Bond Selectivity Modification Of Recombinant β-Glucuronidase

Engineering enzymes to meet special requirements of diverse industrialapplications is an important task in Protein engineering, because not all wild typeenzymes have the desired qualities for specific applications. In this paper，in order tocreate biocatalysts suit able for the biosynthesis of GAMG (a high-potency sweeteneras well as a novel anticancer agent)， PGUS-E, a Promising recombinant β-glucuronidase from P. purpurogenum Li-3for bioteehnologica1applications，wasengineered by protein engineering (semi-rational directed evolution and rationaldesign). One mutant primarily produced GAMG was created, whereas the parentenzyme (PGUS-E) primarily formed end-product GA.The mutant producing GAMGwith purity may be a good starting point for the biosynthesis of GAMG.The reactionprocess and the molecular mechanism of GAMG formation were investigated. Theeffects of the amino acid substitution on the structure and function of PGUS-E relatedto bond specific was dicussed.On the ground of analysis of obtaining crystalline structure of PGUS-E withglucuronic acid (GlcA), learn that the substrate-binding pocket consist of two smallpockets A and B. That is, pocket A contain10amino acid and pocket B contain15amino acid.Pocket A recognize glycol of GAMG and GL, however pocket B onlyrecognizes glycol of GL.Study on spatial conformation, hydrogen bond, hydrophobic force, polarity ofamino acid in pockets B, side-directed saturation mutagenesis was carried out at8sites.Screening of the resulting libraries were carried out through thin layer chromatography(TLC) and high performance liquid chromatography (HPLC)with the difference ratioof products, three mutantsR329K,T369V,V447Q(M42),were found. One of which,mutant V447Q remarkably enhanced bond selectivity. Bond selectivity of M42(V447Q)was71.28%.In order to further improve bond selectivity of M42,two mutants werecreated. Bond selectivity of mutant M42-1(T369V and V447Q) was81.78%, increased10.5%compared with M42. Bond selectivity of mutant M42-2(V447Q/R563K) was95.5%, increased24.22%compared with M42. Characterization of the best mutant pET-M42-2was performed and its kineticparameters and effects of pH as well as temperature on its activity and stabilitydetermined. The best mutant pET-M42-2showed similar optimum pH and temperaturewith the PGUS-E, but the catalytic efficiency was decreased by nearly fourfoldcompared with PGUS-E, mainly because of its lowered affinity of glycol of GL andGAMG. GL as the substrate: The Km, kcatand kcat/Kmwere7365.214uM，10442.81min-1，1.417885uM-1min-1respectively. GAMG as the substrate. The Km, kcatandkcat/Kmcan’t detected.In summary, the mutant pET-M42-2has the potential to produceGAMG as the main product...