| Sucrose isomerase（EC 5.4.99.11）,also known as isomaltulose synthase or trehalulose synthase,can isomerize sucrose into isomaltulose and trehalulose.The reaction product of most sucrose isomerases is mainly isomaltulose.Isomaltulose is an isomer of sucrose,which naturally exists in sugarcane juice,honey,beet and other foods.It has many excellent physiological functions such as low calorie,no caries,no obesity,no stimulation of rapid blood sugar rise,and can be added to various foods as a low-calorie sweetener.Bacillus subtilis has many advantages such as strong protein secretion ability,non endotoxin production,and high-density fermentation,and is widely used for recombinant expression of exogenous proteins.The expression level of sucrose isomerase in food safety microorganisms is generally low,which limits the application of isomaltulose in the food industry.In order to solve this problem,in this study,the sucrose isomerase derived from Pantoea dispersa UQ68J was recombinantly expressed in B.subtilis,and the enzymatic conversion process of the recombinant sucrose isomerase to prepare isomaltulose was optimized,subsequently,the high expression level of sucrose isomerase in B.subtilis was realized by optimizing the expression elements and fermentation conditions of the recombinant strain.The main research findings are as follows:（1）Study on enzymatic properties of recombinant sucrose isomerase and optimization of reaction conditions for preparation of isomaltulose:The sucrose isomerase gene（sim）derived from P.dispersa UQ68J was transferred into B.subtilis WS9 for recombinant expression and the enzymatic properties of the recombinant sucrose isomerase were explored.The results showed that the optimum temperature and p H of the recombinant sucrose isomerase were 30℃and 6.0,respectively.The half-life of recombinant sucrose isomerase at 45°C was 68 min and it had good stability in a wide range of p H 5.0-8.0.Subsequently,the reaction conditions for the preparation of isomaltulose by recombinant sucrose isomerase were optimized.The results show that with p H 6.0,400 g·L-1 sucrose as the substrate,the amount of recombinant enzyme added was 20 U·g-1 sucrose,and the yield of isomaltulose reached the highest 90.61%at 30℃for 10 h.When the substrate concentration was increased to 700 g·L-1,the yield of isomaltulose could still reach 89.20%.（2）Optimization of the expression elements of the recombinant plasmid:Firstly,the extracellular enzyme activity of sucrose isomerase was increased from 9.51 U·m L-1 to 17.12 U·m L-1 by co-expressing the extracellular molecular chaperone Prs A.Subsequently,the effect of 8 promoters(Pfus A,Pylb,Pydz A,Ptuf A,P43,Pspo VG,PHpa II-Pamy QΔ109,Pglv A)on the expression of sucrose isomerase in B.subtilis WS9 host was studied,and it was found that the double-promoter PHpa II-Pamy QΔ109 can better mediate the expression of sucrose isomerase and increase the extracellular enzyme activity of sucrose isomerase from 17.12 U·m L-1 to 27.46 U·m L-1,which was 2.89 times that of the recombinant strain before optimization.（3）Optimization of the fermentation conditions of recombinant strain:Firstly,the culture conditions such as nitrogen source,carbon source,metal ion,medium p H and temperature were optimized at the shake flask level,and the extracellular enzyme activity of sucrose isomerase was increased to 70.87 U·m L-1,which was 2.6 times the enzyme activity of the shake flask before optimization.Based on the results of the shake flask experiment,the culture conditions of the recombinant strain 3-L fermenter were optimized,and the optimal enzyme production conditions of recombinant strain in 3-L fermentor were determined as follows:Temperature 33°C,p H 7.0,dissolved oxygen（DO）value of 30%,10 g·L-1glycerol as the carbon source of the basal medium,20g·L-1 corn syrup and 36 g·L-1 angel yeast powder as the nitrogen source of the basal medium,250g·L-1 glycerol and 250 g·L-1 maltose were the carbon sources of the feed medium,and 23 g·L-1 corn syrup and 41 g·L-1 angel yeast powder were the nitrogen sources of the feed medium.Under these conditions,when the recombinant strain was cultured for 80 hours,the OD600 of the strain was 350.32,and the extracellular enzyme activity of sucrose isomerase reached the highest at 862.86 U·m L-1.The enzyme activity of the recombinant strain 3-L fermenter was the highest level of sucrose isomerase expressed in food safety microorganisms at present.This study improved the expression level of sucrose isomerase in food safety microorganisms and achieved high yield of isomaltulose,which laid a solid foundation for the industrial production of isomaltulose and its application in food field. |
