Heterologous expression and biochemical characterization of a novel thermostable Sclerotinia sclerotiorum GH45 endoglucanase in Pichia pastoris.

Enzymatic saccharification of lignocellulosic biomass has been widely studied. Mainly endoglucanases were found to be a prerequisite for the quick initial biomass liquefaction. In the present study, Pichia pastoris was used as a host for the heterologous expression of a Sclerotinia sclerotiorum GH45 endoglucanase, Endo2. The recombinant plasmid pPICZαA was used to transform Pichia pastoris. Pichia culture supernatants expressing the recombinant Endo2 (rEndo2) were used for the purification and biochemical characterization of this enzyme. Therefore, rEndo2 was purified 6.7 fold to homogeneity with 34% yield and gave 19U/mg specific activity. It also showed maximum activity at pH 7.0 and 60°C (against pH 5.0 and 50°C for the native enzyme) and was thermostable at relatively high temperatures. Furthermore, rEndo2 retained its activity in a wide pH range (from 5 to 8). Besides, the recombinant endoglucanase was produced as an active 47kDa enzyme. This molecular weight differs from the one of the native enzyme (34kDa), which suggested a potential glycosylation of the recombinant enzyme. Moreover, rEndo2 was able to produce fermentable sugars after enzymatic assay on various cellulosic substrates with an interesting yield. Therefore, all these features offer prospects for large-scale production and industrial application of the recombinant endoglucanase.

Recombinant overexpression of camel hepcidin cDNA in Pichia pastoris: purification and characterization of the polyHis-tagged peptide HepcD-His.

Hepcidin, a liver-expressed antimicrobial peptide, has been demonstrated to act as an iron regulatory hormone as well as to exert a wide spectrum of antimicrobial activity. The aim of this work was the expression, as secreted peptide, purification, and characterization of a new recombinant polyHis-tagged camel hepcidin (HepcD-His) in yeast Pichia pastoris. The use of this eukaryotic expression system, for the production of HepcD-His, having 6 histidine residues at its C terminus, was simpler and more efficient compared with the use of the prokaryotic system Escherichia coli. Indeed, a single purification step was required to isolate the soluble hepcidin with purity estimated more that 94% and a yield of 2.8 against 0.2 mg/L for the E coli system. Matrix-assisted laser desorption/ionization time-of-flight (TOF)/TOF mass spectrometry of the purified HepcD-His showed 2 major peaks at m/z 4524.64 and 4634.56 corresponding to camel hepcidin with 39 and 40 amino acids. Evaluation of disulfide bond connectivity with the Ellman method showed an absence of free thiol groups, testifying that the 8 cysteine residues in the peptide are displayed, forming 4 disulfide bridges. Circular dichroism spectroscopy showed that camel hepcidin structure was significantly modified at high temperature of 90°C and returns to its original structure when incubation temperature drops back to 20°C. Interestingly, this peptide showed also a greater bactericidal activity, at low concentration of 9.5µM, against E coli, than the synthetic analog DH3. Thus, the production, at a large scale, of the recombinant camel hepcidin, HepcD-His, may be helpful for future therapeutic applications including bacterial infection diseases.

A Novel Three Domains Glycoside Hydrolase Family 3 from Sclerotinia sclerotiorum Exhibits ß-Glucosidase and Exoglucanase Activities: Molecular, Biochemical, and Transglycosylation Potential Analysis.

The filamentous fungus Sclerotinia sclerotiorum produces a complete set of cellulolytic enzymes. We report here the purification and the biochemical characterization of a new ß-glucosidase from S. sclerotiorum which belongs to the family 3 of glycoside hydrolases and that was named as SsBgl3. After two size-exclusion chromatography steps, purified protein bands of 80 and 90 kDa from SDS-PAGE were subjected to a mass spectrometry analysis. The results displayed four peptides from the upper band belonging to a polypeptide of 777 amino acids having a calculated molecular weight of 83.7 kDa. Biochemical analysis has been carried out to determine some properties. We showed that this SsBgl3 protein displayed both ß-glucosidase and exoglucanase activities with optimal activity at 55 °C and at pH 5. The transglycosylation activity was investigated using gluco-oligosaccharides TLC analysis. The molecular modeling and comparison with different crystal structures of ß-glucosidases showed that SsBgl3 putative protein present three domains. They correspond to an (α/ß)8 domain TIM barrel, a five-stranded α/ß sandwich domain (both of which are important for active-site organization), and a C-terminal fibronectin type III domain. Enzyme engineering will be soon investigated to identify the key residues for the catalytic reactions.

Biochemical characterization, molecular cloning, and structural modeling of an interesting ß-1,4-glucanase from Sclerotinia sclerotiorum.

The filamentous fungus Sclerotinia sclerotiorum produces a complete set of cellulolytic enzymes needed for efficient solubilization of native cellulose, the major component of plants. In this work, we reported the molecular characterization of an important glycosyl-hydrolase enzyme classified as endo-ß-1,4-glucanase. The importance of this enzyme was revealed with the in-gel activity staining, showing a high degradation capacity of cellulose. When purified from native gel and ran in denaturing polyacrylamide gel, the polypeptide has an apparent molecular mass of about 34 kDa called Endo2. For further characterization of this protein, a mass spectrometry approach was carried out. The LC-MS/MS analysis revealed two peptides belonging to this enzyme. The genomic DNA and cDNA sequences were resolved by PCR amplification and sequencing, revealing a gene with two intron sequences. The open reading frame of 987 bp encoded a putative polypeptide of 328 amino acids having a calculated molecular mass of 33,297 Da. Yet, the molecular modeling and comparative investigation of different 3D cellulase structures showed that this endoglucanase isoform has probably two domains. A core domain having a high similarity with endoglucanases family 5 and a cellulose-binding domain having similarities with those of exo-type cellulases of family 1, linked together by a serine-threonine-rich region. These results are with great interests and show new characteristics of S. sclerotiorum glucanase.