Immobilization Of Pichia Pastoris Surface Displayed With ?-glucosidase And Its Application In The Synthesis Of Isomaltooligosaccharides

As a kind of functional food additive,oligomeric malto-oligosaccharide(IMO)has for decades gained special interest in the scientific consortium as well as among consumers.Pichia pastoris yeast cell surface dispaly technology is the enzyme immobilized on the surface of Pichia pastoris to improve the stability and reusability of the enzyme.Enzyme displaying-Pichia pastoris as a catalyst,continuous synthesis IMO in a packed-bed bioreactor,Pichia uniformly dispersed in the reaction solution,make the continuous operation impossible.Therefore further immobilization of the ?-glucosidase displaying-Pichia pastoris is necessary to accelerate the rate of Pichia pastoris settling.In this paper,?-glucosidase from Aspergillus niger was used to catalytic maltose substrate into IMO.Utilizing the high paramagneticity of ferriferrous oxide,chitosan is encapsulated on Fe3O4 microspheres as a carrier for immobilizing Pichia cells.In this paper,chitosan coated magnetic microspheres that treated by glutaraldehyde solution is used for the immobilization of ?-glucosidase displaying-Pichia pastoris cells,and the nature of the immobilized bacteria and catalytic effect were studied.The research contents and results of this research are as follows:(1)The Pichia pastoris that surface displayed with ?-glucosidase(I-Pp-ANGL-GCW61)was immobilized by calcium alginate embedding and glutaraldehyde cross-linking method,respectively,and the I-Pp-ANGL-GCW61 was following used in continous synthesis of IMO in packed-bed bioreactor.When using calcium alginate embedding as the carrier,substrate residence time was 28.26 min,the conversion rate of IMO was 6%(w/w)in packed-bed bioreactor.The cross-linking of Pichia pastoris with glutaraldehyde resulted in 21%(w/w)conversion of IMO in the packed-bed bioreactor and a 70% decrease in glycoside activity of ?-glucosidase.(2)The aldehyde group modified chitosan magnetic microsphere carrier were used to immobilize I-Pp-ANGL-GCW61,The synthesis activity of IMO of freeze-dried I-Pp-ANGL-GCW61 was 43.3 U,which was slightly lower than that of free yeast cells(Pp-ANGL-GCW61),about 85% of the synthesis activity was retained.The thermal stability of the I-Pp-ANGL-GCW61 was good,and the half-life of the synthesis activity was more than 36 h at 55 ?.The immobilization amounts of the Pp-ANGL-GCW61 was optimized.The optimum immobilization amount of the Pp-ANGL-GCW61 was 5.863 g per gram of freeze-dried magnetic microspheres,and the Pp-ANGL-GCW61 immobilization efficiency were 51.83%.(3)In the 10-m L reaction bottle,the effect of reuse-times of immobilized cells on the synthesis activity was studied.After five times of re-use,the Pp-ANGL-GCW61 synthesis activity have show 80% matained compared to the first time.(4)Freeze-dried I-Pp-ANGL-GCW61 were filled in a packed-bed bioreactor to continuous synthesize isomaltooligosaccharides(IMO).The residence time of the substrate was optimized in the packed-bed bioreactor,and the maximum yield of IMO was 12.12 g/h when the residence time was 36.42 min,with a conversion rate of the substrate 36.69%.Compared with the IMO production efficiency of 35.8 g/L/h in the batch reactor,the IMO production efficiency in the packed-bed increased by 19.83% to 42.9 g/L/h.