为了提高木聚糖酶Xyn11A的稳定性及在低聚木糖生产中的应用潜力,该研究利用有机-无机杂化纳米花技术和以氨基乙基-琼脂糖为载体共价结合法固定Xyn11A,制备了固定化酶Xyn11A-Cu3(PO4)2和Xyn11A-Agarose,并比较研究了2种固定化酶的酶学性质,发现2种固定化酶的pH稳定性和热稳定性均有所提高,Xyn11A-Agarose稳定性更佳,在pH 4.0~4.5处理24 h后仍有40%~65%的残余活性,50 ℃处理3 h后仍有60%的残余活性。以榉木木聚糖为底物,与游离酶相比,Xyn11A-Cu3(PO4)2和Xyn11A-Agarose对底物的亲和力和催化效率有所降低,但有较好的重复使用次数。特别是Xyn11A-Agarose循环使用12轮后,仍有90%的残余活性。Xyn11A-Cu3(PO4)2水解甘蔗渣和玉米芯粉生成的低聚木糖中木二糖和木三糖含量最高,木四糖含量最低;而Xyn11A-Agarose催化生成的不同低聚木糖含量相当。2种固定化方法均能显著提高Xyn11A的稳定性,但Xyn11A-Agarose的稳定性和重复性优于Xyn11A-Cu3(PO4)2,表明Xyn11A-Agarose在生产低聚木糖中具有更大应用潜力。
In this study, the immobilization of xylanase Xyn11A using a protein-inorganic hybrid nanoflower system and covalent immobilization with aminoethyl-agarose were assessed to improve the enzyme properties.The enzymatic properties of Xyn11A-Cu3(PO4)2 and Xyn11A-Agarose were compared.It was found that the pH stability and thermal stability of the two immobilized enzymes were improved, and Xyn11A-Agarose was more stable. Xyn11A-Agarose retained 40%-65% residual activity after incubation at pH 4.0-4.5 buffer for 24 h and 60% residual activity at 50 ℃ for 3 h, respectively.Xyn11A-Cu3 (PO4)2 and Xyn11A-Agarose hydrolyzed beechwood xylan with Km of (10.14±3.56) mg/mL and (21.52±2.33) mg/mL, 2.39 and 5.06 times of free Xyn11A, respectively.The Kcat of Xyn11A-Cu3 (PO4)2 was 1.87 times higher than that of Xyn11A, but the Kcat/Km decreased by about 21%.The Kcat of Xyn11A-Agarose was similar to the free enzyme, but the Kcat/Km was only 22% of the free enzyme.After 6 cycles of reuse, Xyn11A-Cu3(PO4)2 retained 30% residual activity, while Xyn11A-Agarose still presented 90% residual activity after 12 cycles of reuse.The xylooligosaccharides produced by the hydrolysis of bagasse and corncob flour by Xyn11A-Cu3(PO4)2 had the highest content of xylobiose and xylotriose, and the lowest content of xylotetraose, while the different xylooligosaccharides catalyzed by Xyn11A-Agarose were almost equivalent in content.The stability and reusability of Xyn11A-Agarose were better than those of Xyn11A-Cu3(PO4)2, indicating that Xyn11A-Agarose has greater application potential in the production of xylooligosaccharides.
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