Trichoderma viride cellulase induces resistance to the antibiotic pore-forming peptide alamethicin associated with changes in the plasma membrane lipid composition of tobacco BY-2 cells

<p>Abstract</p> <p>Background</p> <p>Alamethicin is a membrane-active peptide isolated from the beneficial root-colonising fungus <it>Trichoderma viride</it>. This peptide can insert into membranes to form voltage-dependent pores. We have previously shown that alamethicin efficiently permeabilises the plasma membrane, mitochondria and plastids of cultured plant cells. In the present investigation, tobacco cells (<it>Nicotiana tabacum </it>L. cv Bright Yellow-2) were pre-treated with elicitors of defence responses to study whether this would affect permeabilisation.</p> <p>Results</p> <p>Oxygen consumption experiments showed that added cellulase, already upon a limited cell wall digestion, induced a cellular resistance to alamethicin permeabilisation. This effect could not be elicited by xylanase or bacterial elicitors such as flg22 or elf18. The induction of alamethicin resistance was independent of novel protein synthesis. Also, the permeabilisation was unaffected by the membrane-depolarising agent FCCP. As judged by lipid analyses, isolated plasma membranes from cellulase-pretreated tobacco cells contained less negatively charged phospholipids (PS and PI), yet higher ratios of membrane lipid fatty acid to sterol and to protein, as compared to control membranes.</p> <p>Conclusion</p> <p>We suggest that altered membrane lipid composition as induced by cellulase activity may render the cells resistant to alamethicin. This induced resistance could reflect a natural process where the plant cells alter their sensitivity to membrane pore-forming agents secreted by <it>Trichoderma spp</it>. to attack other microorganisms, and thus adding to the beneficial effect that <it>Trichoderma </it>has for plant root growth. Furthermore, our data extends previous reports on artificial membranes on the importance of lipid packing and charge for alamethicin permeabilisation to <it>in vivo </it>conditions.</p

Recombinant production and characterization of six novel GH27 and GH36 alpha-galactosidases from Penicillium subrubescens and their synergism with a commercial mannanase during the hydrolysis of lignocellulosic biomass

alpha-Galactosidases are important industrial enzymes for hemicellulosic biomass degradation or modification. In this study, six novel extracellular alpha-galactosidases from Penicillium subrubescens were produced in Pichia pastoris and characterized. All alpha-galactosidases exhibited high affinity to pNP alpha Gal, and only AglE was not active towards galacto-oligomers. Especially AglB and AglD released high amounts of galactose from guar gum, carob galactomannan and locust bean, but combining alpha-galactosidases with an endomannanase dramatically improved galactose release. Structural comparisons to other alpha-galactosidases and homology modelling showed high sequence similarities, albeit significant differences in mechanisms of productive binding, including discrimination between various galactosides. To our knowledge, this is the first study of such an extensive repertoire of extracellular fungal alpha-galactosidases, to demonstrate their potential for degradation of galactomannan-rich biomass. These findings contribute to understanding the differences within glycoside hydrolase families, to facilitate the development of new strategies to generate tailor-made enzymes for new industrial bioprocesses.Peer reviewe

Boosting of enzymatic softwood saccharification by fungal GH5 and GH26 endomannanases

Background: Softwood is a promising feedstock for lignocellulosic biorefineries, but as it contains galactoglucomannan efficient mannan-degrading enzymes are required to unlock its full potential. Results: Boosting of the saccharification of pretreated softwood (Canadian lodgepole pine) was investigated for 10 fungal endo-β(1→4)-mannanases (endomannanases) from GH5 and GH26, including 6 novel GH26 enzymes. The endomannanases from Trichoderma reesei (TresMan5A) and Podospora anserina (PansMan26) were investigated with and without their carbohydrate-binding module (CBM). The pH optimum and initial rates of enzyme catalysed hydrolysis were determined on pure β-mannans, including acetylated and deacetylated spruce galactoglucomannan. Melting temperature (Tm) and stability of the endomannanases during prolonged incubations were also assessed. The highest initial rates on the pure mannans were attained by GH26 endomannanases. Acetylation tended to decrease the enzymatic rates to different extents depending on the enzyme. Despite exhibiting low rates on the pure mannan substrates, TresMan5A with CBM1 catalysed highest release among the endomannanases of both mannose and glucose during softwood saccharification. The presence of the CBM1 as well as the catalytic capability of the TresMan5A core module itself seemed to allow fast and more profound degradation of portions of the mannan that led to better cellulose degradation. In contrast, the presence of the CBM35 did not change the performance of PansMan26 in softwood saccharification. Conclusions: This study identified TresMan5A as the best endomannanase for increasing cellulase catalysed glucose release from softwood. Except for the superior performance of TresMan5A, the fungal GH5 and GH26 endomannanases generally performed on par on the lignocellulosic matrix. The work also illustrated the importance of using genuine lignocellulosic substrates rather than simple model substrates when selecting enzymes for industrial biomass applications

The GH5 1,4-β-mannanase from <i>Bifidobacterium animalis</i> subsp. <i>lactis </i>Bl-04 possesses a low-affinity mannan-binding module and highlights the diversity of mannanolytic enzymes

β-Mannans are abundant and diverse plant structural and storage polysaccharides. Certain human gut microbiota members including health-promoting Bifidobacterium spp. catabolize dietary mannans. Little insight is available on the enzymology of mannan deconstruction in the gut ecological niche. Here, we report the biochemical properties of the first family 5 subfamily 8 glycoside hydrolase (GH5_8) mannanase from the probiotic bacterium Bifidobacterium animalis subsp. lactis Bl-04 (BlMan5_8)

Crystal structure and substrate interactions of an unusual fungal non-CBM carrying GH26 endo-β-mannanase from Yunnania penicillata

Endo-β(1 → 4)-mannanases (endomannanases) catalyse degradation of β-mannans, an abundant class of plant polysaccharides. This study investigates structural features and substrate binding of YpenMan26A, a non-CBM carrying endomannanase from Yunnania penicillata. Structural and sequence comparisons to other fungal family GH26 endomannanases showed high sequence similarities and conserved binding residues, indicating that fungal GH26 endomannanases accommodate galactopyranosyl units in the −3 and −2 subsites. Two striking amino acid differences in the active site were found when the YpenMan26A structure was compared to a homology model of Wsp.Man26A from Westerdykella sp. and the sequences of nine other fungal GH26 endomannanases. Two YpenMan26A mutants, W110H and D37T, inspired by differences observed in Wsp.Man26A, produced a shift in how mannopentaose bound across the active site cleft and a decreased affinity for galactose in the −2 subsite, respectively, compared to YpenMan26A. YpenMan26A was moreover found to have a flexible surface loop in the position where PansMan26A from Podospora anserina has an α-helix (α9) which interacts with its family 35 CBM. Sequence alignment inferred that the core structure of fungal GH26 endomannanases differ depending on the natural presence of this type of CBM. These new findings have implications for selecting and optimising these enzymes for galactomannandegradation

β-Mannanase BoMan26B from Bacteroides ovatus produces mannan-oligosaccharides with prebiotic potential from galactomannan and softwood β-mannans

Galactomannan (GM) in legumes and acetyl-galactoglucomannan (AcGGM) in softwoods are wide-spread β-mannans. Their depolymerisation is catalyzed by β-mannanases. We have investigated a cell-surface exposed and galactose-tolerant β-mannanase (BoMan26B) from the abundant gut bacterium Bacteroides ovatus. Glycosidases from the gut microbiota have potential for production of prebiotics, such as dietary saccharides that would promote beneficial bacteria in the gut. BoMan26B was explored for production of potential prebiotics. Using the above β-mannans as substrate we investigated the product profiles using a herein developed new high-resolution anion-exchange chromatography procedure. The produced linear and galactosyl-decorated β-mannan-oligosaccharides (MOS/GMOS) were mainly of degree of polymerization (DP) 2–6, consistent with the glycan-binding subsites of BoMan26B. Some GM and AcGGM products were acetylated. DP 2–6 MOS were produced at a yield of 30 and 33% (w/w) from GM and AcGGM, respectively. In addition, about as much DP 2–6 GMOS were produced, assessed using guar α-galactosidase as analytical aid. Growth studies using the human gut bacteria Bifidobacterium adolescentis ATCC 15703 (acetate producer) and Roseburia hominis DSMZ 6839 (butyrate producer) revealed significant differences in utilization of specific MOS/GMOS. The prebiotic potential of the MOS/GMOS generated by BoMan26B was further underlined by the observation that both bacterial strains produced short-chain fatty acids