Construction And Optimization Of Coupling System For Enzymatic Preparation Of Linear Maltooligosaccharides

Linear maltooligosaccharides are usually linear oligosaccharides composed of 3～10glucose molecules combined with α-1,4-glycosidic bonds.They have unique physical and chemical properties and physiological functions and they are widely used in food,medicine,chemical and other fields.At present,linear maltooligosaccharides in industry are mainly prepared by enzymatic method.However,the production and purification technology of linear maltooligosaccharides has been monopolized by foreign countries for a long time.Meanwhile,there are still shortcomings in the enzymatic process,such as low production efficiency,long reaction time,and much energy loss by enzymatic elimination and purification after production.While the application of enzymatic membrane reactor(EMR)in the traditional process can solve the problems mentioned above to a certain extent.Therefore,it is necessary to optimize the production process of linear maltooligosaccharides prepared by traditional enzymatic method to determine the basic production conditions for the efficient preparation.In the meanwhile,the use of an enzyme membrane reactor to construct a coupling system on this basis can shorten reaction time,improve the efficiency and obtain the separated and purified product.The laboratory has successfully constructed and expressed the MFA enzyme(Bst-MFA)derived from Bacillus stearothermophilus STB04 in the early stage,and obtained two mutants(W139Y,linear maltopentaose-producing enzyme,G109 D,linear malt six Carbohydrate generating enzyme).This topic constructed a coupling system on the basis of the traditional process of preparing linear maltopentaose with corn starch as a enzymatical substrate and those two mutants as production enzymes.The conditions were systematically optimized to obtain a new and efficient-preparation production system of linear maltopentaose with which linear maltohexaose could be prepared and membrane fouling mechanism and cleaning scheme studied.First,under the condition of 20%(w/v)corn starch concentration,the traditional process of enzymatic preparation of linear maltopentaose was systematically optimized.The best process was obtained when the conditions were optimized as follow: corn starch 60°C,pulping time 5 min,Glucoamylase(W139Y)plus enzyme amount 30 U/g dry starch,liquefaction time15 min.MFA enzyme can play a role in liquefaction with no more enzyme added in subsequent saccharification process.The reaction temperature was 60 °C with pullulanase added at 20 h after the reaction began,and continued debranching for 4 h until the end.The amount of debranching enzyme added was 2 U/g dry starch.Under the conditions above,the yield of linear maltooligosaccharides(G1～G7)was 94.29%.The target product linear maltopentaose(G5)41.17%,and the proportion of G5 in all products of G1～G7 was stable at 43% ～45%.Secondly,based on the enzymatic preparation of the linear maltopentaose production system,a cyclic enzyme membrane reactor was used to construct a coupling system in which the membrane modules selected molecular weight cut-off(MWCO)of 50 KDa multi-channel tubular ceramics(average pore size 20 nm).The membrane’s rejection rate to MFA enzyme protein was 97.01%.The optimal process conditions were determined as follow through system optimization: liquefaction time 15 min,continuous reaction time 5 h,reaction temperature65 °C,glucoamylase(W139Y)plus enzyme amount 35 U/g dry starch,membrane pressure 0.3MPa,material circulation flow rate 900 L/h.In the reaction process,pullulanase was added 3 h after the reaction started with the addition amount 6 U /g dry starch.The yield of G1～G7reached 95.81%,in which the main product G5 42.64%.The recovery rate of the permeate end product was more than 95% after 5 h reaction.Compared with the traditional reaction for 5 h,the yield of G1～G7 and G5 had been increased by 89.99% and 153.36% respectively,meaning an achievement and exceedment against the traditional 24 h reaction process and an omit of the subsequent steps of enzyme inactivation.The product has been purified by ceramic membrane during the production process to achieve the effect of preliminary separation and purification,the reduction of the follow-up production cost and improvement of production efficiency.Thirdly,in order to obtain a production process for efficiently preparing linear maltohexaose,the production enzyme in the coupling system was replaced by linear maltohexaose generating enzyme(G109D).The production process for preparing linear maltohexaose was optimized.In the best production process,since the molecular weight of the product and the product system of the two systems were similar to each other,both of them owned the same conditions.The reaction time,the molecular weight cut-off of the membrane module and the reaction temperature were 5 h,50 KDa and 65°C,respectively.The difference of them were shown as follow: the amount of glucoamylase(G109D)added 30 U/g dry starch,the membrane pressure 0.2 MPa,and the material circulation flow rate 700 L/h,which were slightly lower than the former.While the adding time of the enzyme was 2 h after the reaction started,1 h earlier than W139 Y.And the addition amount was lower than 4 U/g dry starch.Under these conditions,the yield of G1～G7 reached 96.53%,in which the main product linear maltohexaose(G6)46.95%,accounted for nearly 50% of all products.The permeate end product recovery rate reached more than 95%.By optimizing production conditions,the effect of high-efficiency production of linear maltohexaose in a short time is also achieved,which greatly shortened the reaction time and improved production efficiency.Finally,in view of the pollution of membrane modules in preparing linear maltooligosaccharides using ceramic membrane reactors,the pollution model was fitted through STATISTICA 12.In the meanwhile,different pollution resistances were calculated to determine that the pollution mechanism was dominated by completely blocked with the other three pollution modes coexisted.On this basis,pure water,citric acid,sodium hydroxide,a mixed solution of sodium hydroxide and sodium hypochlorite,and α-amylase were used to clean and regenerate the membrane under the conditions of low pressure and high flow rate.The results showed that different cleaning he membrane flux recovery rates of the corresponding methods were 71.53%,86.92%,95.70%,99.61% and 92.31%,respectively.Alkali cleaning,oxidant cleaning and enzyme cleaning can restore membrane flux to more than90%.Oxidation cleaning agent can restore membrane flux to close to 100%,indicating that this method has good regeneration for membrane pollution caused by starch and polysaccharides effect.