Evaluation of β-1,3-glucanase and β-1,4-glucanase enzymes production in some Trichoderma species

Trichoderma species have become the important means of biological control for fungal diseases. This research was carried on to access the high β-1,3-glucanase and β-1,4-glucanase enzyme producer of Trichoderma species isolates using two different carbon sources for finding a method to obtain more concentrate culture filtrates. Therefore, 14 Trichoderma isolates belonging to species: Trichoderma ceramicum, T. virens, T. pseudokoningii, T. koningii, T. koningiosis, T. atroviridae, T. viridescens, T. asperellum, T. harzianum1, T. orientalis, T. harzianum2, T. brevicompactum, T. viride and T. spirale were cultured in Wiendling’s liquid medium plus 0.5% glycerol or 0.5% Phytophthora sojae-hyphe as the carbon source in shaking and non-shaking (stagnant) statuses. Enzyme activity rate and total protein were evaluated in raw, acetony and lyophilized concentrated culture filtrates and the specific enzyme activity of β-1,3-glucanase and β-1,4-glucanase were measured by milligramme glucose equivalent released per minute per milligramme total protein in culture filtrates. The results showed that using Phytophthora – hyphe in medium increased the enzyme activities as compared to glycerol at all Trichoderma species which suggested that these substrates can also act as inducer for synthesis of lytic enzymes, in addition the most enzymes activity was observed in the lyophilised concentrated culture filtrate. The most successful species in β-1,3-glucanase and β-1,4-glucanase enzymes activities were T. brevicompactum and T. virens and these species can be used for mass production of these enzymes which are supposed to be used in commercial formulation and also will be able to control P. sojae directly.     

Structural analyses and yeast production of the β-1,3-1,4-glucanase catalytic module encoded by the licB gene of Clostridium thermocellum

A thermophilic glycoside hydrolase family 16 (GH16) β-1,3-1,4-glucanase from Clostridium thermocellum (CtLic16A) holds great potentials in industrial applications due to its high specific activity and outstanding thermostability. In order to understand its molecular machinery, the crystal structure of CtLic16A was determined to 1.95 Å resolution. The enzyme folds into a classic GH16 β-jellyroll architecture which consists of two β-sheets atop each other, with the substrate-binding cleft lying on the concave side of the inner β-sheet. Two Bis–Tris propane molecules were found in the positive and negative substrate binding sites. Structural analysis suggests that the major differences between the CtLic16A and other GH16 β-1,3-1,4-glucanase structures occur at the protein exterior. Furthermore, the high catalytic efficacy and thermal profile of the CtLic16A are preserved in the enzyme produced in Pichia pastoris, encouraging its further commercial applications.     

The effect of ethylene on the cell-type-specific and intracellular localization of β-1,3-glucanase and chitinase in tobacco leaves

We have studied the effect of ethylene on the localization of the basic isoforms of glucan endo-1,3--glucosidase (-1,3-glucanase, EC 3.2.1.39) and endo-chitinase (chitinase, EC 3.2.1.14) in leaves of Nicotiana tabacum L. cv. Havana 425. Comparisons of the enzyme contents of the lower epidermis of the leaf, leaf expiants with the lower epidermis removed, and intercellular wash fluid indicate that both enzymes are localized inside epidermal cells of untreated leaves. Ethylene treatment (20 l·l^-1, 4d) induced a marked -10- to 30-fold-coordinated accumulation of the enzymes. This was due primarily to induction of the basic isoforms inside chlorenchyma cells of the leaf interior. The localization of basic -1,3-glucanase was confirmed by immunofluorescence histochemistry and immunogold cytochemistry. Immunolabelling was confined to electron-dense bodies of the cell vacuole. No extracellular immunolabelling was detected in control or ethylene-treated leaves. We conclude that ethylene changes the cell-type-specific distribution but not the intracellular compartmentation of the two enzymes. These results support the generalization that basic isoforms of chitinase and -1,3-glucanase are intracellular whereas the acidic isoforms are secreted into the extracellular space.Abbreviations IgG immunoglobulin G - IWF intercellular wash fluid - PBS 0.14 M NaCl, 0.1 M K₂HPO₄, pH 7.5 - TMV tobacco mosaic virus We thank Monique Seldran and Alfred Milani for expert technical help, Patricia Ahl-Goy, Ciba-Geigy, AG, Basel for supplying IWF from TMV-infected leaves, and our colleagues Thomas Boller and Lilian Sticher for their comments and criticism.     

Effect of oxygen on the rate of β-1,3-glucan microbial exopolysaccharide production

Summary Examination of the relationship between the rate of oxygen transfer and the rate of polymer production revealed an unexpectedly high requirement for oxygen. At a cell density of about 3 g (dry wt)/L, the threshhold value for OTR for optimal synthesis of polymer is about 50 mmoles O₂/L.hr. Whereas Rushton turbines are effecient at transfering oxygen to solution, their use reduces the quality of the recovered polymer. Although better quality polymer can be produced in a reactor employing an agitator which causes less shear stress, the productivity can be compromised due to the inefficiency in OTR. The present study describes operating conditions for the provision of sufficient OTR in a system compatible with the production of high-quality polymer whereby turbine impellers were replaced with a marine-type propeller and mass transfer was assisted by means of a gas dispersion device.     

Antifungal effect of Trichoderma spp. β-1,3-glucanase on Phytophthora parasitica: Hyphal morphological distortions

Phytopathogenic fungi devastate agricultural crops worldwide. The biological agents, such as Trichoderma spp., antagonize phytopathogenic fungi by secreting various cell wall-degrading enzymes, for example, endochitinase and β-1,3-glucanase that target glycosidic linkages in β-glucan and chitin polymers of fungal cell walls, thus inhibiting pathogen growth. In this study, two antifungal genes endochitinase and β-1,3-glucanase cloned from local Trichoderma spp. were ligated in pET28a+ expression vector individually to generate two recombinant vectors. The vectors were mobilized into Escherichia coli host strain Rosetta-gami 2 for protein expression, and the 6xHis-tagged recombinant proteins were purified through Ni-NTA affinity chromatography. The purified proteins were individually confronted in vitro with pure cultures of Phytophthora parasitica (destructive pathogen affecting several hundred plant species worldwide) for analyzing their effect on pathogen growth. In vitro confrontation assay revealed P. parasitica growth inhibition by purified β-1,3-glucanase. The pathogen growth inhibition was due to hyphal morphological distortions, such as breakages, swelling, and holes evinced through electron micrography confirming direct role of β-1,3-glucanase in pathogen structural degradation.     

Statistical optimization of production medium for β-1,3-glucanase synthesis by Trichoderma harzianum

Response surface methods were used to determine the optimum concentration of medium nutrients for extracellular -1,3-glucanase production by Trichoderma harzianum in shakeflask culture. A Plackett-Burman design was used to screen the important variables which were then grouped and studied by a central composite design. The optimum levels of the medium constituents were determined by the complex algorithm of Box.     

Self-directing optimization of β-1,3-glucanase production by Trichoderma harzianum in batch culture

The self-directing optimization technique was employed to determine the optimal conditions for -1,3-glucanase production by Trichoderma harzianum in batch culture. A maximum -1,3-glucanase production of 0.910 U was obtained at a pH (controlled) of 4.9, an aeration rate of 0.9 1/(1)(min), and an agitator speed of 220 rev/min which were found to be the most suitable.     

Localization of β-1,3-glucanase in Trichoderma harzianum

Studies on the constitutive β-1,3-glucanase were conducted in submerged as well as in the stationary culture conditions, in the presence and in the absence of lactose and glucose as main carbon sources. In the absence of lactose or glucose, expression of β-1,3-glucanase was observed at 96?h in extracellular, periplasmic, cell wall bound and internal fractions during submerged fermentation. In shake flask culture, enzyme was found in all subcellular fractions using optimal glucose concentration. When Trichoderma harzianum was grown on media containing 55?kg lactose/m³ in submerged culture, activity was found in extracellular, cell wall bound and in the periplasmic fractions. The relative distribution of the enzyme in the cell is independent of the nature of the carbon source and its concentration.     

Study on preparation of β-cyclodextrin encapsulation tea extract

Microencapsulation of ethanol extract of tea was performed in this study. In order to microencapsulate, β-cyclodextrin was used as wall material. Ethanol extract of tea was used as the core material. Microcapsules in the solid form were obtained by drying the emulsions. RSM showed that optimal processing parameters were as followings: core material/wall material 1/4, β-cyclodextrin content 16%, stirring time 30 min and stirring temperature 200 °C. Pharmacological activities of β-cyclodextrin encapsulation tea extract were determined. It was found that β-cyclodextrin encapsulation tea extract could enhance BMD, BMC and bone Ca, Zn and Cu contents. In addition, β-cyclodextrin encapsulation tea extract could still reduce blood Ca contents. These results indicated that β-cyclodextrin encapsulation tea extract was useful for improving bone quality in aged animals.     

Improvement of the lytic properties of a β-1,3-glucanase by directed evolution

BGLII is a bacterial endoglucanase that hydrolyzes the β-1,3-glucan present in yeast cell walls, resulting in lysis of Saccharomyces cerevisiae. As a result of this property, BGLII is considered a potential tool for downstream processing and recovery of biotechnological products produced in yeast. Here we describe the improvement of the yeast lytic activity of BGLII, achieved by a directed evolution approach involving random mutagenesis and screening for variants with improved catalytic activity, combined with site-directed mutagenesis. A BGLII variant having three times the wild-type hydrolytic activity on laminarin was identified. The purified enzyme also exhibited higher lytic activity on yeast cells. Mutations causing the improvements are located very close to each other in the amino acid sequence, suggesting that the region should be considered as a target for further improvements of the glucanase activity. These results demonstrate the feasibility of molecular evolution methods for the improvement of the BGLII hydrolytic activity, and open a window for further improvement of this or other properties in glycosyl hydrolases in general.     

Chitinase and β-1,3-glucanase in the lutoid-body fraction of Hevea latex

The lutoid-body (bottom) fraction of latex from the rubber tree (Hevea brasiliensis) contains a limited number of major proteins. These are, besides the chitin-binding protein hevein, its precursor and the C-terminal fragment of this precursor, proteins with enzymic activities: three hevamine components, which are basic, vacuolar, chitinases with lysozyme activity, and a β-1,3-glucanase. Lutoid-body fractions from three rubber-tree clones differed in their contents of these enzyme proteins. The hevamine components and glucanase were isolated and several enzymic and structural properties were investigated. These enzymes are basic proteins and cause coagulation of the negatively charged rubber particles. The coagulation occurs in a rather narrow range of ratios of added protein to rubber particles, which indicates that charge neutralization is the determining factor. Differences in coagulation of rubber particles by lutoid-body fractions from various rubber clones can be explained by their content of hevamine and glucanase. Glucanase from the lutoid-body fraction may dissolve callus tissue and this may explain the observation that rubber-tree clones with a high glucanase content in this fraction produce more latex than clones with little glucanase. Sequence studies of two CNBr peptides of the glucanase indicate that this protein is homologous with glucanases from other plants, and that a C-terminal peptide, possibly involved in vacuolar targeting, may have been cleaved off.     

Role of β-1,3-glucanase in postmeiotic microspore release

Stage-specific extracts of Lilium anthers undergoing meiosis exhibited sharp peaks of both endolytic and exolytic β-1,3-glucanase activity at the time of in situ callose breakdown. The endo- and exo-β-1,3-glucanase activities, attributable to different enzymes, were found to have molecular weights of 32,000 and 62,000, respectively. The majority of exoglucanase activity was found in the outer somatic layers of the anther, whereas the majority of endoglucanase activity was located in the immediate surroundings of the meiocytes. The action of both glucanase activities on callose wall removal was monitored. It was shown that endo-β-1,3-glucanase, but not exoglucanase, was able to effect callose wall removal. To the extent that detection of glucanase activity in extracts reflects its activity in vivo, the endoglucanase enzyme may be considered as the immediate agent of callose wall breakdown and, hence, as a critical regulator in the initiation of the development of the gametophyte stage.     

Study of the influence of temperature on the dynamics of the catalytic cleft in 1,3-1,4-β-glucanase by molecular dynamics simulations

The dependence of some molecular motions in the enzyme 1,3-1,4-β-glucanase from Bacillus licheniformis on temperature changes and the role of the calcium ion in them were explored. For this purpose, two molecular dynamics simulated trajectories along 4 ns at low (300 K) and high (325 K) temperatures were generated by the GROMOS96 package. Several structural and thermodynamic parameters were calculated, including entropy values, solvation energies, and essential dynamics (ED). In addition, thermoinactivation experiments to study the influence of the calcium ion and some residues on the activity were conducted. The results showed the release of the calcium ion, which, in turn, significantly affected the movements of loops 1, 2, and 3, as shown by essential dynamics. These movements differ at low and high temperatures and affect dramatically the activity of the enzyme, as observed by thermoinactivation studies. The first two authors contributed equally to this work     

Some properties of A β-1,3-glucanase from rye

Molecular-sieve chromatography of an extract from ungerminated rye indicated the presence of enzymes which hydrolysed cellobiose, laminaribiose and the β-glucans cellodextrin, laminarin and barley β-glucan. A purified endo-β-1,3-glucanase was prepared from the extract by ammonium sulphate fractionation and molecular-sieve chromatography on Biogel P60. The substrate specificity and some properties of the enzyme are reported and the in vivo role of the enzyme is discussed.     

Evolution of the βGRP/GNBP/β-1,3-glucanase family of insects

The βGRP/GNBP/β-1,3-glucanase protein family of insects includes several proteins involved in innate immune recognition, such as the β-glucan recognition proteins of Lepidoptera and the Gram-negative bacteria-binding proteins of Drosophila. A phylogenetic analysis supported the existence of two distinct subfamilies, designated the pattern recognition receptor (PRR) and glucanase subfamilies, which originated by gene duplication prior to the origin of the Holometabola. In the C-terminal region (CTR) shared by both subfamilies, the PRR subfamily has evolved significantly more rapidly at the amino acid sequence level than has the glucanase subfamily, implying a relative lack of constraint on the amino acid sequence of this region in the PRR subfamily. PRR subfamily members also include an N-terminal region (NTR), involved in carbohydrate recognition, which is not shared by glucanase subfamily members. In comparisons between paralogous PRR subfamily members, there were no conserved amino acid residues in the NTR. However, when pairs of putatively orthologous PRR subfamily members were compared, the NTR was most often as conserved as the CTR or more so. This pattern suggests that the NTR may be important in functions specific to the different paralogs, while amino acid sequence changes in the NTR may have been important in functional differentiation among paralogs, specifically with regard to the types of carbohydrates that they recognize.     

Epoxiconazole-induced stimulation of the antifungal hydrolases chitinase and β-1,3-glucanase in wheat

Young plants of wheat (Triticum aestivum L. cv. Star), which were treated hydroponically with the triazole fungicide epoxiconazole (BAS 480 F) over a period of 8 days, showed a dose-dependent stimulation of the enzyme activities of the two antifungal hydrolases chitinase and -1,3-glucanase in the shoot tissue. In the root tissue, no significant rise in the enzyme activities was found. As shown by immunoblot analysis and enzyme-linked immunosorbent assay (ELISA) using antisera against tobacco acidic and basic chitinases and -1,3-glucanases, the obeserved increase in the activities coincided with an accumulation of enzyme proteins. This possibly indicates the induction of a de novo synthesis of chitinases and -1,3-glucanases by epoxiconazole. To our knowledge, this effect of a synthetic fungicide on antifungal hydrolases in an intact plant is demonstrated for the first time.     

Developmental and hormonal regulation of β-1,3-glucanase in tobacco

A highly sensitive and specific rocket immunoassay was used to measure the content of an endo-type -1,3-glucanase (EC 3.2.1.39) in tissues of Nicotiana tabacum L. cv. Havana 425. We show that the accumulation of -1,3-glucanase in cultured pith-parenchyma tissue is blocked by combinations of the auxin, -naphthaleneacetic acid (NAA), and the cytokinin, kinetin. When tissues pre-incubated for 7 d on complete medium containing 2.0 mg·l^-1 NAA and 0.3 mg·l^-1 kinetin are transferred onto medium without hormones or with either hormone added separately, the -1,3-glucanase content expressed per mg soluble protein increases approx. ten fold over a 7-d period. Under these inductive conditions, up to approx. 5% of the soluble protein is -1,3-glucanase. The induction is inhibited by >90% when tissues are cultured over the same period on medium containing both hormones. This -1,3-glucanase is developmentally regulated in the intact plant. It is a major component of the soluble protien in the lower leaves and roots but is not detectable in leaves near the top of the plant.Abbreviation NAA -naphthaleneacetic acid     

Characterization and expression of β-1,3-glucanase genes in peach

Beta-1,3-glucanase is one of the pathogenesis-related (PR) proteins involved in plant defense responses. A peach beta-1,3-glucanase gene, designated PpGns1, has been isolated and characterized. The deduced amino acid sequence of the product of PpGns indicates that it is a basic isoform (pI 9.8), and contains a putative signal peptide of 38 amino acids but has no C-terminal extension. Amino acid sequence comparisons revealed that PpGns1 is 69% and 67% identical to citrus and soybean beta-1,3-glucanases, respectively. Southern analysis of total genomic DNA also indicates that at least three genes for beta-1,3-glucanases exist in peach, forming a small gene family. Characterization of four additional clones by PCR has identified a second beta-1,3-glucanase gene, PpGns2. PpGns2 has been partially sequenced, and when compared to PpGns1, it shows high sequence homology, 96% and 99% nucleotide identity in the first and (partial) second exons, respectively. The deduced partial sequence of the PpGns2 product displays only two differences from PpGns1 in the signal peptide and one in the (partial) mature protein (141 amino acids). The 5'-flanking promoter regions of these two genes share 90% identity in nucleotide sequences interrupted by five major gaps (4-109 nt long). The promoter region contains various sequences similar to cis-regulatory elements present in different stress-induced plant genes. In leaves and stems of peach shoot cultures grown in vitro, PpGns1 is induced within 12 h after exposure to a culture filtrate of Xanthomonas campestris pv. pruni or ethephon. However, it is not induced following treatment with mercuric chloride.     

Proteomic analysis of β-1,3-glucanase in grape berry tissues

Grape berries are considered recalcitrant materials in proteomic analysis, because berry tissues contain large amounts of secondary metabolites, especially phenolic compounds, which severely interfere with protein extraction and electrophoresis separation. We report hereby a PVPP/TCA-based protein extraction protocol for grape berries. Phenolic compounds in berry extracts were removed with repeated PVPP cleanups, and proteins were recovered with TCA precipitation. Protein resolution in 2-D gels was gradually improved with the increase of PVPP cleanup steps. By the protocol, about 760 protein spots of berry tissues were clearly resolved in 2-D gels with CBB staining. This protocol was also used to analyze β-1,3-glucanase (EC 3.2.1.39) in berry tissues. An anti-synthetic peptide antibody was prepared against 15 amino acid sequence residing on the surface of β-1,3-glucanase molecule. It detected two major spots in 2-D blots of berry extracts. The spots were identified by MALDI-TOF analysis as β-1,3-glucanase. The present study validates that β-1,3-glucanase is present in higher abundance in berry skins than in pulps, and in red berries than in white berries. Therefore, β-1,3-glucanase displays a tissue-specific expression. The preferential accumulation of β-1,3-glucanase in skins may be relevant to berry ripening.