Characterization and use of a recombinant CGTase to produce maltooligosaccharides from alternative flours for gluten-free bakeries

CAMINATA LANDRIEL, SOLEDAD; CASTILLO DE LAS MERCEDES, JULIETA; COSTA, HERNÁN

Workshop; Tercer encuentro de la Red Argentina de Tecnología Enzimática - Primer workshop RedTEz; 2021

Red Argentina de Tecnología Enzimática (RedTEz)

The enzyme cyclodextrin glucanotransferase (CGTase; EC 2.4.1.19), a member of the GH13 family, biotransforms starch into mixtures of cyclic and linear oligosaccharides, named cyclodextrins (CD) and maltooligosaccharides (MOS), respectively. These oligosaccharides, consisting of glucose residues linked by α-1,4 bonds, are used in different industries. In the bakery industry, MOS are used as hydrocolloids, since they delay the retrogradation of starch. This process occurs during the storage of the baked product and causes crumb hardening.Celiac disease is a chronic autoimmune enteropathy that prevents people from eating gluten. This protein is found in wheat, oat, barley, and rye flours. Therefore, these flours must be avoided in the preparation of bread suitable for people suffering from celiac disease. As an alternative, cassava, potato, rice, corn, quinoa, and amaranth flours, among others, can be used, but they lack the viscoelastic properties of gluten. The aim of this work was to characterize a recombinant CGTase and to evaluate its potential use to produce MOS from alternative flours for gluten-free bakeries. The CGTase from Paenibacillus barengoltzii was expressed in Pichia pastoris, a GRAS (Generally Recognized As Safe) yeast suitable for making products for human consumption.The recombinant enzyme was purified by affinity chromatography to a-CD. It showed an increased molecular mass due to N-glycosylations introduced by P.pastoris, as demonstrated by enzymatic treatment with PNGase F, followed by SDS-PAGE.The highest CD formation activity was obtained in 50 mM phosphate buffer, pH 6.0, with an optimum temperature between 50 and 60 ºC. The enzyme was stable for one hour at pH 6 to 11 and temperatures below 50 ºC. The Km and Vmax values did not show differences from wild-type values.To analyze enzyme microheterogeneity, a second purification step was performed by FPLC with a MonoQ 5/50 GL ion exchange column. Two fractions (A and B) with high amylolytic activity were obtained. Each fraction was analyzed by SDS-PAGE, Mass Spectrometry (MS), and EDMAN degradation before and after deglycosylation with endoglucanase Endo H. The same amino-terminal sequence was observed for each fraction, but they both differed in their molecular mass, 87286 Da (fraction A) and 84277 Da (fraction B), from the wild type enzyme (74470 Da). This increase suggests the presence of six to seven glycosylated sites with high mannose residues in each fraction.Finally, the production of MOS was determined by employing the affinity-purified recombinant CGTase on different gluten-free substrates. The highest production of MOS was obtained with starch from cassava, rice, corn, and potato.In sum, the recombinant CGTase obtained from P. pastoris shows microheterogeneity due to N-glycosylations introduced by the host. However, the enzyme is active over a wide range of pH and temperatures. The highest production of MOS, employed in the bakery industry to delay the retrogradation of the starch, is obtained when cassava, potato, corn, and rice flours are used as substrates of the recombinant enzyme. The potential application of this recombinant enzyme in the production of gluten-free bakeries shall be explored further.