Engineering the affinity of a family 11 carbohydrate binding module to improve binding of branched over unbranched polysaccharides.

Carbohydrate binding modules (CBMs) are non-catalytic domains within larger multidomain polypeptides. The CelH from Ruminoclostridium (Clostridium) thermocellum contains a family 11 CBM (RtCBM11) with high binding affinity for the linear polysaccharide ß-glucan, and low affinity for the branched xyloglucan. Screening a random RtCBM11 mutant phage library created by error prone PCR for xyloglucan binding identified RtCBM11 mutants with enhanced xyloglucan affinity. Subsequent recombination of the selected variants by site-directed mutagenesis generated the H102L/Y152F and Y46N/G52D/H102L/Y152F mutants. Fusion of the quadruple RtCBM11 mutant with the xyloglucanase from Aspergillus niveus increased the catalytic efficiency of the enzyme by 38%. Isothermal titration calorimetry demonstrated increased xyloglucan affinity for both mutants and reduced affinity for ß-glucan in the H102L/Y152F mutant. Molecular dynamics simulations indicated that the increased xyloglucan specificity results both from formation of a xylosyl binding pocket in the carbohydrate binding cleft, and via modulation of a hydrogen bond network between the oligosaccharide ligand and the protein. These results explain the improved xyloglucan binding in the RtCBM11 H102L/Y152F mutant and advance the understanding of the structural determinants of CBMs binding that discriminate between branched and unbranched polysaccharides.

Disorder-to-order transitions in the molten globule-like Golgi Reassembly and Stacking Protein.

BACKGROUND: Golgi Reassembly and Stacking Proteins (GRASPs) are widely spread among eukaryotic cells (except plants) and are considered as key components in both the stacking of the Golgi cisternae and its lateral connection. Furthermore, GRASPs were also proved essential in the unconventional secretion pathway of several proteins, even though the mechanism remains obscure. It was previously observed that the GRASP homologue in Cryptococcus neoformans has a molten globule-like behavior in solution. METHODS: We used circular dichroism, synchrotron radiation circular dichroism and steady-state as well as time-resolved fluorescence. RESULTS: We report the disorder-to-order transition propensities for a native molten globule-like protein in the presence of different mimetics of cell conditions. Changes in the dielectric constant (such as those experienced close to the membrane surface) seem to be the major factor in inducing multiple disorder-to-order transitions in GRASP, which shows very distinct behavior when in conditions that mimic the vicinity of the membrane surface as compared to those found when free in solution. Other folding factors such as molecular crowding, counter ions, pH and phosphorylation exhibit lower or no effect on GRASP secondary structure and/or stability. GENERAL SIGNIFICANCE: 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.

Overexpression of a Cellobiose-Glucose-Halotolerant Endoglucanase from Scytalidium thermophilum.

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-1 NaCl, respectively. Hydrolysis of medium viscosity carboxymethylcellulose (CMC) by Egst was stimulated 1.77-, 1.84-, 1.64-, and 1.8-fold by dithiothreitol, ß-mercaptoethanol, cysteine, and manganese at 10, 10, 10, and 5 mmol L-1 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-1, 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.

Lignocellulose binding of a Cel5A-RtCBM11 chimera with enhanced ß-glucanase activity monitored by electron paramagnetic resonance.

BACKGROUND: The Bacillus subtilis endo-ß-1,4-glucanase (BsCel5A) hydrolyzes ß-1,3-1,4-linked glucan, and the enzyme includes a family 3 carbohydrate-binding module (CBM3) that binds ß-1,4-linked glucan. METHODS: Here we investigate the BsCel5A ß-1,3-1,4 glucanase activity after exchanging the CBM3 domain for the family 11 CBM from Ruminiclostridium thermocellum celH (RtCBM11) having ß-1,3-1,4 glucan affinity. RESULTS: The BsCel5A-RtCBM11 presents a 50.4% increase in Vmax, a 10% reduction in K0.5, and a 2.1-fold increase in catalytic efficiency. Enzyme mobility and binding to barley ß-1,3-1,4 glucan and pre-treated sugarcane bagasse were investigated using Electron Paramagnetic Resonance (EPR) with Site-Directed Spin Labeling (SDSL) of the binding site regions of the CBM3 and RtCBM11 domains in the BsCel5A-CBM3 and BsCel5A-RtCBM11, respectively. Although higher mobility than the RtCBM11 was shown, no interaction of the spin-labeled CBM3 with ß-1,3-1,4 glucan was observed. In contrast, a Ka value of 0.22 mg/mL was estimated from titration of the BsCel5A-RtCBM11 with ß-1,3-1,4 glucan. Enzyme binding as inferred from altered EPR spectra of the BsCel5A-RtCBM11 was observed only after xylan or lignin extraction from sugarcane bagasse. Binding to xylan- or lignin-free lignocellulose was correlated with a 4.5- to 5-fold increase in total reducing sugar release as compared to the milled intact sugarcane bagasse, suggesting that xylan impedes enzyme access to the ß-1,3-1,4 glucan. CONCLUSIONS: These results show that the non-specific binding of the BsCel5A-RtCBM11 to the lignin component of the cell wall is minimal, and represent the first reported use of EPR to directly study the interaction of glycoside hydrolyse enzymes with natural insoluble substrates.

Lipid membranes and acyl-CoA esters promote opposing effects on acyl-CoA binding protein structure and stability.

Acyl-CoA Binding Proteins (ACBP) form a housekeeping family of proteins that is responsible for the buffering of long chain acyl-coenzyme A esters (LCFA-CoA) inside the cell. Even though numerous studies have focused on the characterization of different members of the ACBP family, the knowledge about the impact of both LCFA-CoA and phospholipids on ACBP structure and stability remains scarce. Besides, there are still controversies regarding the possible interaction of ACBP with biological membranes, even though this might be essential for the cargo capture and delivery. In this study, we observed that LCFA-CoA and phospholipids play opposite roles on protein stability and that the interaction with the membrane is dictated by electrostatic interaction. Furthermore, the results support the hypothesis that the LCFA-CoA delivery is driven by the increase of the negative charge on the membrane surface. The combined influence played by the different molecules on ACBP structure is discussed on the light of cargo capture/delivery giving new insights about this important process.

Thermostabilization of Bacillus subtilis GH11 xylanase by surface charge engineering.

Aiming to improve thermostability of the mesophilic xylanase A from Bacillus subtilis (XynA), five single mutants (S22E, S27E, N32D, N54E and N181R) were used to construct a random combinatorial library, and screening of this library for thermostable XynA variants identified a double mutant (S22E/N32D). All 6 mutants were expressed in Escherichia coli (BL21) and purified. Xylanase activity showed all mutants have an optimum catalytic temperature (Topt) of 55°C, and with the exception of the S27E mutant, a higher specific activity than the wild-type XynA. The time for loss of 50% activity at 55°C (t50) decreased in the order S22E/N32D>N181R>S22E>Wild-type>S27E=N32D≈N54E. The values of the van't Hoff denaturation enthalpy change (ΔHND), melting temperature (Tm) and heat capacity at constant pressure (ΔCp) between the native and denatured states were estimated from thermal denaturation curves monitored by circular dichroism ellipticity changes. The decreasing order of Gibbs free energy change at 328K (ΔG328) S22E/N32D>N181R>S22E>Wild-type>S27E≈N54E>N32D correlates well with the thermotolerance results, and is dominated by changes in ΔHND which is consistent with increased in hydrogen bonding in the thermostable mutants.

Beauveria bassiana Lipase A expressed in Komagataella (Pichia) pastoris with potential for biodiesel catalysis.

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 ß-sheet, similar to α/ß 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.

Biochemical properties of glycosylation and characterization of a histidine acid phosphatase (phytase) expressed in Pichia pastoris.

Phytases catalyze the cleavage of phosphate groups from phytic acid. Here, we have studied the effects of glycosylation on the properties of Aspergillus japonicus C03 phytase expressed in Pichia pastoris. The enzyme ORF of 1338 nucleotides was cloned from genomic DNA, and encoded a secreted mature protein of 446 amino acids, which included the sequence motif RHGXRX and dipeptide HD, classifying the phytase as a histidine acid phosphate. After transformation and 72h of induction, P.pastoris GS115 expressed a 75kDa protein showing 526U/mg phytase activity and 143mg/L of protein. The amino acid sequence showed 8 and 3 potential N- and O-glycosylation sites, respectively. Analysis by ESMS showed two glycoform masses of 75,467 and 72,793, which after deglycosylation decreased to 54,327 and 54,128, respectively, indicating a carbohydrate content of 27-30%. A single GlcNAc was assigned at Asn6, Asn38, Asn84, Asn99, Asn209, Asn218, Asn355 and Asn367. The recombinant phytase showed maximum activity at 50°C, a half-life of 40min, and farUVCD spectroscopy indicated a secondary structure rich in α-helix. Thermal denaturation analyses reveal the melting temperature varied from 50°C at pH 6 to a maximum of 66°C at pH 3 and pH 4.

Engineering the pattern of protein glycosylation modulates the thermostability of a GH11 xylanase.

Protein glycosylation is a common post-translational modification, the effect of which on protein conformational and stability is incompletely understood. Here we have investigated the effects of glycosylation on the thermostability of Bacillus subtilis xylanase A (XynA) expressed in Pichia pastoris. Intact mass analysis of the heterologous wild-type XynA revealed two, three, or four Hex(8-16)GlcNAc2 modifications involving asparagine residues at positions 20, 25, 141, and 181. Molecular dynamics (MD) simulations of the XynA modified with various combinations of branched Hex9GlcNAc2 at these positions indicated a significant contribution from protein-glycan interactions to the overall energy of the glycoproteins. The effect of glycan content and glycosylation position on protein stability was evaluated by combinatorial mutagenesis of all six potential N-glycosylation sites. The majority of glycosylated enzymes expressed in P. pastoris presented increased thermostability in comparison with their unglycosylated counterparts expressed in Escherichia coli. Steric effects of multiple glycosylation events were apparent, and glycosylation position rather than the number of glycosylation events determined increases in thermostability. The MD simulations also indicated that clustered glycan chains tended to favor less stabilizing glycan-glycan interactions, whereas more dispersed glycosylation patterns favored stabilizing protein-glycan interactions.

The bactericidal effect of human secreted group IID phospholipase A2 results from both hydrolytic and non-hydrolytic activities.

The Human Secreted Group IID Phospholipase A(2) (hsPLA2GIID) may be involved in the human acute immune response. Here we have demonstrated that the hsPLA2GIID presents bactericidal and Ca(2+)-independent liposome membrane-damaging activities and we have compared these effects with the catalytic activity of active-site mutants of the protein. All mutants showed reduced hydrolytic activity against DOPC:DOPG liposome membranes, however bactericidal effects against Escherichia coli and Micrococcus luteus were less affected, with the D49K mutant retaining 30% killing of the Gram-negative bacteria at a concentration of 10µg/mL despite the absence of catalytic activity. The H48Q mutant maintained Ca(2+)-independent membrane-damaging activity whereas the G30S and D49K mutants were approximately 50% of the wild-type protein, demonstrating that phospholipid bilayer permeabilization by the hsPLA2GIID is independent of catalytic activity. We suggest that this Ca(2+)-independent damaging activity may play a role in the bactericidal function of the protein.