Abstract: Through promoter engineering and medium optimization strategies, sucrose isomerase with high expression level regulated by non-inducible promoter was constructed, and the effects of different promoters on sucrose isomerase expression of recombinant strain Bacillus subtilis SCK6 were investigated. Unrestricted cloning method was used to construct 6 non-inducible single promoters （P_Hpal Ⅰ, P₄3, P_gsiB, P_srfA, P_amyQ and P_aprE） and 3 double promoters （P₄3-P_gsiB, 2P₄3 and 2P_gsiB） regulated sucrose isomerase recombinant strains, respectively. The results showed that the activity of sucrose isomerase under the control of 2P₄3 was the highest (19.08±0.09) U/mL, which was 1.37 times of that under the control of the original promoter P_grac. The expression level of sucrose isomerase was further improved by optimizing the medium. The fermentation medium of recombinant strain regulated by 2P₄3 was optimized from carbon source, nitrogen source and inorganic salt by single factor method. The optimal fermentation medium formula was 25 g/L glycerol, 30 g/L yeast extract powder and 20.75 g/L soybean peptone. The activity of Pal Ⅰ enzyme was increased to （80.36±9.32） U/mL. It was 4.2 times of the original medium (19.08±0.09) U/mL. In this experiment, the expression system of double-promoter regulated Bacillus subtilis sucrose isomerase with high product activity was successfully constructed, which laid a theoretical foundation for subsequent research and production of sucrose isomerase available for food, and provided a theoretical basis for industrial production and application of Isomaltulose.

Key words: sucrose isomerase, initiator project;Bacillus subtilis, medium optimization, full-scale experiment