Semi-rational Design And Immobilization Of Two Sugar Aminotransferases

As an important lead compound for the treatment of second-type diabetes,valienamine has a wide range of applications in the field of medicine.Currently,there are a variety of related drugs on the market,so the efficient synthesis of valienamine has been a hot issue in the industry.Our laboratory found that a more efficient biosynthetic pathway of valienamine could be reconstructed by introducing heterologous sugar aminotransferase Wec E into the chassis Streptomyces hygroscopicus 5008.In the previous work,we used protein engineering to initially improve the catalytic activity of Wec E on valienone,but a big gap still existed from the target activity.In this paper,sugar aminotransferase Wec E has been further redesigned on the basis of previous work.Firstly,the loop 7 of Wec E dimer was reconstructed by homology modelling,and the external aldimine form of valienone was docked into the binding pocket.We selected 24 hot-spot sites for site-saturation mutagenesis from following four aspects:binding pocket analysis,AA conservation evaluation,virtual alanine scanning and substrate channel analysis,followed by screening using the optimized L-GDH coupling method.After first and second round screening,five positive mutants C174A,V318R,F319V,Y321F and I322T were identified;on this basis,we combined these positive sites by the way of iterative saturation mutagenesis,and obtained five positive mutants（M1,M2,M3,M4 and M5）with the activity gradually improved.The activity of the best mutant M5 increased by 21 folds compared with the wild type,15-fold higher than the former mutant Var A（6-fold higher than the wild type）.The kinetic parameters of Wec E and the five mutants were measured,showing that as the catalytic activity increased from M1 to M5,the binding affinity with valienone decreased instead.According to the result of MD simulations,the distance between valienone external aldimine and loop 7 as well as loop 9 in M5 was farther than that in Wec E,but distance between the carbonyl group of valienone and the catalytic lysineε-amino group become closer in M5.This result corresponded with the trends of kinetic parameters K_mand k_cat.Finally,we studied the thermal stability of the enzymes,and the result showed that the half-life of M5 at 40℃was 50.5-folds higher than Wec E.The T_mvalue of M5 was also 4℃higher than Wec E.Our work realized the co-evolution of catalytic activity and thermal stability in sugar aminotransferase for the first time.Meanwhile,it also indirectly verified the"Active Center Stabilization"strategy proposed by our laboratory in the early stage,expanding its scope of application.Similar to valienamine,its enantiomerβ-valienamine is an important potential drug for the treatment of diseases caused by the malfunction ofβ-glucosidase.Due to the multiple chiral centers,the traditional chemical synthesis has many drawbacks,and the biosynthesis also bears the disadvantages of complex metabolic regulation and low yield.Therefore,we immobilized the sugar aminotransferase Btr R,which could convert the pro-chiral ketone to optically pureβ-valienamine.By comparing the immobilization effect of six different epoxy resins,we determined MC-150EP as the optimal immobilization carrier for sugar aminotransferase Btr R,followed by optimizing the immobilization process using single-factor tests and response surface methodology.The final immobilization conditions were as follows:the mass ratio of carrier to protein 21,p H 8.2,temperature 17°C,salt ion concentration 460 m M（referred to the sodium ion concentration in the immobilization system）,PLP concentration 0.2 m M,and immobilization time 15 h.In this condition,the specific activity of immobilized enzyme was determined as 20.6 U·g^-1（wet support）with activity recovery 66.5%.The obtained immobilized enzyme maintained high stereoselectivity and stability,and could be easily adapted to industrial application.To sum up,this thesis mainly focused on two sugar aminotransferases that used for the preparation of two important C₇N aminocyclitol compounds:valienamine and its epimerβ-valienamine.On the one hand,we coevolved the catalytic activity and thermal stability of sugar aminotransferase Wec E by semi-rational design,laying the foundation for structure-fuction analysis of Wec E and artificial biosynthesis of valienamine.On the other hand,sugar aminotransferase Btr R was successfully immobilized with epoxy resins,which provided a new choice for the preparation of optical pureβ-valienamine.