The Heterologous Expression,Molecular Modification And Coupled Catalysis Of Levansucrases And Levanases

In recent years,with increasing consciousness of health by the public,the prebiotics with important immunomodulatory effects on human body have attracted increaseed attention.As an important prebiotic and functional food,oligosaccharide syrup has lots of special characteristics,such as high sweetness,excellent hygroscopicity and strong osmotic pressure.In addition,the fruit oligosaccharide syrup is also suitable for special populations like diabetes because it contains no glucose or sucrose.Thus,oligosaccharide syrup,as a new sweetener for replacing sucrose,should have an increasing application prospect.However,many problems exist in the current production process of oligosaccharides syrup,such as the low efficiency of producing fructan by current enzymes and the difficulty of removing glucose from oligosaccharide syrup,etc.In this research,we had used different technical strategies to slove these problems,such as molecular cloning and heterologous expression,of novel and efficient enzymes,deeply analysis the catalysis mechanism of enzymes followed by molecular modification,enzyme immobilization and coupling catalysis.As a result,we have lay the foundation for solving these technical problems in preparation of oligosaccharide syrup.Firstly,we have cloned,expressed novel levansucrase CA-Sac B from bacillus Clostridium,and analyzed its enzyme characteristics.The results showed that this enzyme had good specificity on catalytic product,which can only produce levan and fructose,but not produce fructo-oligosaccharide.The Km and Vmax values of thisenzyme were 64 mmol/L and 190 umol/min/mg,respectively.The conversion rate of sucrose substrate was up to 60%.Therefore,this enzyme may have application value in industrial production.This enzyme is the first bacterial levan synthase cloned from the strict anaerobic bacteria,which has the highest amino acid homology of only 53% with the reported levansucrases until now.In order to study catalytic mechanism,spatial structure and the evolution strategies of this enzyme,and to discover novel bacteria levansucrases,modification of CA-SacB was performed by a novel method of metagenomics DNA region shuffling.This novel method may also provide a novel strategy and new idea for the researches in the related fields.Secondly,in order to deeply reveal the expression characteristics,catalytic mechanism and molecular evolution of levansucrase,the strategy of conditional lethal phenomenon of the levansucrase was studied.It is well-known that Escherichia coli harboring Bacillus amyloliquefaciens levansucrase BA-SacB1 cannot grow on the medium containing sucrose.Thus,BA-SacB1 is usually used as a negative screening marker,and is widely applied in molecular biology.However,the mechanism of such conditional lethality is not clear so far.In this research,through deletion of signal peptideand molecular localization of BA-Sac B1 with or without signal peptide,we have,for the first time,proved that this conditional lethality was directly related to the expression of BA-SacB1 and the formation of levan in periplasmic space.Besides,molecular modification of the enzyme was carried out by point mutation and section deletion.The properties of the enzyme before and after the mutation analyzed to provide a theoretical basis to reveal the catalysis mechanism and molecular evolution of this enzyme.Thirdly,some novel bacterial levanase genes derived from B.amyloliquefaciens,B.subtilis and B.licheniformiswere cloned,heterologous expressed and analyzed enzyme characteristics.The results showed that two levanases(BL-LEV and BS-LEV2)from B.licheniformis and B.subtilis should be excision enzyme whose catalytic products contained only fructose but no oligosaccharides.The Km values of these two enzymes were 16 mmol/L and 28 mmol/L,respectively.The optimum reaction temperatures were 30 and 20 ?,and pH value were 8 and 7 respectively.Another two levanases(BA-LEV and BS-LEV1)from B.amyloides and B.subtilis,expressed endonuclease activities and their catalytic products were mostly the oligosaccharides with molecular weight around 1200 Da but no fructose was detected.The Km values of these two enzymes were 69 mmol/L and 82 mmol/L,respectively.The optimum reaction temperature of these enzymes were between 30-70 ?.BA-LEV had a wide range of pH value and had good activities from pH 3 to 7,and the optimum pH of BS-LEV1 was pH 5.The satisfactory catalytic efficiencies of these enzymes make them promising for inductry application.Finally,a high purity fructo oligosacaccharide was produced with sucrose as the substrate by the coupled reaction with levansucrase and levanase.This strategy can obviously reduce the production cost and increasethe yield of the oligosaccharide syrup.A mixture of levan,fructan and sucrose was obtained by catalysis of sucrose using levansucrase.Then simple and less expensive strategy was used to purify levan.The levanase was used to hydrolyze levan into oligosacaccharide.The strategy of coupled catalysis could introduce high purity of oligosaccharide syrup(nearly 100%)with simple process.More importantly,it is possible to avoid complex processes required by traditional methods to eliminate glucosefrom oligosacaccharide.In addition,new systems of nanoscale zinc oxide and immobilization cell membrane based immobilization were established to immobilize the levansucrase and levanase respectively,which could significantly improve the properties of the enzymes.In addition,comprehensive analysis of various factors in the process of coupling catalysis(including reaction temperature,pH and time)was carried out using response surface analysis.As a result,when the concentration of substrate sucrose was 200 g/L,the reaction temperature was 45 ?,the reaction time was7 hours,and the solution p H was7 under such conditions,the best levan yield was 47.44%.In addition,when the concentration of levan was 105 g/L,the optimum conditions for oligosaccharide were as follows: the reaction temperature was 42 ?,the reaction time was 7 hours,and the solution pH was 7.Under such conditions,the yield of oligosaccharide syrup was 88.28.This research was of great significance for improving the production mode of oligosaccharide syrup,and has important reference value for related research.