Purification and biochemical characterization of a novel thermophilic exo-ß-1, 3-glucanase from the thermophile biomass-degrading fungus Thielavia terrestris Co3Bag1

Background: The aim of this work was to purify and characterize exo-ß-1,3-glucanase, namely, TtBgnA, from the thermophilic fungus Thielavia terrestris Co3Bag1 and to identify the purified enzyme. Results: The thermophilic biomass-degrading fungus T. terrestris Co3Bag1 displayed ß-1,3-glucanase activity when grown on 1% glucose. An exo-ß-1,3-glucanase, with an estimated molecular mass of 129 kDa, named TtBgnA, was purified from culture filtrates from T. terrestris Co3Bag1. The enzyme exhibited optimum activity at pH 6.0 and 70°C and half-lives (t1/2) of 54 and 37 min at 50 and 60°C, respectively. Substrate specificity analysis showed that laminarin was the best substrate studied for TtBgnA. When laminarin was used as the substrate, the apparent KM and Vmax values were determined to be 2.2 mg mL-1 and 10.8 U/mg, respectively. Analysis of hydrolysis products by thin-layer chromatography (TLC) revealed that TtBgnA displays an exo mode of action. Additionally, the enzyme was partially sequenced by tandem mass spectrometry (MS/MS), and the results suggested that TtBgnA from T. terrestris Co3Bag1 could be classified as a member of the GH-31 family. Conclusions: This report thus describes the purification and characterization of TtBgnA, a novel exo-ß-1,3-glucanase of the GH-31 family from the thermophilic fungus T. terrestris Co3Bag1. Based on the biochemical properties displayed by TtBgnA, the enzyme could be considered as a candidate for potential biotechnological applications.

Purification and characterization of a novel ß-1,3-glucanase from Arca inflata and its immune-enhancing effects.

A novel ß-1,3-glucanase from Arca inflata was purified using chromatography methods. It was determined as a glycoprotein comprising 23.65% carbohydrate content with O-linked glycan and showed specific activity of 90.01 ±â€¯1.2 U/mg against laminarin. The optimal pH and temperature for the activity of the glucanase were 6.0 and 40 °C, respectively. The affinity parameter of the glucanase using laminarin was determined as Kd = 13.09 µM. The activity of the glucanase was 27 ±â€¯2.6% enhanced by 2-mM Mn2+ ions and inhibited by 40-50% using 2-mM Zn2+, Cu2+, or Ba2+ ions. The glucanase showed an endo-type cleavage mode and hydrolyzed laminarin into glucoses, disaccharides, trioligosaccharides, and tetraoligosaccharides. Otherwise, the glucanase exhibited immune-enhancing effects via significantly increasing the phagocytic activity of macrophages and inducing the release of nitric oxide, tumor necrosis factor α, and interleukin-6 in RAW264.7 cells. It might be used as a bifunctional additive for the food industry.

Biochemical and genetic characterization of ß-1,3 glucanase from a deep subseafloor Laceyella putida.

A ß-1,3-glucanase (LpGluA) of deep subseafloor Laceyella putida JAM FM3001 was purified to homogeneity from culture broth. The molecular mass of the enzyme was around 36 kDa as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). LpGluA hydrolyzed curdlan optimally at pH 4.2 and 80 °C. In spite of the high optimum temperature, LpGluA showed relatively low thermostability, which was stabilized by adding laminarin, xylan, colloidal chitin, pectin, and its related polysaccharides. The gene for LpGluA cloned by using degenerate primers was composed of 1236 bp encoding 411 amino acids. Production of both LpGluA and a chitinase (LpChiA; Shibasaki et al. Appl Microbiol Biotechnol 98, 7845-7853, 2014) was induced by adding N-acetylglucosamine (GluNAc) to a culture medium of strain JAM FM3001. Construction of expression vectors containing the gene for LpGluA and its flanking regions showed the existence of a putative repressor protein.

Purification, characterization and synergism in autolysis of a group of 1,3-ß-glucan hydrolases from the pilei of Coprinopsis cinerea fruiting bodies.

Using a combined chromatography method, we simultaneously purified three protein fractions (II-2, II-3 and II-4) with 1,3-ß-glucanase activity from extraction of pilei of Coprinopsis cinerea fruiting bodies. MALDI-TOF/TOF amino acid sequencing showed that these three fractions matched a putative exo-1,3-ß-glucanase, a putative glucan 1,3-ß-glucosidase and a putative glycosyl hydrolase family 16 protein annotated in the C. cinerea genome, respectively; however, they were characterized as a 1,3-ß-glucosidase, an exo-1,3-ß-glucanase and an endo-1,3-ß-glucanase, respectively, by analysis of their substrate specificities and modes of action. This study explored how these three 1,3-ß-glucoside hydrolases synergistically acted on laminarin: the endo-1,3-ß-glucanase hydrolysed internal glycosidic bonds of laminarin to generate 1,3-ß-oligosaccharides of various lengths, the exo-1,3-ß-glucanase cleaved the longer-chain laminarioligosaccharides into short-chain disaccharides, laminaribiose and gentiobiose, and the 1,3-ß-glucosidase further hydrolysed laminaribiose to glucose. The remaining gentiobiose must be hydrolysed by other 1,6-ß-glucosidases. Therefore, the endo-1,3-ß-glucanase, exo-1,3-ß-glucanase and 1,3-ß-glucosidase may act synergistically to completely degrade the 1,3-ß-glucan backbone of the C. cinerea cell wall during fruiting body autolysis. These three 1,3-ß-glucoside hydrolases share a similar optimum pH and optimum temperature, supporting the speculation that these enzymes work together under the same conditions to degrade 1,3-ß-glucan in the C. cinerea cell wall during fruiting body autolysis.

Expression, purification and crystallization of a family 55 ß-1,3-glucanase from Chaetomium thermophilum.

A ß-1,3-glucanase from the thermophilic fungus Chaetomium thermophilum was overexpressed in Pichia pastoris, purified and crystallized in the presence of 1.8 M sodium/potassium phosphate pH 6.8 as a precipitant. Data to 2.0 Å resolution were collected in-house at 293 K from a single crystal. The crystal was found to belong to space group P2(1), with unit-cell parameters a = 64.1, b = 85.8, c = 68.5 Å, ß = 93.1° and one molecule in the asymmetric unit.

The yeast Wickerhamomyces anomalus AS1 secretes a multifunctional exo-ß-1,3-glucanase with implications for winemaking.

A multifunctional exo-ß-1,3-glucanase (WaExg2) was purified from the culture supernatant of the yeast Wickerhamomyces anomalus AS1. The enzyme was identified by mass spectroscopic analysis of tryptic peptide fragments and the encoding gene WaEXG2 was sequenced. The latter codes for a protein of 427 amino acids, beginning with a probable signal peptide (17 aa) for secretion. The mature protein has a molecular mass of 47 456 Da with a calculated pI of 4.84. The somewhat higher mass of the protein in SDS-PAGE might be due to bound carbohydrates. Presumptive disulphide bridges confer a high compactness to the molecule. This explains the apparent smaller molecular mass (35 kDa) of the native enzyme determined by electrophoresis, whereas the unfolded form is consistent with the theoretical mass. Enzymatic hydrolysis of selected glycosides and glycans by WaExg2 was proved by TLC analysis of cleavage products. Glucose was detected as the sole hydrolysis product from laminarin, underlining that the enzyme acts as an exoglucanase. In addition, the enzyme efficiently hydrolysed small ß-linked glycosides (arbutin, esculin, polydatin, salicin) and disaccharides (cellobiose, gentiobiose). WaExg2 was active under typical wine-related conditions, such as low pH (3.5-4.0), high sugar concentrations (up to 20% w/v), high ethanol concentrations (10-15% v/v), presence of sulphites (up to 2 mm) and various cations. Therefore, the characterized enzyme might have multiple uses in winemaking, to increase concentrations of sensory and bioactive compounds by splitting glycosylated precursors or to reduce viscosity by hydrolysis of glycan slimes.

Protocol for simultaneous isolation of three important banana allergens.

Banana fruit (Musa acuminata) has become an important food allergen source in recent years. So far, 5 IgE reactive banana proteins have been identified, and the major allergens are: Mus a 2 (a class I chitinase, 31kDa), Mus a 4 (thaumatin-like protein, 21kDa), and Mus a 5 (ß-1,3-glucanase, 33kDa). Due to variations in allergen expression levels, diagnostic reagents for food allergy can be improved by using individual allergen components instead of banana allergen extracts. The purpose of this study was to optimize the purification protocol of the three major allergens present in banana fruit: Mus a 2, Mus a 4 and Mus a 5. By employing a three-step purification protocol (a combination of anion-exchange, cation-exchange and reversed-phase chromatography) three important banana allergens were obtained in sufficient yield and high purity. Characterization of the purified proteins was performed by both biochemical (2-D PAGE, mass fingerprint and N-terminal sequencing) and immunochemical (immunoblot) methods. IgE reactivity to the purified allergens was tested by employing sera of five allergic patients. The purified allergens displayed higher sensitivity in IgE detection than the routinely used extracts. The three purified allergens are good candidates for reagents in component-based diagnosis of banana allergy.

Recombinant Arthrobacter ß-1, 3-glucanase as a potential effector molecule for paratransgenic control of Chagas disease.

BACKGROUND: Chagas disease is most often transmitted to humans by Trypanosoma cruzi infected triatomine bugs, and remains a significant cause of morbidity and mortality in Central and South America. Control of Chagas disease has relied mainly on vector eradication. However, development of insect resistance has prompted us to develop a paratransgenic strategy to control vectorial transmission of T. cruzi. Here, the potential role of recombinant endoglucanases as anti-trypanosomal agents for paratransgenic application is examined. The surface of T. cruzi is covered by a thick coat of mucin-like glycoproteins that have been proposed to play a role in the binding of T. cruzi to the membrane surface of the vector gut. We hypothesize that disruption of these glycoconjugates could arrest parasite development in the vector and abort the transmission cycle. In this work, we examine the effects of recombinant Arthrobacter luteus ß-1, 3-glucanase expressed via Rhodococcus rhodnii on T. cruzi Sylvio II strain. METHODS AND RESULTS: The coding sequence for ß-1, 3-glucanase was cloned in-frame to a heterologous promoter/signal sequence from the Mycobacterium kansasii alpha antigen gene resident in an E. coli/R. rhodnii shuttle vector. The resulting construct was confirmed by sequencing, and electroporated into R. rhodnii. Expression products from positive clones were purified from log phase cultures followed by dialysis into physiological buffers. Lysates and media were quantitated by ELISA against rabbit antibody specific to ß-1,3-glucanase. Glucanase-positive samples were applied to live T. cruzi parasites in culture and viability accessed by spectrophotometric and fluorescent microscopic measurements. R. rhodnii-expressed ß-1,3-glucanase exhibited toxicity against T. cruzi compared to controls when applied at 5 and 10% of the total culture volume. The decrease in cell viability ranged from a maximum of 50% for the media treatments to 80% for the filtered lysates. CONCLUSIONS: These results suggest that recombinant ß-glucanase could be a powerful addition to the arsenal of effector molecules for paratransgenic control of Chagas disease. In future studies, the ability of ß-glucanase to function in combination with other effector molecules will be explored. Dual targeting of T. cruzi should not only slow resistance but also permit synergistic or additive lethal effects on T. cruzi.

Novel sources of ß-glucanase for the enzymatic degradation of schizophyllan.

Schizophyllan is a homoglucan produced by the fungus Schizophyllum commune, with a ß-1,3-linked backbone and ß-1,6-linked side chains of single glucose units at every other residue. Schizophyllan is commercially produced for pharmaceutical and cosmetics uses. However, surprisingly little information is available on the biodegradation of schizophyllan. Enzymes that attack schizophyllan could be useful for controlled modifications of the polymer for novel applications. Enrichment cultures were used to isolate 20 novel fungal strains from soil samples, capable of growing on schizophyllan as a sole carbon source. Three additional strains were isolated as contaminants of stored schizophyllan solutions. Strains showing the highest levels of ß-glucanase activity were identified as Penicillium simplicissimum, Penicillium crustosum, and Hypocrea nigricans. ß-glucanases also showed activity against the similar ß-glucans, laminarin and curdlan. By comparison, commercial ß-glucanase from Trichoderma longibrachiatum and laminarinase from Trichoderma sp. showed lower specific activities toward schizophyllan than most of the novel isolates. ß-glucanases from P. simplicissimum and H. nigricans exhibited temperature optima of 60°C and 50°C against schizophyllan, respectively, with broad pH optima around pH 5.0. Partial purifications of ß-glucanase from P. simplicissimum and P. crustosum demonstrated the presence of multiple active endoglucanase species, including a 20-25 kD enzyme from P. simplicissimum.

Biocontrol of anthracnose in pepper using chitinase, beta-1,3 glucanase, and 2-furancarboxaldehyde produced by Streptomyces cavourensis SY224.

A strain of Streptomyces cavourensis subsp. cavourensis (coded as SY224) antagonistic to Colletotrichum gloeosporioides infecting pepper plants was isolated. SY224 produced lytic enzymes such as chitinase, beta-1,3-glucanase, lipase, and protease in respective assays. To examine for antifungal activity, the treatments amended with the nonsterilized supernatant resulted in the highest growth inhibition rate of about 92.9% and 87.4% at concentrations of 30% and 10%, respectively. However, the sterilized treatments (autoclaved or chloroform treated) gave a lowered but significant inhibitory effect of about 63.4% and 62.6% for the 10% supernatant concentration, and 75.2% and 74.8% for the of 30% supernatant concentration in the PDA agar medium, respectively, indicative of the role of a nonprotein, heat stable compound on the overall effect. This antifungal compound, which inhibited spore germination and altered hyphal morphology, was extracted by EtOAc and purified by ODS, silica gel, Sephadex LH-20 column, and HPLC, where an active fraction was confirmed to be 2-furancarboxaldehyde by GS-CI MS techniques. These results suggested that SY224 had a high potential in the biocontrol of anthracnose in pepper, mainly due to a combined effect of lytic enzymes and a non-protein, heatstable antifungal compound, 2-furancarboxaldehyde.

Endo-ß-1,3-glucanase GLU1, from the fruiting body of Lentinula edodes, belongs to a new glycoside hydrolase family.

The cell wall of the fruiting body of the mushroom Lentinula edodes is degraded after harvesting by enzymes such as ß-1,3-glucanase. In this study, a novel endo-type ß-1,3-glucanase, GLU1, was purified from L. edodes fruiting bodies after harvesting. The gene encoding it, glu1, was isolated by rapid amplification of cDNA ends (RACE)-PCR using primers designed from the N-terminal amino acid sequence of GLU1. The putative amino acid sequence of the mature protein contained 247 amino acid residues with a molecular mass of 26 kDa and a pI of 3.87, and recombinant GLU1 expressed in Pichia pastoris exhibited ß-1,3-glucanase activity. GLU1 catalyzed depolymerization of glucans composed of ß-1,3-linked main chains, and reaction product analysis by thin-layer chromatography (TLC) clearly indicated that the enzyme had an endolytic mode. However, the amino acid sequence of GLU1 showed no significant similarity to known glycoside hydrolases. GLU1 has similarity to several hypothetical proteins in fungi, and GLU1 and highly similar proteins should be classified as a novel glycoside hydrolase family (GH128).

Sensitization profiles to purified plant food allergens among pediatric patients with allergy to banana.

Banana fruit allergy is well known, but neither immunoglobulin E recognition patterns to purified plant food allergens nor true prevalences of putative banana allergens have been established. This study aimed to characterize ß-1,3-glucanase and thaumatin-like protein (TLP) as banana allergens, testing them, together with other plant food allergens, in 51 children with allergic reactions after banana ingestion and both positive specific IgE and skin prick test (SPT) to banana. Banana ß-1,3-glucanase and TLP were isolated and characterized. Both banana allergens, together with kiwifruit TLP Act d 2, avocado class I chitinase Pers a 1, palm pollen profilin Pho d 2 and peach fruit lipid transfer protein (LTP) Pru p 3, were tested by in vitro and in vivo assays. Banana ß-1,3-glucanase (Mus a 5) was glycosylated, whereas banana TLP (Mus a 4) was not, in contrast with its homologous kiwi allergen Act d 2. Specific IgE to both banana allergens, as well as to peach Pru p 3, was found in over 70% of sera from banana-allergic children, and Mus a 4 and Pru p 3 provoked positive SPT responses in 6 of the 12 tested patients, whereas Mus a 5 in only one of them. Both peptidic epitopes and cross-reactive carbohydrate determinants were involved in the IgE-binding to Mus a 5, whereas cross-reactivity between Mus a 4 and Act d 2 was only based on common IgE protein epitopes. Profilin Pho d 2 elicited a relevant proportion of positive responses on in vitro (41%) and in vivo (58%) tests. Therefore, Mus a 4 and LTP behave as major banana allergens in the study population, and profilin seems to be also a relevant allergen. Mus a 5 is an equivocal allergenic protein, showing high IgE-binding to its attached complex glycan, and low in vivo potency.

Recomendaciones sobre el diagnóstico de la enfermedad fúngica invasora de la Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica (SEIMC). Actualización 2010 / Spanish Society of Clinical Microbiology and Infectious Diseases (SEIMC) guidelines for the diagnosis of invasive fungal infections. 2010 update

Este texto incluye una actualización de las recomendaciones sobre el diagnóstico de la infección fúngica invasora de la Sociedad Española de Microbiología Clínica y Enfermedades Infecciosas (SEIMC) elaboradas en 2004 (Enferm Infecc Microbiol Clin. 2004, 22:32-9). En la actualización de 2010 se incluye una revisión exhaustiva de las novedades tecnológicas de los últimos años, así como los niveles de evidencia para recomendar cada una de las técnicas de diagnóstico. En primer lugar, se analizan los métodos convencionales, examen microscópico y cultivo, con sus limitaciones, lo que ha llevado a desarrollar métodos alternativos como la cuantificación de antígenos y de ADN fúngico. Las indicaciones de los métodos alternativos se analizan para las diferentes infecciones fúngicas, candidiasis, aspergilosis y micosis invasoras por otras especies. Por último, se incluye una breve descripción de los métodos de identificación molecular y se revisan las pruebas para realizar estudios de sensibilidad a los antifúngicos, los procedimientos de referencia, las técnicas comerciales y sus indicaciones (AU)

These guidelines are an update of recommendations for the diagnosis of invasive fungal infections by the Spanish Society of Clinical Microbiology and Infectious Diseases (SEIMC) published in 2004 (Enferm Infecc Microbiol Clin. 2004, 22:32-9). In this updated version of the guidelines, a comprehensive review of the most recent diagnostic innovations and levels of evidence to recommend those diagnostic procedures are included. We first analyse conventional diagnostic methods, microscopic examination and culture, underlining their limitations which have led to the development of alternative methods, such as fungal antigen and DNA quantification. Those alternative methods of diagnosis are analysed by fungal infection. We also briefly review the methods for molecular identification of fungal species and recommendations for carrying out susceptibility tests for antifungal drugs, including reference procedures, commercial techniques and their indications (AU)

Purification of exo-1,3-beta-glucanase, a new extracellular glucanolytic enzyme from Talaromyces emersonii.

The moderately thermophilic aerobic ascomycete Talaromyces emersonii secretes, under selected growth conditions, several ß-glucan hydrolases including an exo-1,3-ß-glucanase. This enzyme was purified to apparent homogeneity in order to characterise its biochemical properties and investigate hydrolysis of different ß-glucans, including laminaran, a 1,3-ß-glucan from brown algae. The native enzyme is monomeric with a molecular mass of ~40 kDa and a pI value of 4.3, and is active over broad ranges of pH and temperature, with optimum activity observed at pH 5.4 and 65 °C. At pH 5.0, the enzyme displays strict specificity for laminaran (apparent K(m) 1.66 mg mL⁻¹; V(max) 7.69 IU mL⁻¹) and laminari-oligosaccharides and did not yield activity against 1,4-ß-glucans, 1,3;1,4-ß-glucans or 4-nitrophenyl- and methylumbelliferyl-ß-D: -glucopyranosides. Analysis of hydrolysis products formed during time-course hydrolysis of laminaran by high-performance anion exchange chromatography with pulsed amperometric detection revealed a strict exo mode of action, with glucose being the sole reaction product even at the initial stages of hydrolysis. The T. emersonii exo-1,3-ß-glucanase was inhibited by glucono-δ-lactone (K(i) 1.25 mM) but at significantly higher concentrations than typically inhibitory for exo-glycosidases such as ß-glucosidase. 'De novo' sequence analysis of the purified enzyme suggests that it belongs to family GH5 of the glycosyl hydrolase superfamily. The results clearly show that the exo-1,3-ß-glucanase is yet another novel enzyme present in the ß-glucanolytic enzyme system of T. emersonii.

Characterization of a ß-1,3-glucanase active in the alkaline midgut of Spodoptera frugiperda larvae and its relation to ß-glucan-binding proteins.

Spodoptera frugiperda ß-1,3-glucanase (SLam) was purified from larval midgut. It has a molecular mass of 37.5 kDa, an alkaline optimum pH of 9.0, is active against ß-1,3-glucan (laminarin), but cannot hydrolyze yeast ß-1,3-1,6-glucan or other polysaccharides. The enzyme is an endoglucanase with low processivity (0.4), and is not inhibited by high concentrations of substrate. In contrast to other digestive ß-1,3-glucanases from insects, SLam is unable to lyse Saccharomyces cerevisae cells. The cDNA encoding SLam was cloned and sequenced, showing that the protein belongs to glycosyl hydrolase family 16 as other insect glucanases and glucan-binding proteins. Multiple sequence alignment of ß-1,3-glucanases and ß-glucan-binding protein supports the assumption that the ß-1,3-glucanase gene duplicated in the ancestor of mollusks and arthropods. One copy originated the derived ß-1,3-glucanases by the loss of an extended N-terminal region and the ß-glucan-binding proteins by the loss of the catalytic residues. SLam homology modeling suggests that E228 may affect the ionization of the catalytic residues, thus displacing the enzyme pH optimum. SLam antiserum reacts with a single protein in the insect midgut. Immunocytolocalization shows that the enzyme is present in secretory vesicles and glycocalyx from columnar cells.

[Inhibition of adherence of Corynebacterium diphtheriae to human buccal epithelium by glycoside hydrolases from marine hydrobiontes].

A possibility of adhesion inhibition of Corynebacterium diphtheriae to human buccal epithelium by glycoside hydrolases of marine hydrobiontes was investigated using alpha-galactosidase from marine bacterium Pseudoalteromonas sp. KMM 701, total enzyme preparation and beta-1,3-glucanase from marine fungi Chaetomium, total enzyme preparation and beta-1,3-glucanase from marine mollusk Littorina kurila, and total enzyme preparation from crystalline style of marine mollusk Spisula sachalinensis were used. The enzymes were added to test-tubes containing buccal epithelial cells and/or the toxigenic bacterial strain C. diphtheriae No 1129, v. gravis. All the investigated enzymes were able to abort C. diphtheriae adherence, to human buccal epithelocytes. Inhibition of adhesion was more pronounced in the case of treatment of epithelocytes with highly purified enzymes of marine hydrobiontes in comparison with total enzyme preparations. The significant inhibition of C. diphtheriae adhesion was observed when the enzymes were added to the epithelocytes with the attached microorganisms. The results obtained show that glycoside hydrolases of marine hydrobiontes degrade any carbohydrates expressed on cell surface of bacterium or human buccal epithelocytes, impair unique lectin-carbohydrate interaction and prevent the adhesion.

A novel cold-adapted cellulase complex from Eisenia foetida: characterization of a multienzyme complex with carboxymethylcellulase, beta-glucosidase, beta-1,3 glucanase, and beta-xylosidase.

Clostridium sp. and some bacterial cellulases exist as an enzyme complex with cellulolytic, and hemicellulolytic enzymes, so called "cellulosome". In this article, we report that EF-CMCase25 occurs as a complex with beta-glucosidase, beta-1,3 glucanase, and beta-xylosidase. The multienzyme complex had a molecular mass of 150 kDa on gel filtration under non-reducing condition. After the gel filtration, the enzyme complex was purified to homogeneous state on BN-PAGE. The SDS-PAGE demonstrated that the purified protein is a complex with at least one CMCase (25 kDa), one beta-glucosidase (32 kDa), and one beta-1,3 glucanase (40 kDa) components. The CMCase activity in the purified enzyme complex at 15 degrees C was 44% of that obtained at the optimal temperature. The optimum pH of the EF-CMCase25 in the purified enzyme complex was pH 5.0 and stable at pH 7.0-9.0.

Isolation and characterization of two types of beta-1,3-glucanases from the common sea hare Aplysia kurodai.

Two types of beta-1,3-glucanases, AkLam36 and AkLam33 with the molecular masses of 36kDa and 33kDa, respectively, were isolated from the digestive fluid of the common sea hare Aplysia kurodai. AkLam36 was regarded as an endolytic enzyme (EC 3.2.1.6) degrading laminarin and laminarioligosaccharides to laminaritriose, laminaribiose, and glucose, while AkLam33 was regarded as an exolytic enzyme (EC 3.2.1.58) directly producing glucose from polymer laminarin. AkLam36 showed higher activity toward beta-1,3-glucans with a few beta-1,6-linked glucose branches such as Laminaria digitata laminarin (LLam) than highly branched beta-1,3-glucans such as Eisenia bicyclis laminarin (ELam). AkLam33 showed moderate activity toward both ELam and LLam and high activity toward smaller substrates such as laminaritetraose and laminaritriose. Although both enzymes did not degrade laminaribiose as a sole substrate, they were capable of degrading it via transglycosylation reaction with laminaritriose. The N-terminal amino-acid sequences of AkLam36 and AkLam33 indicated that both enzymes belong to the glycosyl hydrolase family 16 like other molluscan beta-1,3-glucanases.

Isolation and partial purification of the Saccharomyces cerevisiae cytokinetic apparatus.

Cytokinesis is the process by which a cell physically divides in two at the conclusion of a cell cycle. In animal and fungal cells, this process is mediated by a conserved set of proteins including actin, type II myosin, IQGAP proteins, F-BAR proteins, and the septins. To facilitate biochemical and ultrastructural analysis of cytokinesis, we have isolated and partially purified the Saccharomyces cerevisiae cytokinetic apparatus. The isolated apparatus contains all components of the actomyosin ring for which we tested-actin, myosin heavy and light chain, and IQGAP-as well as septins and the cytokinetic F-BAR protein, Hof1p. We also present evidence indicating that the actomyosin rings associated with isolated cytokinetic apparati may be contractile in vitro, and show preliminary electron microscopic imaging of the cytokinetic apparatus. This first successful isolation of the cytokinetic apparatus from a genetically tractable organism promises to make possible a deeper understanding of cytokinesis.

beta-1,3-glucanase inhibits activity of the killer toxin produced by the marine-derived yeast Williopsis saturnus WC91-2.

The marine-derived Williopsis saturnus WC91-2 was found to produce very high killer toxin activity against the pathogenic yeast Metschnikowia bicuspidata WCY isolated from the diseased crab. It is interesting to observe that the purified beta-1,3-glucanase from W. saturnus WC91-2 had no killer toxin activity but could inhibit activity of the WC91-2 toxin produced by the same yeast. In contrast, the WC91-2 toxin produced had no beta-1,3-glucanase activity. We found that the mechanisms of the inhibition may be that the beta-1,3-glucanase competed for binding to beta-1,3-glucan on the sensitive yeast cell wall with the WC91-2 toxin, causing decrease in the amount of the WC91-2 toxin bound to beta-1,3-glucan on the sensitive yeast cell wall and the activity of the WC91-2 toxin against the sensitive yeast cells. In order to make W. saturnus WC91-2 produce high activity of the WC91-2 toxin against the yeast disease in crab, it is necessary to delete the gene encoding beta-1,3-glucanase.