Cell-surface Display Of Maltotriose-producing ?-amylase In Pichia Pastoris And The Application On The Synthesis Of Isomalto-oligosaccharides From Starch

With the development of society and economy,new products such as functional sugar prepared with starch as a substrate are very popular among consumers.Isomalto-oligosacc-harides?IMOs?are a kind of prebiotics,with low sweetness and low calories,high moisturizing properties.And it can be used by beneficial bacteria such as bifidobacterial to improve the intestinal microflora and so on.The long-chains of IMOs are better than short-chains.Therefore,it can be added to food,medicine and feed as food additives due to its many advantages.?-glucosidase is a key enzyme in the preparation of IMOs.The?-glucosidase from Aspergillus niger has specificity for short-chains substrates.And it has a higher k_cat/K_m value for maltotriose.So the maltotriose-producing?-amylase that specifically produces maltotriose is added to the saccharification reaction of starch to prepare IMOs.It can provide more maltotriose for?-glucosidase.In this study,we got the maltotriose-producing?-amylase gene from Thermobifida fusca NTU22 by gene synthesis after being optimized as the Pichia pastoris preferred codons.And we displayed maltotriose-producing?-amylase on the GS115 cell surface to get recombinant strain.Then we found that it had the ability to produce maltotriose.And we explored the enzymatic properties and reusability.Subsequently,the effect of whole-cell catalyst on producing maltotriose with maltodextrin as substrate was explored.At the same time,the cell-surface display maltotriose-producing?-amylase and?-glucosidase from Aspergillus niger were added the reaction system to prepare IMOs.Then we analyzed the effect for IMOs yield and component yield by adding maltotriose-producing?-amylase.The research content is listed below:?1?Construction of Pichia pastoris displaying Thermobifida fusca NTU22 maltotriose-producing?-amylase:Recombinant Pichia pastoris GS115/p PIC9K-tfa-flag-GCW61 were constructed using Pichia pastoris GPI wall protein GCW61 as the anchoring protein.The experiment result showed the display of foreign protein had no significant effect on the growth of the recombinant strain.The whole-cell catalyst was made by fermentation and lyophilization.And the enzyme activity reaches 260 U/g,The optimal temperature is 55?and the p H is 6.0.95%residual enzyme activity remains at 55°C for 1 h.It proves that the enzyme has good thermal stability.Preferably,the whole-cell catalyst still has 80%activity after being reused three times.When the substrate was 30%maltodextrin,it achieved 60.13%conversion rate of dextrin.The main component of the enzymatic hydrolysis is maltotriose,accounting for 50.24%.And the concentration of maltotriose is 90.62 g/L.?2?Cell-Surface display maltotriose-producing?-amylase involved in synthesis of IMOs:The cell-surface display maltotriose-producing?-amylase Pp-TFA-GCW61 participated in the reaction process of preparing IMOs from soluble starch.It is conducive to the increase of the IMOs yield.And the cell-surface display?-glucosidase Pp-ANGL-GCW?21+19?is higher than commercial?-glucosidase for the yield of IMOs under the experimental conditions of this study.It is conducive to the increase the yield of isomaltose,isomaltotriose and isomaltotetraose adding the maltotriose-producing?-amylase,especially for the commercial?-glucosidase.But it is not conducive to the accumulation of panose.?3?Preparation of IMOs in the 2 L reactor kettle:After the soluble starch was liquefied,add?-amylase,cell-surface display maltotriose-producing?-amylase Pp-TFA-GCW61 and cell-surface display?-glucosidase Pp-ANGL-GCW?21+19?to the reaction system.The highest conversion of IMOs achieves 48%,and the yield is 144 g/L.It has a greater conversion rate of IMOs than maltose as the substrate,and its components are more abundant.