【摘要】 对相同表达系统中产生的黑曲霉植酸酶(r-Anp)与烟曲霉植酸酶(r-Afp)的酶学特性进行了比较.两者的Km值都较低,但r-Anp的Vmax值远高于r-Afp(102.5 vs.29.5μmol.mg-1.min-1,P<0.05).r-Anp在pH 2.5仍具有植酸酶活力,而r-Afp则丧失全部活性.差示扫描量热法(DSC)研究发现r-Afp的蛋白质解链温度(Tm)低于r-Anp(59.1℃vs.62.1℃),但经热处理(90℃,20 min)后,前者残余更多的相对酶活(81%vs.38%,P<0.05).两种植酸酶对胃蛋白酶都有较高的耐受性,但r-Anp对胰蛋白酶的耐受性却不如r-Afp.当溶液中胰蛋白酶与植酸酶的质量之比为0.02时,r-Anp完全失活,而r-Afp则仍保留76.9%的相对酶活.结果表明两种植酸酶的性质具有明显的互补性.更多还原

【Abstract】 Artifical genes encoding Aspergillus niger NRRL3135 phytase(rAnp) and Aspergillus fumigatus ATCC 13073 phytase(r-Afp) were cloned into yeast expression vector pGAPZαA and then transformed into Pichia pastoris X-33 strain,respectively.The expressed r-Anp and r-Afp by Pichia pastoris under the same condition were purified and characterized.Both phytases exhibit low Km values,which represents high substrate affinity for phytate.However,r-Anp possesses a much higher Vmax value than r-Afp(102.0 vs.29.5 μmol·mg-1·min-1) at pH 5.0.pH optima measurement showed that r-Anp is still active around pH 2.5(72.6 U·mg-1 protein),while r-Afp is totally inactivated at this pH.DSC measurement indicated that the protein unfolding temperature(Tm) of r-Afp is lower than that of r-Anp (59.0 ℃ vs.62.1 ℃).However,the relative residue activity after thermo denaturation(90 ℃,20 min) of r-Afp is higher than that of r-Anp(81% vs.38%).r-Anp possesses much less resistance toward trypsin than r-Afp.It is thoroughly inactivated at the trypsin/phytase ratio of 0.02,while r-Afp keeps most activity at the same ratio.Both phytases are resistant to pepsin.SDS-PAGE results also support their different sensibilities to these proteases.Results showed that both phytases are quite different and mutually complementary in enzymatic properties.更多还原