Cloning And Expression Of The Gene And Structure-function Analysis Of Inulinases From Aspergillus Ficuum

Inulinases, which can be divided into exo-and endoinulinase according to their hydrolysis patterns,hydrolyze β-2,1-glycosidic linkages of the fructan. Endoinulinase （2,1-β-D-fructan fructanohydrolase, EC3.2.1.7） acts only on inulin and hydrolyze internal linkages of inulin to yield fructooligosaccharidesrandomly. Exoinulinase （β-D-fructan fructohydrolase, EC3.2.1.80） can hydrolyze terminal linkages ofinulin to yield fructose successively. Iinulinase broadly exists in various microorganisms and has beenwidely used in feed, food, medicine and energy industries.A strain of Aspergillus ficuum JNSP5-06which could secrete endoinulinase （endo I） and exoinulinase（exo I） was got from the soil in our laboratory. In order to improve the activity of A. ficuum JNSP5-06inulinase and to elucidate the catalytic mechanism of the enzyme, we cloned and expressed the gene. Thenwe studied the catalysis activity centres and the function of C-terminal in the molecular structure ofinulinase through directional mutation technique.According to the information of inulinase in Genbank, the DNA and cDNA of endo I and exo I werecloned from Aspergillus ficuum JNSP5-06. And the coding gene sequences of endo I and exo I wereregistered in the GenBank （FJ984582and HM587130）. The DNA sequence of endo I is1658bp, whichcontains1482bp of protein coding region and176bp of3’ noncoding region. Endo I consists of493aminoacids. Mw and pI are53.2kDa and4.33, respectively. The DNA sequence of exo I is1674bp, whichconsists of57bp signal peptide coding region,1557bp of protein coding region and60bp of intron codingregion. Mw and pI are57.2kDa and4.89, respectively.The encoding sequences of endo I and exo I were inserted into pET-28a（+） respectively. Therecombinant pET28a（+）-endo I and pET28a（+）-exo I were introduced into host E. coli BL21（DE3）. Afterinduced by IPTG, the recombinant JPE21/endo I and JPE21/exo I were successfully expressed. The optimalconditions for expression of JPE21/endo I were IPTG concentration of0.4mM, temperature of23°C andreaction time of6h. And the optimal conditions for expression of JPE21/exo I were IPTG concentration of0.4mM, temperature of26°C and reaction time of7h. After optimization, the specific activities ofJPE21/endo I and JPE21/exo I could reach to22.36u/mg and59.24u/mg, respectively.The recombinant protein was checked by SDS-PAGE, and the Mw of endo I and exo I were59kDaand61kDa, respectively. The optimum pH and temperature of endo were5and60℃. Ag+and Cu²⁺couldinhibit the activity of endo I thoroughly, and Fe²⁺、Fe3+and Al3+cound inhibit activity intensively whileZn²⁺、Mn²⁺and K+could activate endo I. Kmand kcatof endo I with inulin as substrate were （8.9±1.5） mMand（1.38±0.12）×103min-1respectively. Studies on characterization exo I showed optimum pH andtemperature were4and60℃for inulin, whereas the optimum pH and temperature for sucrose were5and55℃. The influence of the metal ions on the exo I activity was related to the substrate. Cu²⁺causeddecrease in exo I activity against inulin and sucrose by100%and90%, respectively, while Ag+causeddecrease in exo I activity against inulin and sucrose by45.6%and82.9%. Mg²⁺、Zn²⁺、Fe²⁺、Al3+and Ni²⁺could inhibit activity intensively while Mn²⁺could activate the activity. Kmand kcatwere （7.1±0.2） mM and（6.1±0.0）×104min-1for inulin, whereas Kmand kcatfor sucrose were （347.6±25.9） mM and （7.34±0.49）×105 min-1.Analysis of enzymatic hydrolysis products by TLC and liquid chromatography showed that endo Iuniquely hydrolyzed inulin, which produced disaccharide, trisaccharide and tetrose. While exo I not onlyhydrolyzed inulin, but also degraded sucrose. In addition, exo I had the weak ability of transfructosylation.Through homology modeling, sequence comparing and site-directed mutagenesis of endo I, it wasproved that Glu-20and Glu-210were catalytically important residues, and Asp-153played an importantrole for substrate recognition in the catalysis reaction.C-terminal domain was processed by deleted and swapped to orthomutation through over-lap PCR orPCR. It was found that enzyme activities were decreased sharply by C-terminal domain deletion andswapping mutants, and in this case endo I had the ability to degraded sucrose. The results showed thatC-terminal domain had the abililty to recognize long-chain inulin, stable enzyme molecule space structureand keep the enzyme catalysis activity.