Fungus Hypocrea jecorina – teleomorph of Trichoderma reesei belongs to the saprotrophic filamentous fungi. The representatives of Hypocrea are known primarily by production of different cellulases and hemicellulases which are currently used in various fields of industry, mainly in paper processing and feed and food productions. As a part of the enzymatic cocktail secreted by H. jecorina during its growth on cellulose, also acetyl esterase was identified. Later it was classified as a first member of newly organized carbohydrate esterase family CE16 (CAZy classification). The enzyme is not capable of deacetylation of polymeric substrates as acetyl glucuronoxylan, but efficiently deacetylates terminally acetylated xylooligosaccharides [1] and shows different positional specificity on mono acetates, e.g. 4-nitrophenyl β-D-xylopyranoside [2]. Further studies showed that the acetyl esterase is crucial for complete deacetylation of naturally acetylated xylans enabling their saccharification by xylanases. To study the relationship between structure and function of acetyl esterase, highly purified recombinant enzyme produced by Trichoderma reesei Rut C-30 was prepared and crystallized. The enzyme is composed of 348 amino acid residues from which the first 19 form a secretion signal peptide. It was proved that the enzyme is glycosylated. The enzyme crystallized very easily, however, the crystals did not diffract. Later, after optimization, also diffracting crystals were obtained and a complete set of diffraction data was collected to 3.98 Å. After crystal annealing another data set was collected to 2.98 Å resolution using synchrotron radiation source at EMBL, DESY, Hamburg. Unfortunately, structure solution by molecular replacement method failed as there is no suitable model structure in PDB. Isomorphous crystals prepared by the native crystal soaking in the solution containing NaBr did not diffract at all. To make the future structure-function study of the enzyme easier, recombinant non-glycosylated enzyme should be prepared. For this purposes synthetic gene optimized for expression in E.coli was designed and synthesized.
The first non-glycosylated acetyl esterase obtained by the expression of its synthetic gene in E.coli cells was mostly insoluble or aggregated; moreover, even the soluble part was not able to bind to Ni-column. Conditions of cell cultivation, induction of gene expression, cells sonication and protein purification using metallo-chelate and size exclusion chromatography were necessary to optimized. At present, after optimization of all steps, the non-glycosylated recombinant CE16 acetyl esterase was prepared in the soluble and active form. All the results were confirmed by SDS-PAGE analysis, analytical FPLC, DLS study and by activity measurement. Preparation of the enzyme in the amounts sufficient for crystallization is under way.
Besides this, bioinformatic studies of the Hypocrea jecorina acetyl esterase as well as all other CE16 members (153 enzymes to this date) were performed. Bioinformatic studies proved that CE16 family members belong to the group of serine hydrolases, more exactly to the group of SGNH lipases/acetyl esterases [3]. They comprise highly conserved GDSL/GDSY motif, typical signature of many esterases and lipases. Alignment of amino-acid sequences showed the most conserved regions, mainly amino-acids of catalytic triad, Ser18, Asp295 and His298, and oxyanion hole, Gly92, Asn148 (part of binding site, numbering according to recombinant acetyl esterase). Also other conserved amino acids were identified; mainly tryptophans and phenylalanines which might be important for substrate binding. These assumptions have to be experimentally confirmed.
The work was supported by the Faculty of Natural Sciences of UCM under the contract No. FPPV-27-2015 and by the Slovak Research and Development Agency grant No. APVV-0602-12.