Purification and Partial Characterization of aβ-1,3-Glucanase Secreted by the MycoparasiteStachybotrys elegans

Abstract

A /I-l,3-glucanase secreted by Stachybotrys elegans, when grown on minimal synthetic medium containing Rhizoctonia solani cell wall fragments, was purified to homogeneity. The purification method involved ammonium sulfate precipitation and ion-exchange and size-exclusion chromatographies. The molecular mass of the enzyme was estimated under denaturing conditions to be about 94 kDa. The enzyme had an optimum pH of 5.0 and was most active between 40 and 50°C. Except for Mn 2 +, the enzyme activity was not sensitive to the metal ions tested and K m of 0.18 mg/ml was estimated for laminarin as a substrate. Cell wall lytic activity of purified /I-l,3-glucanase was tested on actively growing R. solani hyphae. /I-l,3-Glucanase induced morphological changes such as hyphal tip swelling, bursting, leakage of cytoplasm, and formation of numerous septae.Lytic enzymes, particularly chitinases and f3-1,3-glucanases, are essential in the life cycles of higher fungi. They have been implicated in apical growth, 1) wall softening during hyphal branching, germination, hyphal fusion,2,3) and conidia formation. 4 ) Recent studies indicate that during mycoparasitism of plant pathogenic fungi, several of these hydrolytic enzymes are produced by the mycoparasites and are involved in the degradation of fungal host cell walls. 5) Among these enzymes, chitinases and f3-1,3-g1ucanases are considered key cell-wall degrading enzymes of higher fungi. 6, 7) In addition, these enzymes have been shown to be produced by several potential biocontrol agents, and may be an important factor in biological control. 8 -11) Recently Stachybotrys elegans (Pidopl.) W. Gams, a soil fungus, was shown to have a strong mycoparasitic activity against the soilborne pathogen Rhizoctonia solani Kuhn in in vitro studies. Ultrastructural studies of the interaction between the mycoparasite and R. solani,12) as well as biochemical studies on the production of hydrolytic enzymes by S. elegans 13 ) strongly suggest the implication of these enzymes, particularly f3-1,3-g1ucanases, in the degradation of R. solani cell walls. As far as we can tell, nothing was reported about the f3-1 ,3-glucanases of this newly discovered mycoparasite. The objectives of this study were to purify and to partially characterize a f3-1,3-g1ucanase secreted by S. elegans, as well as to study its biological effects on the morphology of R. so/ani hyphae. Materials and MethodsOrganism and culture conditions. The mycoparasite S. elegans was isolated from soil in Izmir, Turkey. S. elegans was grown on sterile oat seeds for 10 days as described by Escande and Echandi. 14) The seeds were stored at 4°C and served as stock cultures. Inoculum for the production of fi-I ,3-glucanases in batch cultures consisted of potato dextrose agar t Corresponding author.(PDA; Difco Laboratories, Detroit, Michigan) disks of IO-day old mycelium of S. elegans grown from colonized oat kernels. Large-scale production of fi-I ,3-glucanases was done in 2-liter and 3~liter Erlenmeyer flasks containing respectively 400 and 60...

Purification and partial characterization of β-1,3-glucanase from Chaetomium thermophilum

“…The optimum temperature was 60°C. Similar pH values were reported for b-1,3-glucanases from other fungi, but the optimum temperature of the b-1,3-glucanase from C. thermophilum was higher than other fungi (Ait-Lahsen et al 2001;Archambault et al 1998;De La Cruz et al 1995;Tweddell et al 1995). The b-1,3-glucanase of C. thermophilum was thermostable at 50°C, and retained 90% activity after 60 min at 60°C.…”

“…1) and 77.6 kDa by gel filtration on Sephacryl S-100 (data not shown), suggesting that the enzyme may be a monomeric protein with a high molecular weight. In fungi, b-1,3-glucanases of high molecular weight were reported in T. harzianum (78 kDa, 78 kDa, 72 kDa) (Ait-Lahsen et al 2001;De La Cruz et al 1995;Gueguen et al 1997) and Stachy- botrys elegans (94 kDa, 75 kDa) (Archambault et al 1998;Tweddell et al 1995). Figure 2 shows the HPLC analysis of the enzymatic products formed from the laminarin by the purified b-1,3-glucanase.…”

Suppression subtractive hybridization and comparative expression of a pore-forming toxin and glycosyl hydrolase genes in Rhizoctonia solani during potato sprout infection

“…For the induction of genes transcript in R. solani AG-3 Rs114, the tester consisted of placing agar plugs from starter cultures on the surface of 100 × 15 mm, plastic Petri plates containing minimal synthetic media (MSM) (Tweddell et al 1995). MSM was supplemented with 1 % phytagel (MSMA; Difco, Detroit, MI) and covered with a cellulose membrane (500 PUT; UCB, North Augusta, USA).…”

Molecular cloning, sequence analysis, expression and characterization of the endochitinase gene from Trichoderma sp. in Escherichia coli BL21

“…strain was grown on potato dextrose agar (PDA) at 28°C for 5 days. Inoculation of potato dextrose broth (PDB) and minimal synthetic medium containing no carbon source (MSM) was done by adding the mycelia to 250 ml Erlenmeyer flasks containing 50 ml of PDB or MSM (Tweddell et al 1995). For induction of endochitinase, purified colloidal chitin from crab shells was added to MSM as the sole carbon source.…”