Gene Cloning Of β-glucuronidase From Fungi And Effect Of Non-conservative Sequence On The Enzyme Properties

Three fungal strains, namely, P. purpurogenum Li-3, A. terreus Li-20and A. ustusLi-62, were screened in our previous studies and represented three modes of GLbiotransformation. In this research, we’ve cloned the β-glucuronidases from A. terreusLi-20and A. ustus Li-62, and sequence effect on the enzyme activity ofβ-glucuronidases have been also studied. Main results are as follows:β-glucuronidase from A. terreus Li-20（Atgus） was cloned consisting of1974bp,encoding637aa with an intron; and two β-glucuronidase from A. ustus Li-62werecloned, which were1917bp and1827bp encoding628aa and608aa, respectively.All of the enzymes shows high similarity with each other, however, C-terminalnon-conservative sequence of AtGUS consisited of65aa and was longer thanPGUS（β-glucuronidase from P. purpurogenum Li-3）, AuGUS1and AuGUS2, whichmay related with enzyme properties. So non-conservative sequence and it propertieswas studied in the following experiments to discover its effect on the enzymeproperies.The partial sequence AtGUS（-3t）（1–592aa） was amplified to determine the effectsof the non-conservative sequence on the enzymatic properties. AtGUS and AtGUS（-3t）were expressed in E. coli BL21, producing AtGUS-E and AtGUS（-3t）-E, respectively.At the similar optimum temperature （55℃） and pH （AtGUS-E,6.6; AtGUS（-3t）-E,7.0）conditions, the thermal stability of AtGUS（-3t）-E was enhanced at65℃, and the metalions Co²⁺, Ca²⁺and Ni²⁺showed opposite effects on AtGUS-E and AtGUS（-3t）-E,respectively. Furthermore, Km of AtGUS（-3t）-E was just nearly one-seventh that ofAtGUS-E （12.9mM）, whereas the catalytic efficiency of AtGUS（-3t）-E was3.2foldhigher than that of AtGUS-E, revealing that the truncation of non-conservativesequence can significantly improve the catalytic efficiency of AtGUS. Conformationalanalysis illustrated significant difference in the secondary structure between AtGUS-Eand AtGUS（-3t）-E by circular dichroism （CD）.In order to discover effect of non-conservative sequence on AtGUS, another twoC-terminal non-conservative sequence truncations [AtGUS（-t1）:1⁶04aa andAtGUS（-t2）:1⁶33aa] were amplified according to analysis of modeler9v7andprotein hydropathic character. Both of them were transformed into E. coli, producingAtGUS（-t1）-E and AtGUS（-t2）-E, respectively. The optimum temperature and pH ofAtGUS（-t1）-E and AtGUS（-t2）-E was similar, which was both55℃and pH5.0, respectively. the effect of metal ions howed similar effects on AtGUS（-t1）-E andAtGUS（-t2）-E, respectively. Km of AtGUS（-t1）-E and AtGUS（-t2）-E was lower thanthat of AtGUS-E. Furthermore, catalytic efficiency of AtGUS（-t1）-E was2.54mM s^-1,while, AtGUS（-t2）-E(8.31mM^-1s^-1) was increased compared to AtGUS-E(2.24mMs^-1). Conformational analysis illustrated significant difference in the secondarystructure between AtGUS（-t1）-E and AtGUS（-t2）-E by circular dichroism.Polar amino acid sequence of non-conservative sequence of AtGUS has beenobtained by gene synthesis and was added right after TIM barrel domain of AtGUS byoverlap extension PCR, named Atgus（-△NP）. Recombinant protein AtGUS（-△NP）-Ewas expressed by E.coli, and was also purified by Ni-NTA. The optimum temperatureand pH of AtGUS（-△NP）-E was50℃and5.6, respectively. The enzyme can beactivated by metal ions Mg²⁺, Mn²⁺, Co²⁺, Ca²⁺and Ni²⁺. Km of AtGUS（-△NP）-Ewas4.85mM, and Kcat/Km was^-1.90mM^-1s^-1. The secondary structure of AtGUS（-△NP）-E analysed by circular dichroism was different from AtGUS-E.So we can conclude that the properties of non-conservative sequence of AtGUS havegreat effect on enzyme activity and structure, and rational design according tonon-conservative sequence characteristic can promote the enzyme properties.