Presence Of Laminarin Research Articles

Plasma β-1,3 Glucan Binding Protein Mediated Opsono-Phagocytosis by Hemocytes In Vitro of Marine Mussel Perna viridis.

We have shown in the past decade, for the first time in a bivalve mollusc, detection, isolation, and purification of β-1,3 glucan binding protein (β-GBP) in the plasma of the marine mussel Perna viridis and demonstrated its role in a nonself-induced activation of plasma prophenoloxidase system. In this study, we present evidence for its ability to function as an opsonin during phagocytosis of trypsinized yeast cells by the hemocytes of P. viridis. The in vitro pretreatment of target cells (trypsinized yeast cells) with β-GBP enhanced the phagocytic response of hemocytes. Such β-GBP-mediated enhanced phagocytic response appeared to be dose dependent. This opsono-phagocytic response could be inhibited by the presence of laminarin (a polymer of β-1,3 glucans), glucose, as well as polyclonal antibodies raised against β-GBP. These observations clearly indicate that the plasma β-GBP can possibly recognize and bind to β-1,3 glucans on the surface of targets and facilitate hemocyte recognition processes possibly by forming a bridge between the hemocytes and the target, consequently leading to opsono-phagocytosis. These observations together with our earlier annotations indicate the multifunctional potential of plasma β-GBP in the marine mussel P. viridis.

Effects of laminarin on the structural properties and in vitro digestion of wheat starch and its application in noodles

In this study, wheat starch (WS) was combined with different concentrations of laminarin (LM) to investigate changes in its microstructure, structure and digestibility. The application of LM to improve the cooking and textural properties of wheat noodles was also investigated. A low concentration of LM (2%, g/100g) reduced the crystallinity and order degree of WS, but a high concentration had the opposite effect. After LM addition, the leached amylose content and swelling power of WS were significantly reduced, indicating that LM coats the starch granule surface, inhibiting swelling and reducing amylose leaching. The addition of LM also significantly changed the microstructure of WS; WS granules aggregated in the presence of LM, forming a denser structure. In vitro digestion indicated that LM strongly inhibits the digestibility of WS, with 6% LM (g/100g) having the strongest inhibitory effect. Cooking and textural analysis of noodles showed that the addition of LM could improve the textural quality of noodles, by reducing cooking loss, increasing water absorption and hardness, and reducing adhesiveness.

Characterization of Aspergillus terreus Accessory Conidia and Their Interactions With Murine Macrophages.

All Aspergillus species form phialidic conidia (PC) when the mycelium is in contact with the air. These small, asexual spores are ideally suited for an airborne dissemination in the environment. Aspergillus terreus and a few closely related species from section Terrei can additionally generate accessory conidia (AC) that directly emerge from the hyphal surface. In this study, we have identified galactomannan as a major surface antigen on AC that is largely absent from the surface of PC. Galactomannan is homogeneously distributed over the entire surface of AC and even detectable on nascent AC present on the hyphal surface. In contrast, β-glucans are only accessible in distinct structures that occur after separation of the conidia from the hyphal surface. During germination, AC show a very limited isotropic growth that has no detectable impact on the distribution of galactomannan. The AC of the strain used in this study germinate much faster than the corresponding PC, and they are more sensitive to desiccation than PC. During infection of murine J774 macrophages, AC are readily engulfed and trigger a strong tumor necrosis factor-alpha (TNFα) response. Both processes are not hampered by the presence of laminarin, which indicates that β-glucans only play a minor role in these interactions. In the phagosome, we observed that galactomannan, but not β-glucan, is released from the conidial surface and translocates to the host cell cytoplasm. AC persist in phagolysosomes, and many of them initiate germination within 24 h. In conclusion, we have identified galactomannan as a novel and major antigen on AC that clearly distinguishes them from PC. The role of this fungal-specific carbohydrate in the interactions with the immune system remains an open issue that needs to be addressed in future research.

A study on β-glucan binding protein (β-GBP) and its involvement in phenoloxidase cascade in Indian white shrimp Fenneropenaeus indicus

The present study reports the purification of novel immune molecule β-1, 3 glucan binding protein from the heamolymph of the Indian white shrimp, Fenneropenaeus indicus (Fiβ-GBP). The purified Fiβ-GBP had 95kDa molecular weight in SDS-PAGE analysis. MALDI-TOF/TOF analysis revealed that the purified Fiβ-GBP showed similarity to various crustacean proteins; 48 and 46% similarity was observed for β-1, 3 glucan binding protein of Chinese white shrimp Fenneropenaeus chinensis and banana shrimp Fenneropenaeus merguiensis, with MOWSE score of 3.11e+12 and 2.05e+8, respectively. The phenoloxidase activity (PO) of Fiβ-GBP was evaluated and, in the presence of laminarin, PO activity increased significantly. Substrate specificity assay demonstrated that Fiβ-GBP had the specific binding site for soluble or insoluble β-glucan (laminarin), since the PO activity increased in the presence of laminarin when compared to other sugars. Enzymatic activities revealed that the optimum temperature and pH for Fiβ-GBP activating PO were 40°C and pH 7–8. Moreover, even at 100°C Fiβ-GBP enhanced PO activity highlighting that Fiβ-GBP was thermostable and thermophilic in nature. Among various divalent metallic ions, Fiβ-GBP significantly promoted the PO activity in presence of Mg2+ and Ca2+. The breakdown of para nitroanilide from Nα−Benzoyl-l-Arginine 4-Nitroanilide hydrochloride showed that serine protease activity was induced by Fiβ-GBP and also increased concentration of Fiβ-GBP evoked the activity. Furthermore, hemolytic activity tests revealed that PO reaction product induced RBC membrane damage and cell shrinkage. Lastly, Baclight bacterial viability assays showed maximum killing effect of PO reaction product on both Gram positive and Gram negative bacteria.

Relative Contributions of Dectin-1 and Complement to Immune Responses to Particulate β-Glucans

Glucan particles (GPs) are Saccharomyces cerevisiae cell walls chemically extracted so they are composed primarily of particulate β-1,3-D-glucans. GPs are recognized by Dectin-1 and are potent complement activators. Mice immunized with Ag-loaded GPs develop robust Ab and CD4(+) T cell responses. In this study, we examined the relative contributions of Dectin-1 and complement to GP phagocytosis and Ag-specific responses to immunization with OVA encapsulated in GPs. The in vitro phagocytosis of GPs by bone marrow-derived dendritic cells was facilitated by heat-labile serum component(s) independently of Dectin-1. This enhanced uptake was not seen with serum from complement component 3 knockout (C3(-/-)) mice and was also inhibited by blocking Abs directed against complement receptor 3. After i.p. injection, percent phagocytosis of GPs by peritoneal macrophages was comparable in wild-type and Dectin-1(-/-) mice and was not inhibited by the soluble β-glucan antagonist laminarin. In contrast, a much lower percentage of peritoneal macrophages from C3(-/-) mice phagocytosed GPs, and this percentage was further reduced in the presence of laminarin. Subcutaneous immunization of wild-type, Dectin-1(-/-), and C3(-/-) mice with GP-OVA resulted in similar Ag-specific IgG(1) and IgG(2c) type Ab and CD4(+) T cell lymphoproliferative responses. Moreover, while CD4(+) Th1 and Th2 responses measured by ELISPOT assay were similar in the three mouse strains, Th17 responses were reduced in C3(-/-) mice. Thus, although Dectin-1 is necessary for optimal phagocytosis of GPs in the absence of complement, complement dominates when both an intact complement system and Dectin-1 are present. In addition, Th-skewing after GP-based immunization was altered in C3(-/-) mice.

Identification and characterization of multiple β-glucan binding proteins in the Pacific oyster, Crassostrea gigas

The present study reports on the characterization of two cDNAs coding β-glucan binding proteins (βGBPs), designated as Cg-βGBP-1 and Cg-βGBP-2, from the Pacific oyster, Crassostrea gigas. Cg-βGBP-1 consists of 555 amino acid residues and possesses two possible integrin recognition sites. The other protein, Cg-βGBP-2, is composed of 447 amino acid residues without integrin recognition sites. Domain structures of both Cg-βGBPs are similar to other invertebrate βGBPs, but phylogenetic positions and major expression tissues for these proteins are different. Cg-βGBP-1 is expressed in circulatory hemocytes and Cg-βGBP-2 in digestive glands. Functional assays using recombinant proteins revealed that Cg-βGBP-2 enhanced the phenoloxidase (PO) activity of hemocyte suspensions under the presence of laminarin, but Cg-βGBP-1 did not show this enhancement. It is suggested that Cg-βGBPs in the Pacific oyster have evolved to obtain different immunological functions. Cg-βGBP-1 possibly evolved for hemocyte-related functions through integrin, and Cg-βGBP-2 for the PO activation system.

Candida albicansinduces cyclo-oxygenase 2 expression and prostaglandin E2 production in synovial fibroblasts through an extracellular-regulated kinase 1/2 dependent pathway

IntroductionSynovial cells are potential sources of inflammatory mediators in bacterial-induced arthritis but their involvement in the inflammatory response to Candida albicans-induced septic arthritis is largely unknown.MethodsPrimary cultures of rat synovial fibroblasts were infected with C. albicans (ATCC90028). Immunocytochemistry, western blotting, and RT-PCR were performed to assess cyclo-oxygenase 2 induction. Phosphorylation of extracellular-regulated kinase (ERK1/2) following infection in the absence or presence of U0126 was assessed by western blotting whilst prostaglandin E2 production was measured by ELISA. Nuclear factor κB (NFκB) translocation was evaluated by an electrophoretic mobility shift assay.ResultsInfection of synovial fibroblasts with C. albicans resulted in cyclo-oxygenase 2 expression and prostaglandin E2 production. Cyclo-oxygenase 2 expression and prostaglandin E2 production was dependent upon extracellular-regulated kinase 1/2 phosphorylation, associated with activation of NFκB and significantly elevated in the presence of laminarin, an inhibitor of dectin-1 activity. Synovial fibroblasts adjacent to C. albicans hyphae aggregates appeared to be the major contributors to the increased levels of cyclo-oxygenase 2 and phosphorylated extracellular-regulated kinase 1/2.ConclusionsC. albicans infection of synovial fibroblasts in vitro results in upregulation of cyclo-oxygenase 2 and prostaglandin E2 by mechanisms that may involve activation of extracellular-regulated kinase 1/2 and are associated with NFκB activation.

Innate immunity in the deep sea hydrothermal vent mussel Bathymodiolus azoricus

The interaction between microorganisms and host defense mechanisms is a decisive factor for the survival of marine bivalves. They rely on cell-mediated and humoral reactions to overcome the pathogens that naturally occur in the marine environment. In order to understand host defense reactions in animals inhabiting extreme environments we investigated some of the components from the immune system of the deep sea hydrothermal vent mussel Bathymodiolus azoricus. Cellular constituents in the hemolymph and extrapallial fluid were examined and led to the identification of three types of hemocytes revealing the granulocytes as the most abundant type of cell. To further characterize hemocyte types, the presence of cell surface carbohydrate epitopes was demonstrated with fluorescent WGA lectin, which was mostly ascribed to the granulocytes. Cellular reactions were then investigated by means of phagocytosis and by the activation of putative MAPKs using the microbial compounds zymosan, glucan, peptidoglycan and lipopolysaccharide. Two bacterial agents, Bacillus subtilis and Vibrio parahaemolyticus, were also used to stimulate hemocytes. The results showed that granulocytes were the main phagocytic cells in both hemolymph and extrapallial fluid of B. azoricus. Western blotting analyses using commercially available antibodies against ERK, p38 and JNK, suggested that these putative kinases are involved in signal transduction pathways during experimental stimulation of B. azoricus hemocytes. The fluorescent Ca 2+ indicator Fura-2 AM was also insightful in demonstrating hemocyte stimulation in the presence of laminarin or live V. parahaemolyticus. Finally, the expression of the antibacterial gene mytilin was analyzed in gill tissues by means of RT-PCR and whole-mount in situ hybridization. Mytilin transcripts were localized in hemocytes underlying gill epithelium. Moreover, mytilin was induced by exposure of live animals to V. parahaemolyticus. These findings support the premise of a conserved innate immune system in B. azoricus. Such system is comparable to other Bivalves and involves the participation of cellular and humoral components.

The role of bacterial symbionts in suppressing the defence reaction in larval hemolymph of the cotton leafworm Spodoptera littoralis (Biosd.)

Spodoptera littoralis hemolymph exhibited a decrease in phenoloxidase activity during the first hour of exposure to alive or dead Xenorhabdus nematophilus and Photorhabdus luminescens even in the presence or absence of laminarin and α-chymotrypsin. Also, as the bacterial numbers in the hemolymph of the larvae of S. littoralis increased, the suppression of the enzyme, phenoloxidase, activity in vivo increased. On the other hand, in the in vitro incubation of the infected hemolymph with X. nematophilus for 30 min with laminarin, the decrease in phenoloxidase activity reached 92% in the infection with the highest dose (1 × 1010 cells/ml) live bacteria, and reached 100% at the same dose in the infection with live bacteria in the absence of laminarin. A two-fold decrease in enzyme activity was recorded in the case of injection of (1 × 106 cells/ml) dead X. nematophilus and the absence of laminarin compared with injection of the same dose in the presence of laminarin. The same trend was also observed by the end of incubation of X. nematophilus-treated hemolymph without α-chymotrypsin. The decrease in phenoloxidase activity was highly significantly different in injection of dead P. luminescens and the absence of laminarin during incubation. In the case of injection of (1 × 108 cells/ml) live P. luminescens a higher degree in the reduction of enzyme activity was recorded in the absence of α-chymotrypsin, where it reached to nearly a two-fold decrease. The results of these studies indicated that both X. nematophilus and P. luminescens alive or dead suppress the phenoloxidase activity in the presence or absence of both laminarin and α-chymotrypsin but, the suppression in absence of both during the in vitro incubation was highly comparable.

Interaction betweenParacoccidioides brasiliensisand Pulmonary Dendritic Cells Induces Interleukin‐10 Production and Toll‐Like Receptor–2 Expression: Possible Mechanisms of Susceptibility

Dendritic cells (DCs) are the most effective antigen-presenting cells for inducing cell-mediated immune responses; it is thus important to investigate the role played by lung DCs in the pathogenesis of paracoccidioidomycosis (PCM) and their potential to initiate an immune response in mice susceptible (B10.A) and resistant (A/J) to PCM. Initially, we observed that lung DCs from susceptible mice were more phagocytic than cells from resistant mice, and we observed that phagocytosis in the presence of laminarin was inhibited only in DCs from susceptible mice. DCs from resistant mice produced a low concentration of interleukin (IL)-10, IL-12, and tumor necrosis factor (TNF)- alpha . In contrast, DCs from susceptible mice produced high concentrations of TNF- alpha and IL-10, but IL-10 production was significantly inhibited in the presence of laminarin. We also observed that DCs from Toll-like receptor (TLR)-2 knockout mice displayed defective production of IL-10. After 15 days of Paracoccidioides brasiliensis infection, DCs from susceptible mice produced IL-10 and expressed costimulatory molecules at a low level. We found that expression of the gene for TLR-2 is increased after infection in susceptible, but not resistant, mice. In conclusion, our data suggest that P. brasiliensis induces regulatory DCs in susceptible mice, which promotes IL-10 production and contributes to the susceptibility of mice to P. brasiliensis infection.

Novel Inhibitors of Phenylalanine Ammonia-lyase: 5-Aryl-1, 3, 4-oxathiazol-2-ones

A series of 5‐aryl‐1,3,4‐oxathiazol‐2‐ones was synthesized and tested for inhibition against phenylalanine ammonia‐lyase (PAL) derived from the yeast Rhodotorula glutinis. All compounds inhibited PAL at 100 µM. Of the compounds tested, 5‐(4‐nitrophenyl)‐1,3,4‐oxathiazol‐2‐one (19) was the most effective, showing IC50 value of 2.5 µM, which was 2 times stronger than that of N‐(aminooxy) acetyl‐2,5‐dichloroaniline (Z302), a known PAL inhibitor. Compound 19 behaved as a noncompetitive inhibitor of PAL. When potato tuber disks were treated with 19 in the presence of laminarin, a β ‐1,3‐glucooligosaccharide elicitor, the amount of trans‐cinnamic acid diminished after 48 hr, being consistent with an effective inhibition of PAL. Most of the compounds inhibited the growth of lettuce seedlings at 100‐500 µM. However, no correlation was observed between the ability of 5‐aryl‐1,3,4‐oxathiazol‐2‐ones to inhibit PAL derived from R. glutinis and growth inhibitory activity against lettuce seedlings.

In vitro acanthamoebicidal activity of a killer monoclonal antibody and a synthetic peptide

To evaluate the in vitro microbicidal activity against Acanthamoeba castellanii of a murine monoclonal anti-idiotypic antibody (KTmAb) and a synthetic killer mimotope (KP), which mimic a yeast killer toxin (KT) characterized by a wide spectrum of antimicrobial activity through interaction with specific cell wall receptors, mainly constituted by beta-glucans. Amoebicidal activity was investigated after incubation of trophozoites under different experimental conditions with laminarinase, KTmAb, KP and a scrambled decapeptide (SP). To confirm the specific interaction of KP with beta-glucans, the experiments were also carried out in the presence of laminarin (beta1-3-glucan) or pustulan (beta1-6-glucan); both glucan molecules were co-incubated with KP or SP. KTmAb and KP exhibited a time-dependent killing activity, in comparison with SP or heat-inactivated KTmAb; this activity was completely abolished by pre-incubation with laminarin, but not by pustulan. Notably, in vitro amoebicidal activity was observed in the presence of laminarinase, an enzyme that specifically hydrolyses beta-glucans. Furthermore, KP specifically inhibited the growth of Acanthamoeba on infected contact lenses and the remaining adherent KP-treated trophozoites appeared strongly damaged. The results indicate that the expression of beta1-3-glucan receptors in the cell membrane is probably modulated during cell growth of A. castellanii and is critical for the killing activity of KT-like molecules. Our data confirm the broad antimicrobial spectra of KTmAb and KP, emphasize the crucial role of beta1-3-glucan in microbial physiology and suggest the potential use of KTmAb and KP in the prevention and therapy of Acanthamoeba infections or in preventing Acanthamoeba contamination during storage of contact lenses.

A recombinant functional variant of the olive pollen allergen Ole e 10 expressed in baculovirus system

Pollens have been reported as important sources of antigens causing type-I allergy and, among them, olive pollen has high clinical relevance in Mediterranean countries. The most recently described olive allergen, Ole e 10, is involved in cross-reactivity phenomena and related to asthma induction in allergic patients. These immunologic features make this allergen a good candidate to be included in diagnosis and therapy of protocols of allergic diseases. Since the availability of Ole e 10 from the olive pollen is limited, the allergen has been efficiently expressed in the baculovirus/insect cell system. The Ole e 10-cDNA inserted into the transfer vector pBacPAK8 allowed the expression of the recombinant protein in cultured Sf21 cells. Recombinant Ole e 10 (rOle e 10) was purified from the culture after dialysis and three chromatographic steps. Mass spectrometry, Edman degradation, IgE- and IgG-binding analyses were employed to characterize the recombinant allergen, which showed molecular and immunological equivalence with the natural protein. Affinity gel electrophoresis in presence of laminarin (1,3-β-glucan) revealed that rOle e 10 retains identical carbohydrate-binding capacity than the natural allergen. In conclusion, the recombinant expression of Ole e 10 in baculovirus/insect cell system produces a homogeneous and biologically active allergen that could be useful for clinical and scientific purposes.

Inhibition of trans-Cinnamate 4-Hydroxylase by 4-Amino-5-aryl-2,3-dihydro-3H-1,2,4-triazole-3-thiones

A number of 4-amino-5-aryl-2,3-dihydro-3H-1,2,4-triazole-3-thiones were synthesized and tested for inhibitory activity against trans-cinnamate 4-hydroxylase (C4H) from Populus kitakamiensis, which was expressed in yeast. Of the compounds tested, 4-amino-5-phenyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (1) was the most effective, having an IC50 value of 0.32 µM. Replacement of the phenyl group with heterocycles or substituted phenyl groups drastically reduced the activity. Compound 1 behaved as a mixed-inhibitor of C4H. When potato tuber disks were treated with 1 (0.2–20 ng/disk) in the presence of laminarin, a β-1,3-glucooligosaccharide elicitor, trans-cinnamic acid was accumulated at levels 3 times higher than in disks treated with laminarin alone. © Pesticide Science Society of Japan

An assay for glucanase activity in wine

A method to measure specific β(1,3)-glucanase activity in wines has been developed, and is based on the measurement of the kinetics of glucose release in the presence of laminarin (a β(1,3)-glucan from Laminaria digitata). The reaction time and substrate amount have been optimised to allow for measurements in wine conditions. The optimised method has been used to measure the efficiency of a commercial enzyme preparation in wines in relation to filterability. The results indicate that despite the inhibition of wine constituents, β-glucanase activities were present in all wines after enzyme addition. The filterability of the wines significantly increased after enzyme addition as measured by a standardised procedure ( V max index).

Carminic acid dye from the homopteran Dactylopius coccus hemolymph is consumed during treatment with different microbial elicitors.

The activation of Dactylopius coccus (Costa) hemolymph with microbial polysaccharide molecules was studied. Hemolymph incubated in the presence of laminarin, zymosan, and N-acetyl glucosamine produced a dark fibrillar precipitated, and the red pigment (carminic acid) was consumed (measured spectrophotometrically at 495 nm). Lipopolysaccharide (LPS) did not induce any response. The reaction was inhibited with millimolar concentrations of serine and cysteine protease inhibitors, EGTA and phenyl thiourea. It was also diminished by prostaglandin synthesis inhibitors: dexamethasone, acetylsalicylic acid, and indomethacin. However, Mg2+ chelator EDTA did not inhibit hemolymph activation. Hemolymph proteins were depleted from soluble phase during treatment with laminarin, but a group of around 34 kDa remained unmodified. These results showed that D. coccus hemolymph is activated by microbial elicitors, its activation depends on eicosanoids, and suggest participation of a prophenoloxidase (PPO)-like activation system that could consume carminic acid. We are currently dissecting the molecular factors involved in D. coccus hemolymph activation to determine homologies and differences with other arthropods immune response pathways.

A β1,3-Glucan Recognition Protein from an Insect, Manduca sexta, Agglutinates Microorganisms and Activates the Phenoloxidase Cascade

Pattern recognition proteins function in innate immune responses by binding to molecules on the surface of invading pathogens and initiating host defense reactions. We report the purification and molecular cloning of a cDNA for a 53-kDa beta1,3-glucan-recognition protein from the tobacco hornworm, Manduca sexta. This protein is constitutively expressed in fat body and secreted into hemolymph. The protein contains a region with sequence similarity to several glucanases, but it lacks glucanase activity. It binds to the surface of and agglutinates yeast, as well as gram-negative and gram-positive bacteria. Beta1,3-glucan-recognition protein in the presence of laminarin, a soluble glucan, stimulated activation of prophenoloxidase in plasma, whereas laminarin alone did not. These results suggest that beta1,3-glucan-recognition protein serves as a pattern recognition molecule for beta1,3-glucan on the surface of fungal cell walls. After binding to beta1,3-glucan, the protein may interact with a serine protease, leading to the activation of the prophenoloxidase cascade, a pathway in insects for defense against microbial infection.

Functional analysis of plasma prophenoloxidase system in the marine mussel Perna viridis

The role of prophenoloxidase (proPO) system in recognition and phagocytosis of yeast cells by hemocytes was examined in vitro using whole plasma and proPO system isolated from the plasma of the marine mussel, Perna viridis. The proPO was isolated from the plasma by ammonium sulphate precipitation and gel filtration. The final proPO preparation was homogeneous in native PAGE, and could be activated by trypsin, α-chymotrypsin and pronase-E. Laminarin (a polymer of β-1, 3-glucan) and lipopolysaccharides (LPS) from diverse bacterial species effectively activated the isolated proPO, demonstrating the ability of this proenzyme to interact directly with microbial surface components. The susceptibility of proPO activation to inhibition by serine protease inhibitors such as soybean trypsin inhibitor (STI) or p-nitrophenyl- p′-guanidinobenzoate ( p-NPGB), indicates that the isolated fraction may contain an integral serine protease domain in an inactive state. The presence of laminarin- or LPS-activated whole plasma of P. viridis facilitated adherence of yeast cells to hemocyte surface as well as eventually stimulated phagocytic uptake of the target cells by hemocytes, and no such hemocytic response was recorded with STI controls. This and other results strongly suggest that the intermediary factors generated during activation of plasma proPO system by non-self molecules play a key role in recognition and opsono-phagocytosis by hemocytes. However, the proPO system isolated from P. viridis plasma, after activation with microbial surface components, failed to show an opsonic effect.

Detection of β‐ 1,3‐glucanase activity after native polyacrylamide gel electrophoresis: Application to tobacco pathogenesis‐related proteins

Beta-1,3-Glucanase (laminarinase) activity was detected after polyacrylamide gel electrophoresis under native conditions by using laminarin as substrate. Following incubation of gels, laminarin was stained with Aniline Blue. Under UV illumination, lysis zones appeared as dark bands against a fluorescent background. As low as 0.001 unit of commercial Penicillium laminarinase could be observed after incubating the polyacrylamide gel for 45 min at pH 5.0. Extracts of commercial Penicillium laminarinase exhibited four bands with lytic activity towards laminarin. Analysis of intercellular fluid extracts of tobacco mosaic virus-infected tobacco leaves revealed four beta-1,3-glucanases corresponding to three acidic pathogenesis-related proteins, b4 (2), b5 (N) and b6b (0), and one basic protein. The presence of laminarin in gels retarded the migration of some proteins with beta 1,3-glucanase activity. This change in electrophoretic mobility could be used as a complementary affinity test for identifying proteins with beta-1,3-glucanase activity.

Production of laminarinase byBacillus polymyxa

Nutritional factors relating to the production of laminarinase (β-glucanase) byBacilluspolymyxa were examined. Studies were carried out in shaken-flask cultures and a 5–1 batch fermenter. Although the enzyme was produced in the presence of a variety of carbon sources, highest activity was detected in the presence of laminarin. Activity induced by starch was 80% of that formed in the presence of the 1,3-β-D-glucan. Of the nitrogen sources examined, peptone provided highest yields of enzyme. Although laminarinase was produced over the range pH 5.0 to 8.5, highest activity was detected after 48 hr with an initial pH of 7.0. However, control of pH at 7.0 during the fermentation resulted in a lower yield of enzyme than that obtained when the culture was allowed to establish its own pH.