SsdchA is a novel secretory cellobiohydrolase driving pathogenicity in Sclerotinia sclerotiorum

The necrotrophic fungus, Sclerotinia sclerotiorum, employs an array of cell wall-degrading enzymes(CWDEs), including cellulase, to dismantle host cell walls. However, the molecular mechanisms through which S. sclerotiorum degrades cellulose remain elusive. Here, we unveil a novel secretory cellobiohydrolase, SsdchA, characterized by a signal peptide and a Glyco_hydro_7(GH7) domain. SsdchA exhibits a robust expression of during early infection stages. Interestingly, colony morphology and growth rates remain unaffected across the wild-type, SsdchA deletion strains and SsdchA overexpression strains on potato dextrose agar(PDA) medium. Nevertheless, the pathogenicity and cellobiohydrolase activity decreased in the SsdchA deletion strains, but enhanced in the SsdchA overexpression strains. Moreover,the heterologous expression of SsdchA in Arabidopsis thaliana leads to reduced cellulose content and heightened susceptibility to S. sclerotiorum. Collectively, our data underscore the pivotal role of the novel cellobiohydrolase SsdchA in the pathogenicity of S. sclerotiorum.

Molecular Cloning and Expression of a Family 6 Cellobiohydrolase Gene <i>cbhII</i>from <i>Penicillium funiculosum</i>NCL1

Aim: Lignocelluloytic enzymes are the largest class of hydrolase enzyme which utilizes the plant biomass to produce renewable sources. Hence practices for larger production of these enzymes at lower cost received much attention for industrial use. Hence this paper deals with expression and purification of cellobiohydrolase gene from Penicillium funiculosum NCL1. Methods & Results: A cellobiohydrolase gene, cbhII of Penicillium funiculosum NCL1 was cloned and expressed in Pichia pastoris X33. Two exons of the cbhII gene were amplified separately and fused by overlap extension PCR. The fused product was cloned in yeast expression vector pPICZαA and expressed in P. pastoris under the control of the AOX1 promoter. P. pastoris transformants expressing recombinant cellobiohydrolase were selected on CMC agar plate and their ability to produce the cellobiohydrolase was evaluated in flask cultures. P. pastoris X33 (pPICbh6) efficiently secreted the recombinant cellobiohydrolase into the medium and produced the cellobiohydrolase activity (5 U/ml) after 96 h of growth. The recombinant cellobiohydrolase produced by P. pastoris (pPICBH6) showed maximum activity at pH 4.0 and temperature 50&degC and higher specificity in hydrolysis of filter-paper.

Constitutive overexpression of cellobiohydrolase 2 in Trichoderma reesei reveals its ability to initiate cellulose degradation

Cellulose degradation results from the synergistic effect of different enzymes,but which enzyme is involved in the initial stage of cellulose degradation is still not well understood.Cellobiohydrolase 2(CBH2)attached to the conidial surface is possibly associated with the initial stage.However,its specific mechanism is still incompletely known.This study explored the potential role of CBH2 in initiating cellulose degradation using a constitutive overexpression strategy.First,the CBH2-overexpression Trichoderma reesei strains Qgc2-5 and Qrc2-40 were constructed using the constitutive promoters P gpd1 and P rpS30,respectively.It was found that cbh2 was ex-pressed at a high level under the glucose conditions and was significantly higher than that of the parental strain QM9414 at the early stage of 29 h when cellulose was used as the carbon source.Particularly,the constitutive overexpression of cbh2 caused the strong expression of major cellulase-encoding genes(cbh1,eg1,and eg2)and the rapid decomposition of cellulosic material.Meanwhile,the scanning electron microscope showed that the groove-like structure of the cellulose surface was eroded seriously owing to CBH2 overexpression,which caused the cellulose surface to be smooth.These results showed that the overexpression of CHB2 caused the major cel-lulase enzymes to be expressed and contributed to cellulose degradation,showing the potential role of CBH2 in the initial stage of the cellulose hydrolytic process.

A novel function for the cellulose binding module of cellobiohydrolase I

A homogeneous cellulose-binding module(CBM)of cellobiohydrolase I(CBHI)from Trichoderma pseudokoningii S-38 was obtained by the limited proteolysis with papain and a series of chromatographs filtration.Analysis of FT-IR spectra demonstrated that the structural changes result from a weakening and splitting of the hydrogen bond network in cellulose by the action of CBMCBHI at 40℃for 24 h.The results of molecular dynamic simulations are consistent with the experimental conclusions, and provide a nanoscopic view of the mechanism that strong and medium H-bonds decreased dramatically when CBM was bound to the cellulose surface.The function of CBMCBHI is not only limited to locating intact CBHI in close proximity with cellulose fibrils,but also is involved in the structural disruption at the fibre surface.The present studies provided considerable evidence for the model of the intramolecular synergy between the catalytic domain and their CBMs.

Mechanism of cellobiose inhibition in cellulose hydrolysis by cellobiohydrolase

An experimental study of cellobiose inhibition in cellulose hydrolysis by synergism of cellobiohydrolyse I and endoglucanase I is presented. Cellobiose is the structural unit of cellulose molecules and also the main product in enzymatic hydrolysis of cellulose. It has been identified that cellobiose can strongly inhibit hydrolysis reaction of cellulase, whereas it has no effect on the adsorption of cellulase on cellulose surface. The experimental data of FT-IR spectra, fluorescence spectrum and circular dichroism suggested that cellobiose can be combined with tryptophan residue located near the active site of cellobiohydrolase and then form steric hindrance, which prevents cellulose molecule chains from diffusing into active site of cellulase. In addition, the molecular conformation of cellobiohydrolase changes after cellobiose binding, which also causes most of the non-productive adsorption. Under these conditions, microfibrils cannot be separated from cellulose chains, thus further hydrolysis of cellulose can hardly proceed.

Structural changes of cellobiohydrolase I (1,4-β-D-glucan-cellobiohydrolase I, CBHI) and PNPC (p-nitro-phenyl-β-D-cellobioside) during the binding process

Conformational changes to 1,4-β-D-glucan cellobiohydrolase I (CBHI) in response to its binding with p-nitrophenyl β-D-cellobioside (PNPC) were analyzed by second-derivative fluorescence spectrometry at the saturation binding point. Irreversible changes to the configuration of PNPC during the course of the binding process were characterized by UV spectral analysis. Isothermal titration calorimetry (ITC) was used to determine the stoichiometry of binding (i.e. the number of molar binding sites) of PNPC to CBHI. Two points on the surface of the CBHI molecule interact with PNPC, and irreversible changes to the configuration of PNPC occur during its conversion to p-nitrophenyl (PNP). The ITC studies demonstrated that the binding of PNPC to CBHI is an irreversible process, in which heat is released, but where there is no reversible equilibrium between PNPC-CBHI and CBHI and PNPC. On the other hand, PNP and cellobiose need to be released from the PNPC-CBHI complex to facilitate the repeated binding of new PNPC molecules to the renewable CBHI molecules. Therefore, we speculate that the energy, which powers the configurational change of PNPC as it is converted to PNP, is generated from cyclic changes in the conformation of CBHI during the binding/de-sorption process. These new insights may provide a basis for a better understanding of the binding mechanism in enzyme-substrate interactions.

Enzymatic Hydrolysis of Cellulose with Different Crystallinities Studied by Means of SEC-MALLS

The reactions of exo-cellulase （cellobiohydrolase, CBH） and endo-cellulase （endoglucanase, EG） were investigated by analyzing the insoluble residues of microcrystalline cellulose （MCC） and filter paper cellulose （FPC） during enzymatic hydrolysis. Molecular parameters including molecular weight and its distribution, degree of polymerization, and radii of gyration were measured by size exclusion chromatography coupled with multi-angle laser light scattering. No significant change in MCC chains was found during the whole reaction period, indicating that CBH digestion follows a layer-by-layer solubilization manner. This reaction mode might be the major reason for slow enzymatic hydrolysis of cellulose. On the other hand, the degree of polymerization of FPC chains decreases rapidly in the initial reaction, indicating that EG digestion follows a random scission manner, which may create new ends for CBH easily. The slopes of the conformation plots for MCC and FPC increase gradually, indicating stronger chain stiffness of cellulose during hvdrolvsis

A Novel Exo-Glucanase Explored from a <i>Meyerozyma</i>sp. Fungal Strain

Isolating cellulase-secreting microbes followed-by screening their cellulolytic activities has been an essential approach to discover novel and potential cellulases for cellulolytic industrial applications. This study was aimed to explore competitive exoglucanases by screening avicelase activities for 92 fungal strains isolated from environmental airborne-fungal-spore samples. Results showed that an isolated fungal strain numbered 58 exhibited the best avicelase activity of 0.209 U/mL when cultured for six days at pH 5.0 - 5.3 and 25℃ - 27℃, and was lately identified as a yeast strain of Meyerozyma sp. (96% ITS fragment similar with Meyerozyma caribbica, HG970748). Based on amino acid sequences revealed from LC/MS/MS, the target exoglucanase was identical to 1,4-beta-D-glucan cellobiohydrolases and was named Mc-CBHI which had optimal avicelase reaction conditions of pH 5 and 70℃ and could remain fairly stable after 4hr incubation at acid conditions (pH 3 - 5) or wide temperature ranges (30℃ - 80℃). Additionally, the Mc-CBHI (~70 kDa and ~3.6% of crude enzyme) had specific FPase and avicelase activities of 0.179 U/mg and 0.126 U/mg, respectively (which were about 40% - 50% activities of a commercial cellulase Accellerase-1000). These results demonstrated that the newly-found Mc-CBHI could become one of potential exoglucanase resources for related cellulolytic industrial applications.

The effects of simulated nitrogen deposition on extracellular enzyme activities of litter and soil among different-aged stands of larch

Aims Nitrogen(N)addition could affect the rate of forest litter and soil organic matter decomposition by regulating extracellular enzyme activity(EEa).The impact of N addition on EEa may differ across different age stands with different organic matter quality.We were interested in whether the impact of N addition on EEa in litter and mineral soil during the growing season was dependent on stand age of a larch plantation in North China.Methods We added three levels of N(0,20 and 50 kg N ha^(−1) year^(−1))in three age stands(11,20 and 45 years old)of Larix principis-rupprech-tii plantation in North China.We measured potential activities of β-1,4-glucosidase(b),cellobiohydrolase(Cb),β-1,4-N-acetyl-glucosaminidase(Nag)and phenol oxidase(Po)in litter(organic horizon)and mineral soil(0-10 cm)during the second growing sea-son after N amendment.We also measured C and N concentrations,microbial biomass C and N,and KCl-extractable ammonium and nitrate in both litter and mineral soil.Important Findings We observed unimodal patterns of EEa during the growing season in all three stands,consistent with the seasonal variations of soil temperature.stand age had a strong effect on EEa in both litter and mineral soil,and this effect differed between litter and mineral soil as well as between different enzymes.N addition did not significantly affect the activities of b or Cb but significantly suppressed the activity of Nag in litter.We also found stand age-specific responses of Po activity to N addition in both litter and mineral soil.N addition suppressed Po activity of the high C:N ratio litters in 20-and 45-year-old stands but had no significant effect on Po activity of the low C:N ratio litter in 11-year-old stand.moreover,N addition inhibited Po activity of the high C:N ratio soil in 20-year-old stand but had no significant impact on Po activity of the low C:N ratio soils in 11-and 45-year-old stands.overall,stand age had a greater effect on EEa in litter and mineral soil compared to 2 years of N addition.moreover,the effect of N addition on Po activity is stand age dependent,which may affect the long-term soil carbon storage in this forest.

Phenological Stage, Plant Biomass, and Drought Stress Affect Microbial Biomass and Enzyme Activities in the Rhizosphere of Enteropogon macrostachyus

Indigenous grasses have been effectively used to rehabilitate degraded African drylands. Despite their success, studies examining their effects on soil bioindicators such as microbial biomass carbon(C) and enzyme activities are scarce. This study elucidates the effects of drought stress and phenological stages of a typical indigenous African grass, Enteropogon macrostachyus, on microbial biomass and enzyme activities(β-glucosidase, cellobiohydrolase, and chitinase) in the rhizosphere soil. Enteropogon macrostachyus was grown under controlled conditions. Drought stress(partial watering) was simulated during the last 10 d of plant growth, and data were compared with those from optimum moisture conditions. The rhizosphere soil was sampled after 40 d(seedling stage), 70 d(elongation stage), and 80 d(simulated drought stress). A high root:shoot ratio at seedling stage compared with elongation and reproduction stages demonstrated that E. macrostachyus invested more on root biomass in early development, to maximise the uptake of nutrients and water. Microbial biomass and enzyme activities increased with root biomass during plant growth. Ten-day drought at reproduction stage increased the microbial biomass and enzyme activities, accompanying a decrease in binding affinity and catalytic efficiency. In conclusion, drought stress controls soil organic matter decomposition and nutrient mobilization, as well as the competition between plant and microorganisms for nutrient uptake.