GRASPs are proteins involved in cell processes that seem paradoxical: responsible for shaping the Golgi cisternae and involved in unconventional secretion mechanisms that bypass the Golgi. Despite its physiological relevance, there is still a considerable lack of studies on full-length GRASPs. Our group has previously reported an unexpected behavior of the full-length GRASP from the fungus C. neoformans: its intrinsically-disordered characteristic. Here, we generalize this finding by showing that it is also observed in the GRASP from S. cerevisae (Grh1), which strongly suggests it might be a general property within the GRASP family. Furthermore, Grh1 is also able to form amyloid-like fibrils either upon heating or when submitted to changes in the dielectric constant of its surroundings, a condition that is experienced by the protein when in close contact with membranes of cell compartments, such as the Golgi apparatus. Intrinsic disorder and fibril formation can thus be two structural properties exploited by GRASP during its functional cycle.

Background: The molecular basis of increased protein stability by N-glycosylation is incompletely understood. Results: Glycosylation position rather than number is more important for protein thermostability in the xylanase A from Bacillus subtilis. Conclusion: Glycans contribute both to stabilizing protein-glycan and less favorable glycan-glycan interactions. An extensive protein-glycan interface favors protein stability. Significance: Formation of a protein-glycan interface provides a conceptual framework to understand glycoprotein stabilization.

17GRASPs are proteins involved in cell processes that seem paradoxical, such as being responsible 18 for shaping the Golgi cisternae and also involved in unconventional secretion mechanisms that 19 bypass the Golgi, among other functions in the cell. Despite its involvement in several relevant 20 cell processes, there is still a considerable lack of studies on full-length GRASPs. Our group has 21 previously reported an unexpected behavior of the full-length GRASP from the fungus C. showing that is also observed in the GRASP from the yeast S. cerevisae (Grh1), which strongly 24 suggests it may be a general property within the GRASP family. Furthermore, Grh1 is also able

To the best of our knowledge, this is the first study focusing on understanding the disorder-to-order transitions of a molten globule structure without the need of any mild denaturing condition. A model is also introduced aiming at describing how the cell could manipulate the GRASP sensitivity to changes in the dielectric constant during different cell-cycle periods.

Enzyme reaction products and by-products from pretreatment steps can inhibit endoglucanases and are major factors limiting the efficiency of enzymatic lignocellulosic biomass hydrolysis. The gene encoding the endoglucanase from Scytalidium thermophilum (egst) was cloned and expressed as a soluble protein in Pichia pastoris GS115. The recombinant enzyme (Egst) was monomeric (66 kDa) and showed an estimated carbohydrate content of 53.3% (w/w). The optimum temperature and pH of catalysis were 60-70 °C and pH of 5.5, respectively. The enzyme was highly stable at pH 3.0-8.0 with a half-life in water of 100 min at 65 °C. The Egst presented good halotolerance, retaining 84.1 and 71.4% of the control activity in the presence of 0.5 and 2.0 mol L NaCl, respectively. Hydrolysis of medium viscosity carboxymethylcellulose (CMC) by Egst was stimulated 1.77-, 1.84-, 1.64-, and 1.8-fold by dithiothreitol, β-mercaptoethanol, cysteine, and manganese at 10, 10, 10, and 5 mmol L concentration, respectively. The enzyme hydrolyzed CMC with maximal velocity and an apparent affinity constant of 432.10 ± 16.76 and 10.5 ± 2.53 mg mL, respectively. Furthermore, the Egst was tolerant to reaction products and able to act on pretreated fractions sugarcane bagasse demonstrating excellent properties for application in the hydrolysis of lignocellulosic biomass.

Lipases (EC 3.1.1.3) comprise a biotechnologically important group of enzymes because they are able to catalyze both hydrolysis and synthesis reactions, depending on the amount of water in the system. One of the most interesting applications of lipase is in the biofuel industry for biodiesel production by oil and ethanol (or methanol) transesterification. Entomopathogenic fungi, which are potential source of lipases, are still poorly explored in biotechnological processes. The present work reports the heterologous expression and biochemical characterization of a novel Beauveria bassiana lipase with potential for biodiesel production. The His-tagged B. bassiana lipase A (BbLA) was produced in Komagataella pastoris in buffered methanol medium (BMM) induced with 1% methanol at 30°C. Purified BbLA was activated with 0.05% Triton X-100 and presented optimum activity at pH 6.0 and 50°C. N-glycosylation of the recombinant BbLA accounts for 31.5% of its molecular weight. Circular dichroism and molecular modeling confirmed a structure composed of α-helix and β-sheet, similar to α/β hydrolases. Immobilized BbLA was able to promote transesterification reactions in fish oil, demonstrating potential for biodiesel production. BbLA was successfully produced in K. pastoris and shows potential use for biodiesel production by the ethanolysis reaction.

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