Researching The Catalytic Mechanism Of Bifidobacterium Longum Sucrose Phosphorylase Probed With Site-directed Mutagenesis

Sucrose phosphorylase is not only specific enzyme that can transfers glucosidebond, but also an important glycosyltransferase. It can use sucrose and glucose1-phosphate as the donor, catalyze transglycosylation of various sugars andnon-carbohydrate receptors into a series of different functions glycosides. As glyco-sides widely used in food, medicine, cosmetics and other industries, to obtain ahigher catalytic activity, good stability sucrose phosphorylase become a research fo-cus.On the basis of Bifidobacterium longum crystal structure of sucrose phosphory-lase which published in the NCBI reference,we analyzed the sucrose phosphorylaseby bio-informatics. Then we performed Phe156Ala(F156A)、Tyr196Ala(Y196A)、Asp342Ala(D342A)、Gln345Ala(Q345A) mutants thereof within the conserved do-main of gene by one-step site-directed PCR mutagenesis. The protein was amenableto expression in Escherichia coli and detected by SDS-PAGE. Experimental resultsshowed that the mutants thereof soluble expression did not change dramatically, andthe soluble protein that constituted60%of the total cell protein content. As the fusionprotein His6.Tag of four mutants thereof genes, the soluble proteins were purified bynickel affinity chromatography method.Characterization of enzymatic properties show that the specific activity ofwild-type SPase was201U/mg on500mM sucrose at30℃and pH7.0. Which were169.0、21.8、775.4and50.25times of mutants thereof F156A、Y196A、D342A、Q345A, respectively. Kinetic data analysis showed that the glycosylation reaction Kmof mutants compared to wild-type SPase is significantly increased, which D342A isapproximately27times of the wild-type SPase, the remaining mutants thereof were7times. Simultaneously, the Kcat/Km of Wild-type SPase were2700、1086、125and15000-fold of mutants thereof, respectively. When the inorganic phosphate as a varia-ble, constanted at500mM sucrose solution for transglycosylation reaction. The Kmof Wild-type SPase were12、40and4-fold of mutants thereof F156A, Y196A, andQ345A，However, the Km of D342A did not change dramatically.The transition-state energies were got from kinetic data. Substitution of Phe156、Tyr196、Asp342、Gln345caused a large destabilization of the transition state forenzyme glucosylation by sucrose, compared to wild-type SPase the G#were+4.2kcal/mol、+2.9kcal/mol、+5.8kcal/mol and+3.4kcal/mol. The transition state for glucosylation by Glc1P was destabilized by+2.9kcal/mol in Y196A and even more so(≈+4.0kcal/mol、≈+3.0kcal/mol、≈+3.8kcal/mol) in F156A、 D342A and Q345A.This can explain why the catalytic activity of mutants thereof are reducing, it is be-cause the process of mutants thereof catalyticed substrate from the ground state intotransition state desired more transition-state energies than wild-type SPase.The properties of the mutants thereof were studied, which including temperature,pH and temperature, pH or metal ion on the stability. The experimental result showsmutants thereof D342A and Q345A have the same optimization pH, and F156A andY196A shifted1.5units. Relative to wild-type SPase for four mutants which haveshown poor pH tolerance. Influence of temperature on enzyme obtained mutantF156A, Y196A, D342A optimum reaction temperature is lower than with thewild-type SPase, while Q345A optimum temperature is5℃higher than the wild-typeSPase. After incubated for1h at different temperatures,tested the thermal stability ofenzyme showed that F156A, Y196A, D342A, Q345A half-life temperatures were9℃,9.5℃,6℃and10℃higher than the wild-type SPase. Tested to obtain abetter metal ion stability of mutants thereof than wild-type SPase, wherein Cu, Mnions on the enzyme stability of the most significant difference.In summary,"alanine scanning" confirmed that amino acids Phe156, Tyr196,Asp342, Gln345were important active sites in phosphorylation reactions catalyzed byBifidobacterium longum sucrose phosphorylase. These amino acids had obviouslysignificant influence on the catalytic activity of the mutant. The result laid the founda-tion for the research on sucrose phosphorylase catalytic reaction mechanism of phos-phorylation and more efficient strains.