Expression And Fermentation Optimization Of Sucrose Phosphorylase From Bifidobacterium Longum ZJ0521 In Recombinant Escherichia Coli

Sucrose phosphorylase (EC 2.4.1.7) (SPase) catalyzes the reaction between sucrose and inorganic phosphate to synthesize a-D-glucosel-phosphate (G1P) and D-fructose and its reverse reaction. SPase, mainly prodocued by lactobacillus and Bifidobacterium, is commonly used for oligosaccharide synthesis, modification of compound containing alcohol-OH groups or carboxyl group, and derivatives synthesis of some unstable compounds. For its catalytic characteristics, SPase shows good potential application in food and cosmetics industry. However, the further application has been constrained for its very limited yield-in wild type strains. It is in urgent need of genetic engineering to overexpress SPase for industrialized production. In this study, the SPase gene from Bifidobacterium longum was cloned and recombinantly expressed in Escherichia coli.Based on the SPase gene informations from NCBI database, the SPase gene from B. longum ZJ0521 was cloned by PCR. This spase gene is 1521 bp, coding 507 amino acids without a signal peptide. The GC content of the spase gene is 61.93%. Then the spase gene was cloned to the plasmid pET-22b(+) and transformed to E. coli Rosetta(DE3). SDS-PAGE analysis exhibited that the SPase was successfully expressed with the intracellular SPase specific activity of 2.0 U/mg. To realize the secreted expression of SPase, the spase gene was fused with four signal peptides OmpA, MalE, TorA and Sufi, respectively. However, there was still no SPase enzyme activity detected extracellularly.Because of the big difference of the enzymatic properties from different sources, we purified the SPase and studied its property. The recombinant SPase from the recombinant E. coli was purified by cell lysis, dialysis by ammonium sulfate precipitation and Ni2+ affinity chromatography. The specific activity of the purified SPase was 40.38 U/mg. The optimal temperature and pH of the purified SPase were identified to be 37? and 6.7, respectively. The purified SPase was relatively stable in pH 6-7, but it was of thermal unstability. The Km and Vmax value of the purified SPase was 11.26 mM and 0.27 ?mol/(min·mg), respectively. Under the condition of 200 U/mL SPase, hydroquinone/sucrose (mole ratio) 1:5, pH 6.5 and 25?, the conversion rate of hydroquinone was nearly 85%, and the yield of a-arbutin was 65°Through the study of enzymatic properties and its use for alpha arbutin production, we found that a better arbutin yield was obtained with SPase. Therefore, it is necessary to improve the production of SPase by fermentation optimization. On the condition of being cultured in shaker flasks with TB medium, and initially pH 6.5 with 0.4 mM IPTG at 25?, the highest intracellular SPase specific activity was achieved to 7.0 U/mg from 2.0 U/mg.. When cultured in 3 L fermenter with initially induced OD600 at 8 and 12 g/L initially glycerol concentration with feeding glycerol, the yield of SPase was increased to 12.71 U/mg.