Computational Modelling And Production Optimization Of The Thermophilic Glucosidase FnCel5A For The Biotransformation Of Rare Gypenoside X?

The applications of pharmacologically bioactive rare ginsenosides in industrial and pharmaceutical fields have created a tremendous demand for their availability.However,because of their low solubility and slow permeability through the cell membrane,it is very hard for the human digestive system to directly absorb major ginsenosides,although these compounds account for the majority of total ginsenosides in crude ginseng.Therefore,the production of smaller deglycosylated ginsenosides by conversion of main ginsenosides is required not only to facilitate the achievement of various types of minor ginsenosides,but also to accelerate the study of the biological and pharmacological properties of rare ginsenosides.The biotransformation of major ginsenoside Rb1 to the more pharmacologically active rare gypenoside X? is a major challenge for the pharmaceutical industry.In previous work,our lab have cloned FnCel5 A,a GH5-?-glucosidase encoded by the Fnod-1560 gene of F.nodosum Rt17-B1.Here,we found that ?-glucosidases FnCel5 A accelerate the biotransformation of ginsenoside Rb1 into rare gypenosideX? at higher temperatures than any other enzyme.FnCel5 A is important because of its phenomenal characteristics such as higher thermostability,activity and specificity towards various substrates.These salient features make the ?-glucosidases FnCel5 A particularly suitable for the industrial biotransformation of major ginsenosides Rb1 into the rare gypenoside X?,involving longer treatment at high temperatures.This study focused mainly on the enzymatic biotransformation of the main ginsenoside Rb1 into the rare gypenoside X? with ?-glucosidase FnCel5 A of Fervidobacterium nodosum.1.Computational modelling of FnCel5 A for the biotransformation of rare gypX?In order to investigate the binding mechanism and key residues affecting the activity of FnCel5 A,eight unconserved residues within 10? of catalytic residue Glu167 were mutated by using site saturation mutagenesis.The activity of the mutants were checked on the two substrates CMC and Rb1 ginsenoside.Interestingly,the same mutants exhibit significantly different activity on these two substrates.All the eight mutants have same activity except N206 K had reduced activities toward carboxymethyl-cellulose while the three residues I164 N,P202A and N206 K had completely lost the activity towards Rb1 ginsenoside compared to that of wild type FnCel5 A.In order to investigate the mechanism of this phenomenon,the mutants were analyzed by molecular docking and MD simulation study.Through molecular docking and molecular dynamics(MD)simulation,the key role of these three residues related to substrate binding were identified.Both computational and mutational analysis suggested that I164 N,P202A and N206 K positions are critical for enzymatic activity.2.Optimization of the production of FnCel5 A in E.coli using response surface methodologyThe production of thermophilic enzymes on large-scale is a big challenge for the biological industries.In this study,we have enhanced the production of thermophilic glucosidase FnCel5 A through optimization of culture and cell disruption process by using response surface methodology.Firstly,a Box-behnken design was applied to optimize the physiochemical parameters such as isopropyl?-D-1-thiogalactopyranoside(IPTG)concentrations,induction temperatures,and induction times on the heterogeneous expression of FnCel5 A gene in E.coli.Secondly,an economical "thermolysis" cell lysis method for the liberation of the enzymes was also optimized using Box-behnken design.The maximum productivity of FnCel5 A achieved(5772 IU/L)illustrated that its production increased significantly after combining both optimal models.Significance of the studyGlucosidase FnCel5 A has several unique features as compared with other GH5 family members,especially its high catalytic activity and thermostabilty.Strikingly,FnCel5 A functions in biotransformation of Rb1 ginsenosides and other herbal saponins.Our research established the beneficial roles of FnCel5 A in ginsenosides production,and supports future work on FnCel5 A towards applications in nutraceuticals and pharmaceutical industries.