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题名	CTAB反胶团萃取分离工业脂肪酶的研究
作者	沈睿
学位类别	硕士
答辩日期	2004
授予单位	中国科学院过程工程研究所
授予地点	中国科学院过程工程研究所
导师	刘会洲
关键词	脂肪酶 反胶团 蛋白质纯化
其他题名	Extraction of Industrial Lipases with CTAB Reverse Micelles
中文摘要	脂肪酶是工业生产中一种重要的酶类，其分离纯化研究具有重大的应用价值，本 论文利用十六烷基三甲基澳化按（CTAB）反胶团对两种工业脂肪酶（扩展青酶PF868 和lipaseM）进行萃取分离，研究内容如下：（1）采用单一CTAB反胶团体系对两种脂肪酶进行萃取时，国产脂肪酶在pH9.0 左右有最大萃取率，而liPaseM在pH10.0左右有最大萃取率，hpaseM萃取的主要 推动力是静电作用力，而国产脂肪酶萃取的主要推动力是疏水相互作用力；采用 CTAB混合反胶团对国产脂肪酶进行萃取时，PEG400、PEGZO00、Tween85、TBP、 TRPO、BuAc与CTAB形成的混合反胶团的萃取率大于单一CTAB反胶团，lipaseM 也有类似的结果；两种脂肪酶的萃取率随反胶团相中TBP、TRPO以及BuAc含量增 加而增加，而随着Tween的含量增加有一个最大值。（2）采用单一CTAB反胶团体系时，国产脂肪酶的反萃在1 mol／L KBr、反萃 水相pH值6.0左右有最大值，而llpaseM在lmol／L KBr、反萃水相pH值4．5左右有 最大值，这可能与两种酶的等电点不同有关；采用CTAB混合反胶团时，TBP、TRPO 以及BuAc与CTAB形成的混合反胶团对两种脂肪酶的反萃行为不同，这可能是由于 两种酶的反萃推动力不同造成的，在前萃水相pH值为6-10.5的范围内，Tween60、 Tween4O以及PEG2000与CTAB形成的棍合反胶团对国产脂肪酶的反萃率大于单一 CTAB反胶团，而几种混合反胶团对lipaseM的反萃率均没有单一CTAB反胶团效果好。（3）采用单一CTAB反胶团对两种脂肪酶进行萃取分离时，国产脂肪酶在前萃 水相pH值为8.0左右、反萃水相pH值为4.6左右有最高的酶活回收，lipaseM在前 萃水相pH值为8.0左右、反萃水相pH值为5.0左右有最高的酶活回收、采用TBP、 TRPO、BuAc以及Tween与CTAB形成的混合反胶团对两种脂肪酶进行萃取分离时， 在前萃水相pH6.6-10.5的范围内，TRPO、TBP以及Tween60和CTAB形成的混合反胶团对国产脂肪酶的酶活回收高于单一CTAB反胶团，并且T妙O＋cTAB混合反 胶团的酶活回收可以达到72％；对livaseM而言，仅有Tween40和CTAB形成的混 合反胶团的酶活回收高于单-CTAB反胶团。
英文摘要	Lipases [triacyl glycerol acylhydrolases (E.C.3.1.1.3)] occupy a place of prominence among biocatalysts owing to their novel and multifold applications in oieochemistry, organic synthesis, detergent formulation and nutrition. The separation and recovery of lipases with reverse micelles have received wide attention. The effects of some factors such as pH of initial aqueous phase, type and concentration of salt, solvent type, enzyme concentration, phase ratio, etc. on the forward extraction of lipases were studied. The results showed that the forward extraction ratio of the two lipases increased with the increase of pH of initial aqueous phase and concentration of surfactant, while decreased with the increase of ionic strength, enzyme concentration and phase ratio. The forward extraction of lipase M reached stabilization in about 30min, while native lipase only needed 15min. it could be inferred that the forward extraction of lipase M could be mainly explained by the electrostatic interaction, while the forward extraction of native lipase could be mainly explained by the hydrophobic interaction. In the chosen solvents, n-hexane had the highest forward extraction. The forward extractions of lipases have also been studied with mixed reverse micelle systems formed with CTAB (cetyl trim ethyl ammonium bromide) and PEG, Tween, BuAc, TRPO and TBP. The forward extraction of native lipase increased with the increase of pH of the initial aqueous phase slowly. The addition of PEG2000, Tween85, BuAc, TRPO and TBP could improve the extraction of native lipase into CTAB reverse miceliar systems. The backward extraction of the two kinds of lipases increased with the backward extraction time, IP A concentration of backward extraction aqueous phase. The backward extraction of lipase M increased with the n-butanol concentration of organic phase, while the backward extraction of native lipase increased initially and then decreased. The backward extraction of lipase M increased with the pH of backward extraction aqueous phase, while the backward extraction of native lipase increased initially and then decreased. The backward extraction of the two kinds of Upases has also been studied with mixed reverse micelles formed with CTAB and PEG, Tween, BuAc, TRPO and TBP. With the increase of hexane concentration in organic phase, the addition of TRPO could improve the backward extraction of lipase M with CTAB reverse micelles. With the increase of pH of backward extraction, the addition of BuAc, TRPO and TBP increased the backward extraction of native lipase initially, then achieved stability. The addition of TRPO and TBP could increase the backward extraction of lipase M initially and then decreased. With the increase of pH of initial aqueous phase, the backward extraction of native lipase almost kept unchangeable with the addition of Tween into CTAB reverse micelles. The backward extraction of lipase M increased initially and then decreased with the addition o Tween, BuAc, TRPO and TBP. Only the addition of TBP could improve the backwan extraction of CTAB for lipase M. The activity recovery of the two Upases increased initially and then decreased with th pH of initial aqueous phase. The addition of TBP and TRPO could increase the activil recovery of native lipase, and the activity recovery of native lipase could reach 72% wi-the mixed reverse micelles formed with TRPO and CTAB. The addition of BuAc, TRP and TBP decreased the activity recovery of lipase M and the addition of Tween40 cou improve the activity recovery of lipase M. Key words: lipase, reverse micelles, CTAB, protein purification the backward extraction of native lipase increased initially and then decreased. The backward extraction of lipase M increased with the pH of backward extraction aqueous phase, while the backward extraction of native lipase increased initially and then decreased. The backward extraction of the two kinds of Upases has also been studied with mixed reverse micelles formed with CTAB and PEG, Tween, BuAc, TRPO and TBP. With the increase of hexane concentration in organic phase, the addition of TRPO could improve the backward extraction of lipase M with CTAB reverse micelles. With the increase of pH of backward extraction, the addition of BuAc, TRPO and TBP increased the backward extraction of native lipase initially, then achieved stability. The addition of TRPO and TBP could increase the backward extraction of lipase M initially and then decreased. With the increase of pH of initial aqueous phase, the backward extraction of native lipase almost kept unchangeable with the addition of Tween into CTAB reverse micelles. The backward extraction of lipase M increased initially and then decreased with the addition of Tween, BuAc, TRPO and TBP. Only the addition of TBP could improve the backward extraction of CTAB for lipase M. The activity recovery of the two Upases increased initially and then decreased with the pH of initial aqueous phase. The addition of TBP and TRPO could increase the activity recovery of native lipase, and the activity recovery of native lipase could reach 72% with the mixed reverse micelles formed with TRPO and CTAB. The addition of BuAc, TRPO and TBP decreased the activity recovery of lipase M and the addition of Tween40 could improve the activity recovery of lipase M.
语种	中文
公开日期	2013-09-16
页码	84
内容类型	学位论文
源URL	[http://ir.ipe.ac.cn/handle/122111/1428]
专题	过程工程研究所_研究所（批量导入）
推荐引用方式 GB/T 7714	沈睿. CTAB反胶团萃取分离工业脂肪酶的研究[D]. 中国科学院过程工程研究所. 中国科学院过程工程研究所. 2004.

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