Dextran is a homopolysaccharide consisting of glucose units that are linked byα-1,6 bonds.Due to the unique physical and chemical properties of dextran and modified forms,it has a wide range of applications in food,medicine and materials.The traditional methods for preparing dextran comprise microbial fermentation and enzymatic preparation.The enzymes are either directly or indirectly synthesized by dextran sucrase,which uses sucrose as a substrate.Since the synthesis of dextran uses only the glucose unit in sucrose,the highest theoretical conversion rate for the traditional preparation method is only 47.3%,which is even lower in actual production.In this study,we propose a multi-enzyme complex process for the preparation of dextran using starch.The synergistic action of 4,6-α-GT,pullulanase(PUL)and isopullulanase(IPU)enables the synthesis of high-purity dextran without branching modifications using starch-based substrates.The key findings are:(1)The roles of each enzyme were verified using 4,6-α-GT,PUL and IPU in different treatment steps.PUL functions as a starch debranching enzyme,converting starch-branched structures into straight-chain substrates and improving substrate utilization.In contrast,4,6-α-GT uses the straight-chain starch substrate to transglycosylate the linear polysaccharide product IMMP,which is rich inα-1,6 bonds.IPU treats IMMP to obtain continuousα-1,4-bonded orα-1,6-bonded polysaccharides.Finally,glycolytic enzymes digest the former to give dextran.(2)Optimization of the preparation of dextran by combination of multiple enzymes.The optimal concentration of DE2 maltodextrin was 200 g·L-1,and the optimal addition amounts of PUL and IPU were 40 U·g-1 and 50 U·g-1,respectively.Reaction conditions for 4,6-α-GT from different sources,Limosilactobacillus reuteri 121,Limosilactobacillus fermentum NCC 3057,and Bacillus sporothermodurans(named as Lr Gtf B,Lf Gtf B,and Bs Gtf C,respectively),were optimized.The optimal p H was 5.0,and the optimal reaction temperatures were 35°C,40°C,and 40°C,respectively,with the optimal amount of added enzyme being 300 U·g-1,300 U·g-1,and 800 U·g-1,respectively.The amount of added amylase was 1000 U·g-1,and the digestion time was 20 min.The molecular weight of the product was positively correlated with the straight chain starch content of the substrate,and the molecular weight of the product obtained from substrates with different straight chain starch contents ranged from 3 k Da to 20 k Da.In addition,the addition of maltose and isomaltose during the process reduced the polymerization degree of the product.(3)Analysis of the structure-function relationship of 4,6-α-GT on the molecular weight of the product.When using DE2 maltodextrin as the substrate,the molecular weights of the products obtained by Lr Gtf B,Lf Gtf B,and Bs Gtf C were approximately 7 k Da,3 k Da,and 1 k Da,respectively.The+1 subsite of the receptor affects the conformation of the receptor-substrate binding,and the mutants S348V and S346T/S348V of Lf Gtf B can effectively increase the polymerization degree of the product,as well as T920V and S918T/T920V constructed at the same position in Lr Gtf B.Further,the mutants N1019H and H416N constructed at the+1 position of Lr Gtf B and Bs Gtf C,respectively,significantly change the polymerization degree of the product.In addition,the hydrophobic cluster formed by the Y467W mutation at the+3 subsite of Bs Gtf C also plays an important role in receptor-substrate binding.(4)Evaluation of the application effect of dextran.The evaluation of the probiotic effect of small molecule dextran(SDex)prepared with the participation of Bs Gtf C showed that SDex could significantly promote the proliferation of probiotic bacteria such as Bifidobacterium and Lactobacillus and promote more acetic acid production compared with commercial dextran 20,commercial iso-malto-oligosaccharides and blank control.It also does not promote the growth of conditionally pathogenic bacteria Escherichia coli,and has a selective probiotic function.Iron dextran was prepared from dextran prepared with the participation of Lr Gtf B.The purification was carried out using ultrafiltration membranes with a cut-off molecular weight of 3 k Da to obtain dextran with Mw of 5895 and a distribution coefficient of 1.51.The iron content of dextran prepared using this dextran was up to 35.6%,which is much higher than the pharmacopoeia requirement of 25%. |