Expression And Fermentation Optimization Of Leuconstoc Mesenteroides Levansucorse In Bacillus Subtilis

Lactosucrose(LS)is a functional trisaccharide consisting of galactose,glucose and fructose.It has excellent physiological functions such as improving intestinal microenvironment,increasing calcium absorption,regulating immunity and preventing obesity.An effective method to produce lactosucrose is enzymatic biotransformation catalyzed by ?-galactosidase,?-fructofuranosidase or levansucrase,using sucrose and lactose as substrate.At present,the researches on these enzymes are mainly based on the screening of highly productive wild strains,chemical modification,immobilization,molecular transformation and construction of genetic engineered strains.Most studies about levansucrase are using Escherichia coli as the host,which may cause safety problems.Bacillus subtilis has a long history in fermentation products and also recognized as a food-grade bacteria.In this study,the gene encoding levansucrase from Leuconostoc mesenteroides B-512 FMC is expressed in B.subtilis without antibiotic resistance marker.The gene encoding levansucrase from L.mesenteroides B-512 FMC and the gene encoding expression vector from pUB-P43-DPE-dal were amplified using different primers.Three plasmids,named pUB-P43-HPAII-LEME-dal,pUB-P43-LEME-dal,pUB-HPAII-LEME-dal,were constructed by overlap extension PCR.These three plasmids were transformed into B.subtilis 1A751(dal-)and B.subtilis WB600(dal-)respectively,which could be screened with alanine,with six genetic engineered strains obtained,1A-PH,1A-P,1A-H,WB-PH,WB-P,WB-H.Compared with the fermentation enzyme activity of six strains,WB-PH was selected to express recombinant levansucrase.The target protein was purified by Ni2+ affinity chromatography from crude enzyme solution.After the analysis of SDS-PAGE and HPLC result of reaction mixture,the protein was confirmed to be levansucrase which could produce lactosucrase.The characteristics of recombinant levansucrase were studied.The optimum temperature was 40°C and the enzyme was stable under 40°C.When the temperature was more than 50°C,the enzyme was rapidly inactived,which showed a poor thermostability.The optimum pH of levansucrase was 6.5 and it was stable ranging from pH 6.0 to 6.5.The reaction condition of lactosucrose producing was optimized.Results showed that the optimal reaction mixture containing 15%(w/v)sucrose and 15%(w/v)lactose,enzyme dosage 20 U/g substrate,temperature 40°C.The conversion rate of substrate can reach 30%~35% after 1.6 h of reaction.The effects of medium compositions and fermentation conditions were studied.The optimal culture medium consisted of 20 g/L glucose and 20 g/L soy peptone.The optimal conditions for flask fermentation were as following: temperature 37°C,inoculum size 1%(v/v),culture volume 5%(v/v),culture time 18 h.Under these conditions,the highest enzyme activity reached 108.34 U/mL,which showed 25 times higher before optimization.The fermentation of the recombinant B.subtilis WB-PH was conducted in a fermenter.The fermentation enzyme activity was up to 78.4 U/mL after 15 hours at 37°C.