Efficient Synthesis Of Tagatose By Coupling ?-galactosidase And Arabinose Isomerase

Tagatose is a rare sweet substitute with special health care functions.It is widely used in food,medicine,cosmetics and other fields,and the market demand is increasing day by day.In this study,recombinants Escherichia coli BL21 expressing two key enzymes(?-galactosidase and arabinose isomerase)seperately and co-expressing the two enzymes that catalyze the synthesis of tagatose from lactose were constructed,the conditions for the synthesis of tagatose have been optimized.Based on food safety considerations,two recombinants expressing two key enzymes separately were constructed based on B.subtilis168 D1 expression system without resistance genes.After optimizing the catalytic reaction conditions for the coupling of two recombinants,high-value rare tagatose can be synthesized in one-step safely and efficiently from cheap raw material lactose.The main results were as follows:(1)?-galactosidase and arabinose isomerase derived from Escherichia coli K-12 were expressed in E.coli BL21 separately and the recombinants E.coli BL21/p ET28a-lac Z and E.coli BL21/p ET28a-ara A were successfully constructed using overlap extension PCR.The crude enzyme was purified by column affinity chromatography to obtain the purified protein.The enzyme activity of?-galactosidase was 135.5 U·mg^-1 and the enzyme activity of arabinose isomerase was 34.5 U·mg^-1.The catalytic p H,temperature of?-galactosidase and arabinose isomerase were analyzed.(2)The recombinants E.coli BL21/p ET28a-ara A-lac Z and E.coli BL21/p ET28a-lac Z-ara A co-expressing?-galactosidase and arabinose isomerase in E.coli BL21 were constructed using SD sequence as a linker.The crude enzyme of E.coli BL21/p ET28a-ara A-lac Z had better effect of catalyzing lactose to tagatose.Therefore,the whole-cell conversion conditions of E.coli BL21/p ET28a-ara A-lac Z were optimized.Under the conditions of p H 8.0,temperature 50°C,adding 5 mmol·L^-1 Mn²⁺,0.5 mol·L^-1 boric acid and 0.1%SDS,the highest yield of tagatose was 24.0 g·L^-1 from 100 g·L^-1 lactose,the conversion rate was 24.0%,the molar conversion rate was 45.7%.With the concentration of lactose bigger,the yield of tagatose increased in varyied degrees.(3)A B.subtilis 168 D1 expression system without resistance genes was constructed with D-alanine racemase gene as a selection marker using overlap extension PCR and cre/loxP specific recombination theory.The intermediate product galactose accumulated severely when recombinant E.coli BL21/p ET28a-ara A-lac Z synthesis tagatose from lactose.In order to achieve a better catalytic effect,we constructed recombinants B.subtilis 168 D1/p MA5a-lac Z and B.subtilis 168 D1/p MA5a-ara A expressing?-galactosidase and arabinose isomerase respectively using the modified B.subtilis 168 D1 expression system as the host.We also optimized the ratio of the two strains to synthesis tagatose from lactose(B.subtilis 168D1/p MA5a-lac Z:B.subtilis 168 D1/p MA5a-ara A=1:15),which laid the research foundation for the optimization and regulation of the expression of two key enzymes.After optimizied the catalytic conditions for the coupling of two recombinants,the highest yield of was 30.1 g·L^-1,the molar conversion rate was 30.05%,the molar conversion rate was 57.2%(100 g·L^-1 lactose).The highest yield of tagatose was 96.8 g·L^-1 when the high concentration of lactose was 500 g·L^-1,and the molar conversion rate was 36.8%.