Recombinant Expression And Fermentation Optimization Of Bacillus Stearothermophilus Maltogenic Amylase In Bacillus Subtilis

Maltogenic amylase,also known as maltogenic?-amylase?MAase?,which can hydrolyzes maltotriose,starch and some dextrins to maltose.Maltogenic amylase can be used in starch saccharification,food baking,flour modification and other fields.Especially,maltogenic amylase have sigificant application in the field of food baking,it can hydrolyze the starch of baked foods,so,maltogenic amylase can prevents the formation of hinges between starch granules and proteins in bread and prolongs the shelf life of baked goods.In this study,the recombinant plasmid amyM/pHY300PLK containing maltose amylase gene of B.stearothermophilus was constructed.The promoter and signal peptides were optimized on this plasmid,and then these plasmids transferred to the Bacillus subtilis which considered as Generally Recognized As Safe?GRAS?for recombinant expression.At the same time,the enzymatic properties of the recombinant maltogenic amylase were studied,and fermentation conditions were further optimized at shake flask and 3-L fermentor level,which increased the expression level of maltogenic amylase.The main results are as follows:?1?The recombinant plasmid amyM/pHY300PLK containing maltose amylase gene of B.stearothermophilus was constructed.Based on this plasmid,effect of seven constitutive promoters(amyE_?B.S??amyE?aprE?gsiB?Hpa II?SrfA?xylA_?B.S?)and two inducible promoters?xylA?ManP?on the expression of maltogenic amylase were studied in B.subtilis CCTCC M 2016536.The results showed that the recombinant strain was fermented in the TB medium for 48 h at 33?,the expression of maltogenic amylase was the highest with amyE_?B.S?promoter and the enzyme activity reached 145.8 U·mL^-1.?2?Three signal peptides with high expression of maltogenic amylase were screened from 173 signal peptides by the commercial vectors pEB-S and Bacillus subtilis RIK1285 in the Takara signal peptide screening system.Signal peptides of recombinant strains were identified,including YvcE,YncM and Lip A signal peptides.YvcE and YncM direct secretion of the protein from the Sec pathway,and Lip A signal peptide directs secretion of the protein from the Tat pathway.These signal peptides were recombined with the expression vector containing the amyE_?B.S?promoter and transferred into the Bacillus subtilis CCTCC M2016536 expression host for the expression of maltogenic amylase.YvcE,YncM,and LipA were used as signal peptides,these recombinant strains were fermented in TB medium at 33?for 48 h and the enzyme activity reached 250.7 U·m L^-1,194.8 U·mL^-1,and 189.2 U·m L^-1,respectively.It was 1.72,1.33 and 1.29 times before replacing the signal peptide.?3?Enzymatic properties of the recombinant maltose amylase were investigated.When starch was used as the substrate,the results showed as follows:specific hydrolysis activity2646 U·g^-1,K_m 0.95 g·L^-1 and k_cat/K_m 12640.4 s^-1·g·L^-1,optimal pH 5.5,optimum temperature60?.At the same time,the thermal stability was investigated at 60?.The results showed that the recombinant maltogenic amylase was very stable with half-life of 325 h.?4?The fermentation conditions was optimized for recombinant strains,first of all,the medium composition of the recombinant strain was optimized at the shake flask level.The results showed that the optimum medium contains 25 g·L^-1 of yeast extract and 5 g·L^-1 of soybean peptone,5 g·L^-1 glucose,Fe³⁺1 mmol·L^-1.The maltogenic amylase activity reached371.7 U·mL^-1 when the fermentation was conducted in the optimized medium for 48 h.Based on the optimized medium,the conditions were optimized for the production of recombinant enzyme in 3-L fermentor.The results showed that the best fermention conditions as follows:glucose was used as the carbon source and yeast extract was used as the nitrogen source in the feed medium at 37?;the ratio of the C/N is 1:1;and then the maltogenic amylase extracellular enzyme activity reached 2273.6 U·m L^-1.