungal (type 71) and bacterial (type 87). Because of being considered to degrade α-1,3-glucan, α-1,3-glucanases have been purified and characterized from various microbial sources. However, there are few reports on S. mutans biofilm study. For the host cell expression, Brevibacillus system is an effective bacterial expression system for secretory proteins. B. choshinensis is a gram-positive bacterium and easy to handle non-sporulating bacterium, lacking extracellular protease, that has been already shown to provide a high level of recombinant protein expression. Recently, many proteins are produced from this expression system and use for medical treatment, research study (1). Therefore, in this study we attempted to use Brevibacillus expression system to express, purify, and characterize of α-1,3- glucanase. In addition, we aimed to investigate the effect of recombinant enzyme on α-1,3-glucan biofilm produced by S. mutans from the viewpoints of formation and the effect of toothpaste agent on enzyme activity. Two novel catalytic domains of α-1,3-glucanase isozyme genes were cloned from P. glycanilyticus strain FH11 and heterologously expressed in Brevibacillus system. The recombinant isozymes, in termed CatAgl-FH1 and CatAgl-FH2, were purified to homogeneity with specific activity 0.70 U/mg and 0.77 U/mg respectively. The molecular mass of catalytic domain was estimated 62 kDa by SDS-PAGE. Both recombinant enzymes exhibited the different properties. The optimal pH of CatAgl-FH1 and CatAgl-FH2 were 5.5 and 6.0, respectively. The pH stability of CatAgl-FH1 and CatAgl-FH2 were in a range of pH 4.0-11.0 and 4.5-9.0, respectively. The optimal temperature of CatAgl-FH1 and CatAgl-FH2 were 60°C and 55°C, respectively and they were stable until 60°C. Thin Layer chromatography revealed their mode of hydrolysis towards α-1,3-glucan was endo-cleavage pattern. The major products of CatAgl-FH1 were di- and trisaccharide but mainly trisaccharide was for CatAgl-FH2. Both enzymes showed high tolerance against high concentration of sodium fluoride. However, each enzyme activity on surfactants were stepped down when sodium dodecyl sulfate and benzethonium concentration were increased. Please click Additional Files below to see the full abstract

Enhanced propagation of fish nodaviruses in BF-2 cells persitently infected with snakehead retrovirus (SnRV)

Fish nodaviruses are causative agents of viral nervous necrosis causing high mortality in cultured marine fishes around the world. The first successful isolation of fish nodavirus was made with SSN-1 cells, which are persistently infected with snakehead retrovirus (SnRV). In the present study, a BF-2 cell line persistently infected with SnRV (PI-BF-2) was established to evaluate the influence of SnRV on the production of fish nodavirus. The PI-BF-2 cells were slightly more slender than BF-2 cells, but no difference was observed in propagation rate between both cell lines. No difference was observed in production of SnRV between PI-BF-2 and SSN-1 cell lines. Although both PI-BF-2 and BF-2 cell lines showed no cytopathic effect (CPE) after inoculation of striped jack nervous necrosis virus (SJNNV) and redspotted grouper nervous necrosis virus (RGNNV), these fish nodaviruses could be amplified in BF-2 cells, and moreover, production of fish nodaviruses in the PI-BF-2 cell line was more than 40 times higher than in BF-2 cells. Thus, it was concluded that BF-2 cell permissiveness to fish nodaviruses was enhanced by persistent infection with SnRV. Furthermore, homologous cDNA to genomic RNA of SJNNV was detected from both PI-BF-2 and SSN-1 cell lines persistently infected with SnRV. The amount of nodavirus cDNA in SJNNV-inoculated PI-BF-2 cells was clearly lower than that in SJNNV-inoculated SSN-1 cells

Computational Identification and Characterization of a Promiscuous T-Cell Epitope on the Extracellular Protein 85B of Mycobacterium spp. for Peptide-Based Subunit Vaccine Design

Tuberculosis (TB) is a reemerging disease that remains as a leading cause of morbidity and mortality in humans. To identify and characterize a T-cell epitope suitable for vaccine design, we have utilized the Vaxign server to assess all antigenic proteins of Mycobacterium spp. recorded to date in the Protegen database. We found that the extracellular protein 85B displayed the most robust antigenicity among the proteins identified. Computational tools for identifying T-cell epitopes predicted an epitope, 181-QQFIYAGSLSALLDP-195, that could bind to at least 13 major histocompatibility complexes, revealing the promiscuous nature of the epitope. Molecular docking simulation demonstrated that the epitope could bind to the binding groove of MHC II and MHC I molecules by several hydrogen bonds. Molecular docking analysis further revealed that the epitope had a distinctive binding pattern to all DRB1 and A and B series of MHC molecules and presented almost no polymorphism in its binding site. Moreover, using “Allele Frequency Database,” we checked the frequency of HLA alleles in the worldwide population and found a higher frequency of both class I and II HLA alleles in individuals living in TB-endemic regions. Our results indicate that the identified peptide might be a universal candidate to produce an efficient epitope-based vaccine for TB