Fermentation Optimization, Molecular Simulation Of Recombinant Esterase From Microbacterium Chocolate And Molecular Design Of The Aldose Reductase Inhibitors

An Esterase (EstSIT01) from Microbacterium chocolatum catalyzes the asymmetric hydrolysis of meso diesters of D-biotin, generating (4S,5R)-monoesters which are key intermediates for synthesizing D-biotin. To further enhance the expression of EstSIT01, fermentation optimization for recombinant E.coli with the gene of EstSIT01 has been carried out. The contents of fermentation optimization included carbon sources, nitrogen sources, metal ions and so on, which have been inspected one by one. The optimal culture components and condtions include the following:maltose 30 g/L, corn steep liquor 20 g/L, NaCl 5 g/L, Fe2+ 9 mM, pH 7.0; the cells were cultured in 30? for 3 h and the isopropyl-?-D-thiogalactoside (IPTG) was added into flasks with 0.6 mM of final concentration, and cell was induced at 30? for 8 h. The results showed that cell mass and enzyme activity were improved significantly, and the optimized cell mass, specific activity and total activity were increased 2.5-fold,4-fold and 10-fold, respectively, which were 3.47 g/L,242.82 U/g and 842.72 U/L.Esterase EstSIT01 is a typical serine hydrolase, but its spatial structure is unknown. Molecular modeling techniques were used for understanding the structure and reaction mechanism of EstSIT01 esterase with the substrate. Firstly, catalytic triads (Ser110-Asp268-His330) were found out by blasting with other serine hydrolases whose catalytic center has been known; secondly, the structure of EstSIT01 was obtained by homology modeling, and the model is reliable by Ramachandran and Verify 3D; thirdly, molecular docking was carried out to understand the reaction mechanism of esterase EstSIT01 with diester, and interpret the reason why the esterase EstSIT01 has stereoselectivity to diester. Docking results show that a proton transfer chain was formed correctly; moreover, a short distance between groups involved in the reaction and the lower binding energy are the reason of stereoselectivity.The aldose reductase (ALR2, EC1.1.1.21) has been confirmed to be a target for the treatment of chronic diabetic complications and the ALR2 inhibitors (ARIs) are the most effective drugs. In this work, a series of ARIs were utilized to design potent ARIs using a combination of molecular modeling techniques. First, the threedimensional quantitative structure-activity relationship (3D-QSAR) models were built by CoMFA (comparative molecular field analysis), CoMSIA (comparative molecular similarity indices analysis) and Topomer CoMFA. The results show that the best CoMFA model has q2=0.894 and r2=0.994, the best CoMSIA (comparative molecular similarity indices analysis) model has q2=0.907 and r2=0.995, and the Topomer CoMFA analysis has q2=0.752 and r2=0.985, which all exhibited satisfied correlativity and prediction. The results also indicated the steric, electrostatic, H-donor and Hydrophobic fields play key roles in models. Then the molecular docking was performed to explore the conformations of the inhibitors and key amino acid residues at the docking pocket, and the binding relationship between the inhibitors and the receptor protein. Molecular dynamics (MD) simulations further validate the reliability of the docking results. By analyzing the above results, six potential new compounds were identified with good predicted biological activity. The acquired information in this study is believed to provide valuable guidance for the development of potent effective ALR2 inhibitors.