Application Of Dextranase In Dextran Production

Dextran is a type of poly-D-glucose in which the glucose residues are linked to the main chain by means of a?-1,6-glucosidic bond,and the branched chains are linked to the main chain by a glucose residue or glucose chain?simple or complex?via a?-1,2-,?-1,3-or?-1,4-glucosidic bond.The main uses of dextran include plasma,prebiotics and medical substrate.The traditional production technology of dextran includes two-step processes,fermented sucrose into dextran and acid hydrolyzed on the purpose of decreasing the molecular weight of dextran.In this study,a new process was developed in which dextranase was added in the fermentation process to keep the molecular weight of dextran at a low and narrow range at the end of fermentation.The main advantages of the new process are as follows.Firstly,the acid hydrolysis and neutralization process are avoided,so the use of strong acid and strong base is avoided,and the process flow is simplified.Secondly,the viscosity of fermentation broth was decreased and the fermentation was more efficient.Dextranase plays a key role in the new process.DNS method,which measured the increment of reducing end of dextran after the substrate?2 g/d L dextran T-2150 in Na₂HPO₄-KH₂PO₄ buffer?underwent the enzymatic reaction,is applied to determine enzyme activity.In the single-factor test,the optimum reaction temperature,p H and ionic strength were measured as 60/65°C?no significant difference?,5.5 and 0.015 mol/L,respectively.Under simulated fermentation conditions?dextran T-2150,sucrose,bacteriological peptone,Na₂HPO₄ and KH₂PO₄ were 2,8,0.2,0.14 and 0.03 g/d L,respectively,with p H of 6.5?,the enzyme activity was only 18%of the optimal conditions.The K_m value of the dextranase under simulated fermentation conditions and optimal conditions was calculated by Hanes-Woolf diagram as0.006559 and 0.01321 mol/L,respectively.In addition,when the p H of simulated fermentation broth rose to 7.5,8.5 and 9.5,the enzyme activity was only 11.2%,5.7%and 2.5%relative to the optimal conditions.When ethanol was added at 5%,10%,15%and 30%,the enzyme activity was only 15.2%,13.6%,11.1%and 6.8%.However,the dextranase was stored at room temperature for 30 min under these two unfavorable conditions,its effect on enzyme activity was reversible.The change of molecular weight in dextran enzymatic hydrolysis is different from that in acid hydrolysis.Changes of different molecular weight segments of hydrolyzed samples were analyzed by high performance liquid chromatography?HPLC?using two chromatographic columns,OHpak SB-806M HQ and SUGAR KS-803.In the process of decreasing number average molecular weight of dextran?5%?from hundreds of thousands by one order of magnitude,the effect of 4h in sulfuric acid solution at 100°C and p H2 was similar to that of10 U enzyme/g dextran,60°C and p H5.5 solution for 15 min;the efficiency of the acidolysis process is stable,and the dispersion coefficient of the acidolysis sample is lower than that of the enzymatic hydrolysis;the small molecules formed by acidolysis are mainly glucose,and the enzymatic hydrolysis is mainly trisaccharide and tetrasaccharide.In the process of simulated fermentation,the use of dextranase in large amounts for a short period of time can better play the role of the dextranase.In the traditional process,the fermentation broth is low viscosity at 0-12 h,medium viscosity at 12-20 h and high viscosity at 20-31 h.In the new process,dextranase was added at 12 h to extend the low viscosity period to 18 h,and the medium viscosity period from 18 h to the end of fermentation.The time for sucrose conversion rate to reach 90%was shortened from 28.3 h to 25.0 h.There was no significant difference in the mass of substance insoluble in ethanol at 24 h.Dextran was distributed widely at the end of traditional fermentation process,and the target molecular weight ratio was only 54%.The new process increases the target molecular weight ratio to more than 90%,and the distribution is concentrated.