Cloning?Expression And Molecular Modification Of Levansucrase From Bacillus

Fructan include fructosyloligosaccharides and fructosylpolysaccharide,is a kind of polysaccharide mainly formed with fructose residues as monomer.Its molecular weight distuibute in wild range.Fructosylpolysaccharide is usually divided into two categories,one is mainly composed of ?-(2,1)glycosidic bond linkages,classified as inulin;another one is usually composed with ?-(2,6)glycosidic bond linkages,classified as levan or bacterial type fructans.Fructans have been demonstrated to have many biological activities,and showed good effects on antineoplastic,antiviral,strengthen the body immunity and so on.It also can enhanced immune stress,improve gut balance function,and is a very important food additive.Levan can be used for production of prebiotics,dietary fiber,low heat food,slimming products,lipid-lowering products,also as emulsifying agent,humectant and gel agent.It was reported that levan can contribute the same effect in moisturizing properties as the expensive hyaluronic acid,which is needs largely in cosmetics industry.Thus it has great potential of application.Guangxi Province,which is located in subtropical,had become already the largest sucrose production base,occupies more than 60%of countrywide sugar yield in China.But the key problem of sugar industry of Guangxi Province is their product line is too simple hence lack of high additional value products.Using sucrose as raw material to develop high additional value products meet the needs of sustainable development of their local economy,thus has great significance.Levansucrase,(EC2.4.1.10,sucrose:2,6-D-fructan-6-D-fructosyltransferase,FTF,FTase,Lls fructosyltransferase,)also known as fructosyltransferase,is a kind of enzyme which not only can hydrolysis sucrose into glucose and fructose,but also can transfer the fructosyl to levan.The aim of this research is cloning,expression and modification of new levansucrase gene by the modern biological technology,molecular enzyme engineering and other advanced means,in order to raise its activity,stability and substrate tolerance.Study enzymatic properties of the recombinant protein and its catalytic mechanism of levan formation,which would lay foundation for the future scale production of levan.Through the research we also compared and analyzed the differences in structures,enzyme performances of varies levasucrases and their mutants.This work will promote the study of the catalytic domain and the key amino acid residues in glycoside hydrolase family GH-J,which can help to provide theoretical supports to the study of function and mechanism on levansucrase activity,stability and product profiles.We isolated Bacillus subtilis sp.54A from soil and through physical mutagenic method obtained its mutant sp.56A;separated the Gelbacillus sp.HB1 from Yunnan hot spring in Tengchong,and,bought Geobacillus thermoleovorans LMG9823 from the BCCM.According to sequences of levansucrase published in the Genebank,we designed primers and successfully cloned their levansucrase genes.All of these strains were determined their 16s DNA sequences and aligned to constructed the phylogenetic tree.A novel method no reported so far based on the HSAB(High Concentration Sucrose and Aniline Blue Plate)screening plate was builded.According to microorganisms which produce polysaccharide will be stained by aniline blue,with the plate including up to 10%concentrations of sucrose and little aniline blue,we can simultaneously detect hydroloysis and polymerase activity of levansucrase.Thus these double indicators accodina to chromgenic or hydrolysis halo could be very helpful to screening levan-produce wild-type strains.HSAB plate with a high osmotic pressure so that it is not suitable for screening of Escherichia coli recombinants.Another new efficient selection method then was established to resolve this problem,which improved the two step screening method reported in the literature to only one-step.Using SS56A as template,through error-prone PCR we constructed an levansucrase mutants library.Through one-step efficient screening methods,the mutant T305A(also name EPSS1)was obtained,which contained a substition ALA of THR at 305 site.We also succeeded in attached the thermal stability related domain at C-terminal of trehalose synthase of Thermus thermophilus TtHB-8 to levansucrase SS56A,constructed the new fusion protein STHB6.Using pSE380 and pET30a(+)respectively as the basis vectores,the recombinant expression vectors of SS54A,SS56A,T305A,HBR1,9823SB,and STHB6(a fusion protein)was constructed and transformated to Escherichia coli XLIOGOLD and Rossette(DE3),successfully induced to expression.The recombinant proteins then were purified by nickel affinity chromatography purification and demonstrated to reach electrophoretically purity.The hydrolytic properties of recombinant levansucrases were studied.The results indicated that the properties varies in them.Optimum hydrolytic temperature of SS54A is 35?,and its optimum pH is 6.0,its Km value was 5.71mM sucrose,its Vmax is 3.2341?mol/min.mg protein;optimum hydrolytic temperature of SS56A is 45 ?,its optimum pH is 6.0,its Km value is 6.216mM sucrose,its Vmax is 43.29? mol/min.mg protein;optimum hydrolytic temperature of T305A is 50 ?,its optimum pH is 6.5,its Km value is 14.52mM sucrose,its Vmax is 64.52? mol/min.mg protein;iptimum hydrolytic temperature of STHB6 is 50 ?,its optimum pH is 6.5,its Km value is 7.673mM sucrose;,its Vmax is 27.70?mol/min.mg protein.Studies have also shown that levansucrase thermal stability is varies under different pH.Some metal ions and surfactants have significant effect on hydrolysis.MnCl2 showed the strongest activation,and ferric ammonium citrate,CaCl2,DTT also have significant activation.,while AgNO3,MgCl2,SDS showed significant inhibitory effect.Thermal stability of T305A increased slightly,while substrate affinity decreased significantly,but the catalytic speed was improve significantly with a Vmax upgrade to 49%.T305A could maintain high conversion rate of levan production under high temperature and high substrate concentration.Most of the reported levasucrase had the optimum substrate concentration were between 5%-10%,while the conversion rate of polysaccharide on fructose of T305A could reached 71.36%at pH7.0,30 ?,with the 25%sucrose substrate concentration for 24 hours.Although the Km of STHB6 was slightly increased,and the activity of hydrolysis of sucrose decreased,it showed a high efficiency on polymerization of levan at high temperatures.The optimum transfructosylation temperature of STHB6 rised to 40?.When the substrate concentration is 25%,catalyzation at 37? for 30 hours can reach a 80.64%conversion rate(polysaccharide on fructose)Through the analysis of the amino acid residues function of the mutants by homology modeling,ALA-309 amino acid residues of the Bacillus subtilis levansucrase was found for the first time that it take a great role in the thermal stability and catalytic velocity.Compared of SS54A,SS56A and T305A,we found that the three recombinant proteins contained only one amino acid difference between each other.The amino acid sequence of SS54A was hardly complete consistency with SS56,excepting ALA-309 is instead of VAL in SS54.But the optimum hydrolytic temperature of SS54A is only 35? while of SS56A was 45?.The substrate affinity of SS54A(Km=5.71 mM sucrose)was slightly higher than of SS56A(Km=6.216mM),but the Vmax value of SS54A(3.2341 ? mol/min.mg protein)was only 7.5%of SS56A(43.29 ? mol/min.mg protein).The hydrolitic activity decreased significantly.It has not yet been reported so far in the literature about the effections of ALA-309 amino acid residues on the structure and properties of levansucrase.This study also showed that the optimum temperature,pH and substrate concentration were different between our recombinants for their transfructosylation.The mutant T305A and STHB6 we obtained displayed keeping high conversion rate under high temperature and high substrate concentration.These properties were few reported in other levansucrase.The majority reported optimum substrate concentration of lvansucrase polymerization was only about 5%-10%,and some optimum temperature was 4 ?,which is the actual production constraints.Our mutant enzymes showed the potential value in production and application for its high conversion rate(on fructose70%-80%)under the high temperature(40 ?),high substrate concentrations(25%sucrose).In conclusion,we established new efficient screen methods to obtain some levansucrases and mutants constructed by molecular modification with potential value in production of levan,succeeded in expression and purification of them in Escherichia coli hosts.We also found some important amino acid residues related to the levansucrase catalytic function.,which was benificial to further study of levansucrase catalytic domain and other similar structure of the catalytic domain of glycoside hydrolase family GH68.