Stabilizing hyperactivated lecitase structures through physical treatment with ionic polymers
Stabilizing hyperactivated lecitase structures through physical treatment with ionic polymers
复制标题
DOI：
10.1016/j.procbio.2014.05.009
复制
发表时间：
2014-09-01
影响因子：
4.4
通讯作者：
Fernandez-Lafuente, Roberto
中科院分区：
文献类型：
--
作者：
dos Santos, Jose C. S.;Garcia-Galan, Cristina;Fernandez-Lafuente, Roberto
在溴化氰交联4%琼脂糖（CNBr）珠上共价固定了卵磷脂酶Ultra，保持了70%的初始活性。在Triton X-100、十二烷基硫酸钠（SDS）和十六烷基三甲基溴化铵（CTAB）的存在下，固定化酶的活性得到了提高（例如，使用CTAB时，固定化酶的活性可达800%）。而CTAB和Triton X-100即使在低浓度下也会对酶的稳定性产生负面影响，SDS在1%浓度下也不能长期使用。为了在没有洗涤剂的情况下保持酶的超激活构象，在有洗涤剂的情况下，在固定化酶的孵育过程中添加离子聚合物。用聚乙烯亚胺包被固定化酶后，酶活性提高了3倍。然而，在0.1% SDS （v/v）的存在下，该涂层的酶活性提高了50倍。使用PEI和0.01% (v/v) CTAB时，卵磷脂酶活性降至10%。使用不可逆抑制剂，可以证明PEI/SDS-CNBr-Lecitase制备使其催化Ser比未修饰的CNBr-Lecitase更容易被反应介质接近。(C) 2014 Elsevier Ltd.版权所有。
Lecitase Ultra has been covalently immobilized on cyanogen bromide cross-linked 4% agarose (CNBr) beads, maintaining 70% of the initial activity. The activity of the immobilized enzyme was improved in the presence of Triton X-100, sodium dodecyl sulfate (SDS), and cetyltrimethyl ammonium bromide (CTAB) (e.g., up to 800% when using CTAB). However, CTAB and Triton X-100 presented a negative effect on enzyme stability even at low concentrations, and SDS cannot be used for a long time at 1% concentration. To maintain the hyperactivated conformation of the enzyme in the absence of detergent, ionic polymers were added during incubation of the immobilized enzyme in the presence of detergents. Coating the immobilized enzyme with polyethylenimine in aqueous buffer (PEI) produced a 3-fold increase in enzyme activity. However, in the presence of 0.1% SDS (v/v), this coating produced a 50-fold increase in enzyme activity. Using PEI and 0.01% (v/v) CTAB, the Lecitase activity decreased to 10%. Using irreversible inhibitors, it could be shown that the PEI/SDS-CNBr-Lecitase preparation allowed its catalytic Ser to be more accessible to the reaction medium than the unmodified CNBr-Lecitase. (C) 2014 Elsevier Ltd. All rights reserved.