Development of surface plasmon resonance imaging for detection of Acidovorax avenae subsp. citrulli (Aac) using specific monoclonal antibody

An immunosensor based on surface plasmon resonance imaging (SPR imaging) using a specific monoclonal antibody 11E5 (MAb 11E5) was developed for the detection of the seed-borne bacterium Acidovorax avenae subsp. citrulli (Aac), which causes fruit blotch in watermelons and cantaloupes, and compared to the conventional ELISA technique. The 1:40 mixed self-assembled monolayer (mixed SAM) surface was used for the immobilized MAb 11E5 on sensor surface for the detection of Aac. Both whole cells and broken cells of Aac were tested by using direct and sandwich detection assay. The limit of detection (LOD) of Aac using the SPR imaging technique and a direct detection assay was 10(6)cfu/ml and a subsequent amplification of the SPR signal using a polyclonal antibody (PAb) lowered the LOD to 5×10(5) cfu/ml. The LOD for the ELISA technique was 5×10(4) cfu/ml for the detection of Aac, which was slightly better than that for the SPR technique. However, the sensor surface based on SPR imaging offered a major advantage in terms of surface regeneration, allowing at least five cycles with a shorter time assay, multi-channel analysis with an application on multiplex detection, and an ease of the surface usage for the detection of Aac in the naturally infected plant. The surface was tested against the naturally infected sample and showed good selectivity toward the Aac bacteria.     

Essential role of tryptophan residues in toxicity of binary toxin from Bacillus sphaericus

Bacillus sphaericus produces mosquito-larvicidal binary toxin composed of BinA and BinB. While BinB is expected to bind to a specific receptor on the cell membrane, BinA interacts to BinB or BinB receptor complex and translocates into the cytosol to exert its activity via unknown mechanism. To investigate functional roles of aromatic cluster in BinA, amino acids at positions Y213, Y214, Y215, W222 and W226 were substituted by leucine. All mutant proteins were highly produced and their secondary structures were not affected by these substitutions. All mutants are able to insert into lipid monolayers as observed by Langmuir-Blodgett trough and could permeabilize the liposomes in a similar manner as the wild type. However, mosquito-larvicidal activity was abolished for W222L and W226L mutants suggesting that tryptophan residues at both positions play an important role in the toxicity of BinA, possibly involved in the cytopathological process after toxin entry into the cells.     

Heterologous expression of polyhydroxyalkanoate depolymerase from <Emphasis Type="Italic">Thermobifida</Emphasis> sp. in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and catalytic analysis by surface plasmon resonance

A polyhydroxyalkanote depolymerase gene from Thermobifida sp. isolate BCC23166 was cloned and expressed as a C-terminal His₆-tagged fusion in Pichia pastoris. Primary structure analysis revealed that the enzyme PhaZ-Th is a member of a proposed new subgroup of SCL-PHA depolymerase containing a proline–serine repeat linker. PhaZ-Th was expressed as two glycosylated forms with apparent molecular weights of 61 and 70 kDa, respectively. The enzyme showed esterase activity toward p-nitrophenyl alkanotes with V _max and K _m of 3.63 ± 0.16 μmol min⁻¹ mg⁻¹ and 0.79 ± 0.12 mM, respectively, on p-nitrophenyl butyrate with optimal activity at 50–55°C and pH 7–8. Surface plasmon resonance (SPR) analysis demonstrated that PhaZ-Th catalyzed the degradation of poly-[(R)-3-hydroxybutyrate] (PHB) films, which was accelerated in (R)-3-hydroxyvalerate copolymers with a maximum degradation rate of 882 ng cm⁻² h⁻¹ for poly[(R)-3-hydroxybutyrate-co-3-hydroxyvalerate] (12 mol% V). Surface deterioration, especially on the amorphous regions of PHB films was observed after exposure to PhaZ-Th by atomic force microscopy. The use of P. pastoris as an alternative recombinant system for bioplastic degrading enzymes in secreted form and a sensitive SPR analytical technique will be of utility for further study of bioplastic degradation.     

Recombinant expression of BTA hydrolase in Streptomyces rimosus and catalytic analysis on polyesters by surface plasmon resonance

A recombinant polyester-degrading hydrolase from Thermobifida sp. BCC23166 targeting on aliphatic-aromatic copolyester (rTfH) was produced in Streptomyces rimosus R7. rTfH was expressed by induction with thiostrepton as a C-terminal His₆ fusion from the native gene sequence under the control of tipA promoter and purified from the culture supernatant to high homogeneity by a single step affinity purification on Ni-Sepharose matrix. The enzyme worked optimally at 50–55°C and showed esterase activity on C3-C16 p-nitrophenyl alkanoates with a specific activity of 76.5 U/mg on p-nitrophenyl palmitate. Study of rTfH catalysis on surface degradation of polyester films using surface plasmon resonance analysis revealed that the degradation rates were in the order of poly-ε-caprolactone >?Ecoflex^®?>?polyhydroxybutyrate. Efficient hydrolysis of Ecoflex^® by rTfH was observed in mild alkaline conditions, with the highest activity at pH 8.0 and ionic strength at 250 mM sodium chloride, with the maximal specific activity of 0.79 mg^?1min^?1mg^?1 protein. Under the optimal conditions, rTfH showed a remarkable 110-time higher specific activity on Ecoflex^® in comparison to a lipase from Thermomyces lanuginosus, while less difference in degradation efficiency of the two enzymes was observed on the aliphatic polyesters, suggesting greater specificities of rTfH to the aliphatic-aromatic copolyester. This study demonstrated the use of streptomycetes as an alternative expression system for production of the multi-polyester-degrading enzyme of actinomycete origin and provided insights on its catalytic properties on surface degradation contributing to further biotechnological application of this enzyme.     

Insertion behavior of the Bacillus thuringiensis Cry4Ba insecticidal protein into lipid monolayers

Toxicity mechanisms of Bacillus thuringiensis Cry insecticidal proteins involve membrane insertion and lytic pore formation in lipid bilayers of the target larval midgut cell membranes. The B. thuringiensis Cry4Ba mosquito-larvicidal protein has been shown to be capable of permeabilizing liposome vesicles and of forming ion channels in planar lipid bilayers. Here, the membrane interaction of the 65-kDa activated Cry4Ba protein with the lipid monolayers, comprising dipalmitoyl phosphatidylcholine, dioleoyl phosphatidylethanolamine, and cholesterol (Chol), was studied using Langmuir-Blodgett technique. The interactions of the Cry4Ba protein with the lipid monolayers were measured from the surface pressure versus area isotherms of the protein-lipid monolayers. The increase in the mean molecular area was demonstrated as an incorporation of the protein into lipid monolayers. The insertion of the Cry4Ba protein was monitored by measuring as an increase of the surface pressure at constant molecular area. For a given monolayer, the membrane insertion of the Cry4Ba reduced as the initial surface pressure increased. The Cry4Ba protein showed a strong preference of an insertion towards a Chol monolayer. In addition, the mixed monolayers of Chol showed an enhanced effect on the insertion kinetics of Cry4Ba into lipid films, suggesting its involvement in the modulation of the protein insertion. These findings provide the first evidence that the Cry4Ba protein is capable of inserting itself into lipid monolayers, depending on the packing density of the monolayers. Our results also indicate that only a limited part of the protein is likely to be involved in the insertion.