The Exploreation Of β-glucuronidase’s Thermostability Based On Crystal Structural Analysis

The recombinant β-glucuronidase from Penicillium purpurogenum Li-3 expressed in E. coli(PGUS-E) can hydrolyse glycosidic bond of glycyrrhizin(GL) to produce glycyrrhetinic acid monoglucuronide(GAMG)and glycyrrhetinic acid(GA). However, the poor thermal stability of the enzyme made PGUS-E hard to meet the needs of industrial application. Based on the crystal structural analysis, we designed a serious of site-mutations by using homologous sequence alignment methods and we aslo designed a serious of segment-mutations by using a self-built database. 34 mutations were constructed following the designing order “outside-to-inside” and “site-to-segment”, and thermal stability improved mutants were successfully obtained. A special loop near enzyme active center was investigated. The substrate selectivity was changed with the replacement of this loop. Our work provides a new idea for the further research on enzyme structure and function of β-glucuronidase. The main topics and results of this paper were listed as follows:By using the design strategy of homologous sequence alignment and the protocol of site-directed mutagenesis, the thermostability of PGUS-E was enhanced through the rational design. Three mutant enzymes with higher thermostability were obtained: Mutant 13, Mutant 15 and Mutant 16. Compared to PGUS-E, the halftime(T1/2, 65 °C) of Mutant 13, Mutant 15 and Mutant 16 were increased by 6 times, 1.25 times and 4.5 times, respectively. The results show that the introduction of amino acids from thermophilic bacteria’s proteins to the corresponding position, especially to the active center, can effectively improve the thermal stability of the protein.By using PYMOL software, self-writing script and B-factor analysis, the crystal structure data of Glycoside Hydrolase Family 2 in PDB and CAZy database were organized and classified. Nine highly stable Octopus Loops were obtained. The crystal structure of PGUS-E was also analysed and three highly unstable Loop areas were identifed. By using flush-end ligation, stable Octopus Loops were introduced into these unstable Loop areas in PGUS-E. Then ten mutant enzymes with higher thermostability were obtained: Mutant 19, Mutant 21 and Mutant 26. Compared to PGUS-E, the halftime(T1/2, 70 °C) of Mutant 19, Mutant 21 and Mutant 26 were increased by 3.2 times, 11.8 times and 9.4 times, respectively. The Kcat/Km of mutant enzymes remained nearly unchanged. The results show that the introduction of highly stable Octopus Loops to the corresponding position on the surface of protein can effectively improve the thermal stability of the enzyme without influencing the catalytic efficiency.By analyzing the complex crystal structure of PGUS-E and its substrate, an extremely flexible Loop near the active site was found and named for Binding-Loop. SWISS-MODEL and PEP-FOLD online homology modeling were used to compare the structure of At GUS, Au GUS, TM1062 with PGUS-E. These four enzymes share similar catalytic properties and catalytic core structure. An obvious variation was discovered just in this Loop area. Among them PGUS-E own the longest Binding-Loop, while Tm1062 own the shortest. Accordingly, the Binding-Loop of PGUS-E was replaced by using flush-end ligation and 3 mutants were successfully constructed: Mutant 4, Mutant 7 and Mutant 31. Compare to PGUS-E, all these mutant enzymes losted the ability to catalyze GL. What’s more, Mutant 4 losted half of catalyze ability to PNPG and GAMG; Mutant 7 losted the ability to catalyze PNPG, whihe retained the GAMG catalytic activity. Mutant 31 retained all the PNPG and GAMG catalytic activity. The changes of substrate selectivity proved that Binding-Loop do play an important role in process of substrate binding. The enzyme catalytic activity to different substrate can be manipulated by changing this loop area.