Forde Jessica, Tully Elizabeth, Vakurov Alex, Gibson Tim D, Millner Paul, Ó'Fágáin Ciarán
School of Biotechnology & National Centre for Sensors Research, Dublin City University, Dublin 9, Ireland.
Research Institute of Membrane and Systems Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.
Enzyme Microb Technol. 2010 May 5;46(6):430-7. doi: 10.1016/j.enzmictec.2010.01.004. Epub 2010 Jan 25.
Laccase from two different source organisms, Myceliophthora thermophila and Trametes hirsuta, were subjected to chemical modification in solution by (i) two bifunctional reagents, ethylene-glycol-N-hydroxy succinimide (EGNHS) and glutaraldehyde and (ii) by the monofunctional citraconic anhydride. The untreated and chemically modified forms of both enzymes were then immobilised onto three different types of mesoporous silicate (MPS) particle (MCM, CNS and SBA-15). Thermal stabilities of native, modified-soluble and immobilised laccases were then evaluated. Although the two laccases have similar lysine contents, those of M. thermophila are clearly more amenable to chemical modification. Treatment of the M. thermophila enzyme with EGNHS led to a 8.7-fold increase in thermal stability over the free soluble enzyme while glutaraldehyde gave a 5.7-fold increase. Increased activity of M. thermophila laccase occurred only with citraconic anhydride modification (a 3-fold increase), while the glutaraldehyde modification marginally increased the activity of the T. hirsuta enzyme (by 1.2-fold). Upon immobilisation onto MPS, the greatest increase in stability was for the glutaraldehyde-treated M. thermophila preparation on SBA-15 (24-fold over the soluble enzyme). Chemical modification of laccase from T. hirsuta with both glutaraldehyde and EGNHS gave only a 2-fold increase in stability, increasing >4-fold upon immobilisation onto SBA-15 and MCM-41/98.
来自两种不同来源生物嗜热毁丝霉和糙皮侧耳的漆酶，在溶液中通过以下方式进行化学修饰：（i）两种双功能试剂，乙二醇-N-羟基琥珀酰亚胺（EGNHS）和戊二醛；（ii）单功能的柠康酸酐。然后将两种酶的未处理和化学修饰形式固定在三种不同类型的介孔硅酸盐（MPS）颗粒（MCM、CNS和SBA-15）上。接着评估天然、修饰可溶性和固定化漆酶的热稳定性。尽管两种漆酶的赖氨酸含量相似，但嗜热毁丝霉的漆酶显然更易于进行化学修饰。用EGNHS处理嗜热毁丝霉酶导致其热稳定性比游离可溶性酶提高了8.7倍，而戊二醛处理使其提高了5.7倍。嗜热毁丝霉漆酶仅在柠康酸酐修饰时活性增加（提高了3倍），而戊二醛修饰使糙皮侧耳酶的活性略有增加（提高了1.2倍）。固定在MPS上后，稳定性提高最大的是戊二醛处理的嗜热毁丝霉制剂固定在SBA-15上（比可溶性酶高24倍）。用戊二醛和EGNHS对糙皮侧耳漆酶进行化学修饰仅使稳定性提高了2倍，固定在SBA-15和MCM-41/98上后稳定性提高超过了4倍。
