Secretory Expression Of A 4-?-glucanotransferase In Bacillus Amyloliquefaciens

From Thermus Thermophilus HB8(ATTC27634),4-?-glucanotransferases(4?GTase or 4GT,EC 2.4.1.25)is a high-temperature resistant ?-amylase,which can hydrolyze the ?-1,4-glycosidic bond in the donor and transfer to the receptor to form new ?-1,4-glycosidic bond,which can not only improve the properties of starch-based foods,but also catalyze the formation of cycloamylose,thermoreversible gel,resistant starch and other products,which are widely used in food,medicine and health fields.The recombinant expression of 4GT has been realized in genetically engineered bacteria such as Escherichia coli,Bacillus subtilis and Saccharomyces cerevisiae,the later two are also considered as food safety strain(Generally Recognized As Safe,GRAS).However,the enzyme activity reported isn't high enough.Moreover,the relevant patent of 4GT in industrialization has been monopolized by foreign companies.In China,the industrialization of 4GT is still in infancy.Therefore,it's of great signification to improve the production of 4GT to realize the localization process.Bacillus amyloliquefaciens is a GRAS strain.It not only produces ?-amylase,protease,?-glucanase,but also acts as a highly efficient host for extrinsic protein expression.The purpose of this study is to improve the production of 4GT using Bacillus amyloliquefaciens as host.The main research results were as follows:(1)BA23 was determined as a suitable Bacillus amyloliquefaciens strain for 4GT expressionUnder the screening conditions of transformation,high amylase production,low protease production,salt tolerance and high temperature tolerance,and good growth performance,BA23 was selected as the target host from 30 wild strains of Bacillus amyloliquefaciens.(2)Optimize the expression system of 4GT in BA23The expression of 4GT in BA23 was verified by SDS-PAGE and Western-Blotting with 200U/mL extracellular enzyme activity.By constructing different vectors to express 4GT,the optimal vector was pHTNO4,which enzyme activity was increased by 50%,with the maximum enzyme activity up to 300U/mL.The optimal signal peptide SamyQ increased the enzyme activity by 33%,and the highest enzyme activity was 430U/mL.Site-directed mutation of 4GT gene Y54G increased enzyme activity by 14%,and the highest enzyme activity was 492U/mL.Then,the optimal conditions were determined as follows:IPTG concentration 1mM,inoculation amount 1%,carbon source soluble starch(10g/L),nitrogen source soybean meal(5g/L),metal ion Mg2+0.2%.In summary,the optimal combination of soybean meal is 5g/L with 10g/L soluble starch.Starting with 2%inoculation amount,the highest enzyme activity is 575U/mL,which is increased by 16.8%.(3)Breed high yield strain by mutagenesisAfter three rounds of NIT mutagenesis plasmid and three rounds of NTG mutagenesis of recombinant bacteria,and the establishment of iodine and starch color method for high-throughput screening,strain 3T8 was yielded,with the highest enzyme activity of 800U/mL in optimized medium,which was 39.1%higher than that of the original strain,and the unit enzyme activity was increased by 98.2%.(4)Explore the enzymatic properties of recombinant 4GTAnion exchange chromatography was used to purify pHTNO4(Pgrac+SamyQ)-HB4GT(Y54G)/BA23,and the pure enzyme activity was 510U/mg with the purification ratio 7.86 times.The optimum temperature of wildtype HB4GT and mutant Y54G was 60? and 55? respectively.The optimal pH was 7.5 for both enzymes,and pH was stable in 6.5-9 weak alkaline conditions.The half-life of wildtype was about 1.5h longer than that of mutant Y54G at either 55? or 60?.Most metal ions promoted 4GT enzyme activity except Zn2+,Cu2+,Fe3+.It was found that the substract affinity of Y54G was higher than that of wildtype,but its catalytic efficiency(Kcat/Km)was lower,which may be related to the involvement of Y54G in substrate binding.In conclusion,this study achieved the recombinant expression of 4GT in Bacillus amyloliquefaciens.The expression vector pHTNO4 was reconstructed with SamyQ as the signal peptide and Pgrac as the promoter,bearing point mutation at Y54G.By mutagenesis breeding,the maximum enzyme activity of 4GT has reached 800U/mL,which was 3 times increase compared to the baseline.The optimized fermentation condition was determined as 1mM IPTG induction,2%inoculation amount,10g/L soluble starch and 5g/L soybean meal.This paper is a further breakthrough on the recombinant expression of 4GT based on previous studies.Before,Ouyang Xiaoying achieved the highest 4GT enzyme activity of 254.78U/mL in Escherichia coli,and Wan Huihui achieved the highest 4GT enzyme activity of 456.3U/mL in Bacillus subtilis.In this paper,4GT was achieved in Bacillus amyloliquefaciens from the initial enzyme activity of 200U/mL to the highest enzyme activity of 800U/mL,which has achieved the high yield enzyme activity reported in China and lays a foundation for the industrialization of 4GT.