Abstract: Isomalto-oligosaccharieds (IMOs) are a kind of oligosaccharides with α-1,6 glycosidic linkages, mainly including isomaltose, isomaltotriose (IG3) and panose, and they have been widely studied and applied because of the probiotic characteristics such as enhancing human immunity, preventing dental caries, etc. It was reported that IG3 in IMOs mainly contributed to the promotion of Bifidobacterium proliferation. However, due to the low production yield and low probiotics content of traditional preparation process, there is still a bottleneck in the efficient production of IMOs. To enhance IMOs production and increase IG3 proportion, we proposed a two-step method to produce IMOs, which combined dextran synthesis by 4,6-α-glycosyltransferase (4,6-α-GT, LfGT) from Lactobacillus fermentum with the following dextran degradation by isomaltotrio-dextranase (BfIMTD) from Brevibacterium fuscum. Under the condition of 37 ℃ and pH 6.0 for 24 h, using LfGT with pullulanase and isopullulanase, could produce dextran with 66.3% yield from 200 g/L maltodextrins, and the α-1,6 glycosidic linkage ratio of the generated dextran was up to 99.3%. Then 98.3 g/L IG3 was finally produced (65.2% from maltodextrins) when BfIMTD reacted with dextran (100 g/L final concentration) at 40 ℃ and pH of 7.5 for further 10 h. Therefore, this study suggests that the above two-step process can provide not only a new insight into the production of IMOs, but also an innovative strategy for the length controllability of IMOs.
 王良东. 低聚异麦芽糖性质、功能、 生产和应用[J].粮食与油脂, 2008,21(4):43-47. WANG L D.The properties, functions, production and utilizations of isomaltooligosacharide[J].Journal of Cereals and Oils, 2008,21(4):43-47.  SORNDECH W, NAKORN K N, TONGTA S, et al.Isomalto-oligosaccharides:Recent insights in production technology and their use for food and medical applications[J].LWT, 2018, 95:135-142.  KANEKO T, KOHMOTO T, KIKUCHI H, et al.Effects of isomaltooligosaccharides with different degrees of polymerization on human fecal bifidobacteria[J].Bioscience, Biotechnology and Biochemistry, 1994, 58(12):2 288-2 290.  KOHMOTO T, FUKUI F, TAKAKU H, et al.Effect of isomalto-oligosaccharides on human fecal flora[J].Bifidobacteria and Microflora, 1988, 7(2):61-69.  WU Q Q, PI X E, LIU W, et al.Fermentation properties of isomaltooligosaccharides are affected by human fecal enterotypes[J].Anaerobe, 2017, 48:206-214.  SORNDECH W, SAGNELLI D, BLENNOW A, et al.Combination of amylase and transferase catalysis to improve IMO compositions and productivity[J].LWT-Food Science and Technology, 2017, 79:479-486.  LEE H S, AUH J H, YOON H G, et al.Cooperative action of α-glucanotransferase and maltogenic amylase for an improved process of isomaltooligosaccharide (IMO) production[J].Journal of Agricultural and Food Chemistry, 2002, 50(10):2 812-2 817.  GAN W W, ZHANG H B, ZHANG Y Q, et al.Biosynthesis of oligodextrans with different Mw by synergistic catalysis of dextransucrase and dextranase[J].Carbohydrate Polymers, 2014, 112:387-395.  SHUKLA S, SHI Q, MAINA N H, et al.Weissella confusa Cab3 dextransucrase:Properties and in vitro synthesis of dextran and glucooligosaccharides[J].Carbohydrate Polymers, 2014, 101:554-564.  SONG J Y, KIM Y M, LEE B H, et al.Increasing the dietary fiber contents in isomaltooligosaccharides by dextransucrase reaction with sucrose as a glucosyl donor[J].Carbohydrate Polymers, 2020, 230:115 607.  HUANG S X, HOU D Z, QI P X, et al.Enzymatic synthesis of non-digestible oligosaccharide catalyzed by dextransucrase and dextranase from maltose acceptor reaction[J].Biochemical and Biophysical Research Communications, 2020, 523(3):651-657.  GOULAS A K, FISHER D A, GRIMBLE G K, et al.Synthesis of isomaltooligosaccharides and oligodextrans by the combined use of dextransucrase and dextranase[J].Enzyme and Microbial Technology, 2004, 35(4):327-338.  KRALJ S, GRIJPSTRA P, VAN LEEUWEN S S, et al.4,6-α-glucanotransferase, a novel enzyme that structurally and functionally provides an evolutionary link between glycoside hydrolase enzyme families 13 and 70[J].Applied and Environmental Microbiology, 2011, 77(22):8 154-8 163.  BAI Y X, GANGOITI J, DIJKSTRA B W, et al.Crystal structure of 4,6-α-glucanotransferase supports diet-driven evolution of GH70 enzymes from α-amylases in oral bacteria[J].Structure, 2017, 25(2):231-242.  ARDÈVOL A, ROVIRA C.Reaction mechanisms in carbohydrate-active enzymes:Glycoside hydrolases and glycosyltransferases.Insights from ab initio quantum mechanics/molecular mechanics dynamic simulations[J].Journal of the American Chemical Society, 2015, 137(24):7 528-7 547.  LEEMHUIS H, DOBRUCHOWSKA J M, EBBELAAR M, et al.Isomalto/malto-polysaccharide, a novel soluble dietary fiber made via enzymatic conversion of starch[J].Journal of Agricultural and Food Chemistry, 2014, 62(49):12 034-12 044.  BAI Y X, VAN DER KAAIJ R M, LEEMHUIS H, et al.Biochemical characterization of the Lactobacillus reuteri glycoside hydrolase family 70 GTFB type of 4,6-α-glucanotransferase enzymes that synthesize soluble dietary starch fibers[J].Applied and Environmental Microbiology, 2015, 81(20):7 223-7 232.  GANGOITI J, VAN LEEUWEN S S, MENG X F, et al.Mining novel starch-converting glycoside hydrolase 70 enzymes from the nestlé culture collection genome database:The Lactobacillus reuteri NCC 2613 GtfB[J].Scientific Reports, 2017, 7(1):9 947.  宿玲恰, 张悦, 吴敬.嗜热栖热菌海藻糖合酶的分子改造及其应用[J].食品与发酵工业, 2015, 41(12):1-5. SU L Q, ZHANG Y, WU J.Molecular modification and application of trehalose synthase from Thermus themophilus[J].Food and Fermentation Industries, 2015, 41(12):1-5  刘玲玲. β-葡萄糖苷酶转化葡萄糖制备低聚龙胆糖的研究[D].无锡:江南大学, 2009. LIU L L.Studies on production of gentiooligosaccharide from glucose with β-glucosidase[D].Wuxi:Jiangnan University, 2009.  MILLER G L.Use of dinitrosalicylic acid reagent for determination of reducing sugar[J].Analytical Chemistry, 1959, 31(3):426-428.  AKEBOSHI H, KASHIWAGI Y, AOKI H, et al.Construction of an efficient expression system for Aspergillus isopullulanase in Pichia pastoris, and a simple purification method[J].Bioscience Biotechnology and Biochemistry, 2003, 67(5):1 149-1 153.  SAKANO Y, HIGUCHI M, KOBAYASHI T.Pullulan 4-glucanohydrolase from Aspergillus niger[J].Archives of Biochemistry and Biophysics, 1972, 153(1):180-187.  MIZUNO T, MORI H, ITO H, et al.Molecular cloning of isomaltotrio-dextranase gene from Brevibacterium fuscum var.dextranlyticum strain 0407 and its expression in Escherichia coli[J].Bioscience, Biotechnology and Biochemistry, 1999, 63(9):1 582-1 588.