黑曲霉水解京尼平苷β-葡萄糖苷酶的分离纯化及其酶学性质

Purification and characterization of geniposide-hydrolyzing β-glucosidase from Aspergillus niger

黑曲霉Aspergillus niger发酵豆渣产β-葡萄糖苷酶(BGL)。粗酶液依次经过乙醇沉淀和DEAE-Sepharose Fast Flow离子交换层析,获得了两种电泳纯的β-葡萄糖苷酶BGL1和BGL2,它们的相对分子质量分别为102kDa、97kDa,总酶活回收率达到48%。在pH2.5及温度为55℃时,两种BGL水解京尼平苷的速度均达到最大;BGL水解活性受到Na+的激活但不受K+的影响,但Mg2+、Ba2+、Cu2+、Fe2+、Zn2+和Hg+等离子对BGL活性有不同程度的抑制作用(其中对BGL1的抑制普遍强于BGL2),而且同一种离子对不同BGL活性的影响性质不同,Ca2+显著激活BGL1而对BGL2无影响,Fe3+显著抑制BGL1而激活BGL2。在同样条件下,BGL对对硝基苯-β-D吡喃葡萄糖苷(pNPGlu)、对硝基苯-β-D吡喃半乳糖苷(pNPGal)和水杨苷均有催化活性,其中对pNPGlu的Km值最小,BGL1、BGL2分别为0.764和1.934mmol/L;而BGL对京尼平苷水解的催化效率最高,BGL1、BGL2的Kcat/Km值分别为4.28×104和1.04×105L/mol·s。BGL1、BGL2活性的pH稳定范围相近,分别为pH2.0-7.0、pH2.0-8.5,但它们的热稳定性差异较大,55℃时BGL2保温60min,酶活保留了60%,而BGL1的酶活半衰期只有10min。

Using the combination of ethanol precipitation and DEAE-Sepharose Fast Flow chromatography, two forms of extracellular β-glucosidase from Aspergillus niger in bean dregs broth, designated as BGL1 and BGL2, were purified with a recovery of 25% and 23%, respectively. By running SDS-PAGE, their molecular weights were determined to be 102kDa for BGL1 and 97kDa for BGL2. At pH 2.5 and 55 ℃, BGL activity for hydrolyzing geniposide reached maximum, and was activated by Na＋ , but inhibited to varying degrees by Mg^2＋, Ba^2＋, Cu^2＋, Fe^2＋, Zn^2＋and Hg^＋ at 10mmol/L. The inhibition of metal ions on BGL1 were stronger than that on BGL2. For all this, no significant effect of K＋ on BGLs was observed. Ca^2＋ activated BGL1 significantly but had no impact on BGL2, while Fe3＋ inhibited BGL1 but activated BGL2. Though p-nitrophenyl-d-glucopyranoside （pNPGlu） was the best substrate for BGL1 and BGL2 hydrolyzing pNPGlu, p-nitrophenyl-d-galactopyranoside, salicin and geniposide, the hydrolytic efficiencies of BGLs to geniposide were the highest. The Km values of BGLs to pNPGlu were 0.764mmol/L and 1.934mmol/L, and the Kcat/Km values of BGLs to geniposide were 4.28×10^4 and 1.04×10^5L/mol·s, respectively. BGL1 was highly stable over pH range of 2.0-7.0 at 20℃ , and exhibited a half-life of 10min at 55℃, while BGL2 showed a wider range of pH stability （from 2.0 to 8.5） and the activity retained approximately 60% of the original activity after incubation for 60min at 55℃.