Cell-surface Display Of α-glucosidase From Aspergillus Niger In Pichia Pastoris And The Primary Application On The Synthesis Of Isomalto-oligosaccharides

Isomaltooligosaccharide(IMO),a kind of prebiotics,is widely used in health care products,food and feed industry.IMO cannot be digest by yeast and lactobacillus,but is beneficial to intestinal bifidobacterium proliferation.At present,catalytic synthesis of high concentration IMO with maltose by alpha-glycosidase is the most commonly used method in industry.The method was originated in Japan and Aspergillus niger alpha-glycosidase(ANGL)was gained extensive attention because of high yield even in low substrate concentration situation.Pichia pastoris surface display system is a eukaryotic expression system to immobilize enzyme by cell.After inducing expression,cells of Pichia pastoris recombinant strains,which displaying enzyme,can be made into whole-cell catalyst prepared for catalytic reaction.In this study,we got the alpha-glucosidase gene(angl)from Aspergillus niger by gene synthesis after being optimized as the Pichia pastoris preferred codons.And we displayed ANGL on the GS115 cell surface by using the GPI-modified cell wall protein Gcwnp(n=12,19,21,49,61)successfully.Then we explored the synthesis of IMO using whole-cell catalyst in stirred tank reactor.The research content is listed below:(1)Construction of Pichia pastoris displaying Aspergillus niger alpha-glucosidase(ANGL)Recombinant vectors p KANGL-GCWn(n=12,19,21,49,61)and recombinant Pichia pastoris GS115/p KANGL-GCWn(n=12,19,21,49,61)were constructed.Growth curve comparison found that the foreign proteins have no obvious effects on the growth of recombinant strains.Enzyme activity assay revealed that ANGL was successfully displayed on the cell surface.The highest synthesis activity of ANGL displayed on the cell surface were found in the recombinants based on Gcw61 p,Gcw19p,reaching 54 U/g and 48 U/g,respectively。Then,we also tried to construction the co-displaying system by using two different selection marker in Pichia pastoris.We gained 3 strain based on different anchoring proteins harboring 2 copies,and the GS115/p ZANGL-GCW(21+19)had the highest synthesis activity,reaching 75 U/g.(2)2-mL system single factor optimization on IMO synthesis by Pp-ANGL-GCW61The whole-cell catalyst Pp-ANGL-GCW61 was obtained after 120 h induction of methanol.In this study,the p H,temperature,substrate concentration and whole-cell catalyst content of the catalytic reaction were optimized when using maltose as substrate.The optimized 2 ml reaction system: 62.5 mg/ml Pp-ANGL-GCW61,300 mg/ml maltose,2 ml 0.04 M Britton Robinson buffer(p H 4.0),55 ? C,200 rmp.And the yield of IMO is about 45% at 2 h.(3)reused of Pp-ANGL-GCW61 in 2-L reaction systemWe reued Pp-ANGL-GCW61 to produce IMO in 2-m L and 2-L reaction system.The whole-cell catalyst was reused 3 times.Along with increased reused times,the reaction time to reach the highest conversion rate was extended.The yield of IMO is about 30% at 5 h as the enzyme is use for the third time.No significant decline in conversion rate curve by recycling,these result indicated that whole-cell biocatalyst can be reused in reactor on IMO synthesis.