Engineering of an endopolygalacturonase by insertion of different Carbohydrate Binding Modules (CBMs): Chimerogenesis and proximity effects

Author(s)

Sibeli De Carli

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto (PCARP/BC)
• Defense date: 2020-12-10

Examining board members

Richard John Ward; André Ricardo de Lima Damasio; Adriana Grandis; Maria de Lourdes Teixeira de Moraes Polizeli

• Advisor: Richard John Ward

Abstract

The large biomass surplus from agribusiness activity can be used to produce bio-products with high added value. Strategies to improve utilization and reduce costs related to the enzymatic hydrolysis of these lignocellulosic residues include the identification and heterologous expression of enzymes with interesting catalytic properties, the use of protein engineering techniques, to improve the characteristics of the enzymes already identified and finally, the formulation of more efficient enzyme cocktails. The controlled deconstruction of the lignocellulosic matrix is technically challenging and constitutes major barrier for a reasonable production of bioproducts at attractive prices. It is known that the presence of pectins, one of the main components of the medium lamella in plants, hinders the hydrolysis of the plant cell wall and prevents the efficient release of sugars, so it is necessary to develop technologies to optimize the hydrolysis of this polysaccharide. The present work aimed to evaluate the biochemical and kinetic properties of an endopolygalacturonase from Chondrostereum purpureum (EndoPGCp) expressed in Pichia pastoris and Escherichia coli, as well as to evaluate the effect of immobilization on the activity of the enzyme. In addition, the use of chimerogenesis was proposed to evaluate the effect of the proximity of different polysaccharides present in the plant cell wall on the activity of an EndoPGCp_bac, engineered by the addition of carbohydrate binding modules (CBM), as well as the potential for application in the supplementation of a commercial enzymatic cocktail for hydrolysis of different lignocellulosic residues. The enzyme expressed in P. pastoris had a molecular mass of about 60.0 kDa, with an estimated carbohydrate content of 67.0% (w/w). The optimum temperature and pH of the catalysis were 60 - 70 ° C and pH 4.5, respectively. The enzyme was highly stable at pH 6.0 - 8.0 and retained about 60% of its initial activity after incubation at 70 ° C for 30 min. The enzyme hydrolyzed citric pectin with Vmax and KM of 4947.10 ± 393.63 U.mg-1 and 2.45 ± 0.23 mg.mL-1, respectively, and presented catalytic efficiency of 2052.90 ± 193.54 mL.mg-1.s-1. In addition, the enzyme was effective in clarifying apple and passion fruit juices. After immobilization on ferromagnetic nanoparticles, the enzyme showed no significant changes in its biochemical and kinetic characteristics and maintained approximately 65% of the initial activity after 7 cycles of reuse. In supplementing the commercial cocktail Celluclast, EndoPGCp_Pp increased the release of sugars reducing of orange residue by 1.8 times. In addition, the enzyme expressed in E. coli was refolded from the inclusion bodies and the process of chimerogenesis with CBMs did not affect the properties of the enzyme. The enzymes hydrolyzed the citrus pectin with very similar Vmax and KM. However, a large increase was observed in the kcat, since the chimeras 3N, 44N and 77N showed values 1.4, 1.6 and 1.3, times higher, respectively, than that obtained for the isolated catalytic domain. As a result, the catalytic efficiency (kcat/KM) for the hydrolysis of citrus pectin was about 60, 75 and 30% higher for chimeras 3N, 44N and 77N, respectively, compared to that obtained for EndoPGCp_bac. When applied in the supplementation of a commercial cocktail for hydrolysis of lignocellulosic residues, the results indicated that pectinases can improve the efficiency of the biomass saccharification by removing the pectin that prevent the action of other enzymes. In addition, the improved hydrolytic efficiency by chimeric enzymes may be related to the action of CBMs, which can greatly increase the concentration of the enzyme in the vicinity of the substrate, leading to an increase in pectin hydrolysis and, consequently, a decrease in the recalcitrance of the plant cell wall to enzyme hydrolysis. (AU)

• FAPESP's process: 17/13734-3 - Engineering of an endopolygalacturonase by insertion of different Carbohydrate Binding Modules (CBMs): chimerogenesis and proximity effects
• Grantee: Sibeli de Carli
• Support Opportunities: Scholarships in Brazil - Doctorate