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1、Advance and aspect of trypsin improving technology刘子墨胰蛋白酶 Trypsin胰蛋白酶是动物消化道中的一种主要的碱性蛋白水解酶。胰蛋白酶在脊椎动物中，作为消化酶而起作用。在胰脏，胰蛋白酶原作为胰液的成分而分泌，激活后分解为活化胰 蛋白酶。胰蛋白酶能把多肽链中赖氨酸和精氨酸残基中的羧基侧切断。胰蛋白酶 Trypsin胰蛋白酶的年消耗量占到了酶制品市场 的3%左右，被广泛应用于食品业、纺织 业、皮革工业、制药业、畜牧业和现代 生物技术等领域。但是由于天然活性低、稳定性差等原因 ，受到了很大的限制。蛋白质的改性研究可以提高其稳定性， 满足工业生产中
2、酸碱及高温条件的要 求。Trypsin immobilization technology液相胰蛋白酶存在着严重的自溶、解折叠作用，导致生产上直接应用的不便。酶固定化技术克服这一不足，使酶的稳定性增加，并可重复利用。 载体的选择：高分子载体、无机载体、复合载体 固定化方法 壳聚糖 海藻酸钠 珠状琼脂凝胶 纤维素及其衍生物 丙烯酸共聚物 甲基丙烯酸甲酯MMA 聚微球N-乙烯基-苯丙氨酸高分子载 体 玻璃纤维膜 硅凝胶 硅微芯片无机载体 将有机聚合物材料涂在有刚 性结构的无机载体上 磁化涤纶-酰肼颗 粒 涂覆交联壳聚糖的硅胶 多糖-纤维素 碳纳米复合材料复合载体Trypsin immobiliza
3、tion technologyMacromolecular modification大分子修饰剂是通过与酶分子表面的lys残基共价结合，改变酶分子微环境进而影 响其催化特性的。修饰反应随机，没有特异位点，产物不均一。 多糖类大分子及其衍生物 交联剂 聚乙二醇及其衍生物 蔗糖 聚蔗糖 右旋糖酐 肝素 羧甲基纤维素 环糊精多糖类大 分子 戊二醛 聚戊二醛 蔗糖二乙醛交联剂 聚乙二醇1100、2000、 5000 聚乙二醇均三嗪衍生物 聚乙二醇琥珀酰亚胺 聚乙二醇马来酸苷 聚乙二醇胺类衍生物PEGMacromolecular modificationEffects of Polymer Molecu
4、lar Weight on the Size, Activity, and Stability of PEG-Functionalized TrypsinWe therefore pursued the investigation of PEG-functionalized trypsin to higher molecular weights (10 and 20 kDa) to generate polymer- enzyme conjugates in the range of 30 -150 kDa.In addition to activity, we focused our ana
5、lysis on the protein structure and hydrodynamic radius of the conjugates to better determine their ability for exploiting the EPR effect.Effects of Polymer Molecular Weight on the Size, Activity, and Stability of PEG-Functionalized Trypsin，Biomacromolecules 2010Deconvolution of the CD spectra at 25C
6、 indicated that the conjugates have roughly the same amount of secondary structure order as native trypsinTo quantify the change in secondary structure, deconvolutions of CD data taken from 20-85C. The proportion of -helices, -strands, and turns all decreased, while unordered residues increased.The
7、denaturation temperature (Td) increased from 48.7C in free trypsin to 56.3-59.7C in PEG-trypsin conjugates.PEGylation significantly helps to maintain the secondary structure of trypsinCharting the calculated percent of unordered residues for all four conjugates demonstrated that PEG confers thermal
8、stability to trypsin. While unordered residues increased 15% between 20 and 85C for free trypsin, only an increase of 3-5% in PEGylated trypsin conjugates was observed.They have been attributed to increased hydrogen bonding around the enzyme and lead to greater protection from autolysisSystematic st
9、udies of the effect of PEG molecular weight and linking chemistry on the biological activity and particularly the thermal stabilityEffect of PEG molecular weight and linking chemistry on the biological activity and thermal stability of PEGylated trypsin，International Journal of Pharmaceutics， 2008tr
10、ypsin-succinoylated mPEG conjugates，SAtrypsin-cyanurate mPEG conjugates，CCtrypsin-mPEG conjugates，CTPEG-trypsin conjugates containing the higher molecular weight of mPEG (5000 g/mol) were more stable than free trypsinp Although chemical modification of mPEG to trypsin could decrease activity of tryp
11、sin, but the modified trypsin conjugates containing the higher molecular mass mPEG showed higher affinity to binding site of enzyme with substrate, higher thermal stability, more stable against autolysis and had an increased t1/2 compared to the native enzyme. p The trypsin-conjugated mPEG 5000 havi
12、ng cyanurate linker demonstrated the best thermal stability. Stability and conformational change of methoxypolyethylene glycol modification for native and unfolded trypsin ，Food Chemistry，2014PEGylation has been shown to increase thermal stabilityp The stability increase by PEGylation is likely caus
13、ed by either decreased charge repulsion on the surface, due to modification of the lysines or by the amphiphilic nature of PEG. p A second theory is that the increased thermal stability is caused by steric hindrance of the PEG chain, resulting in reduced autolysis and an increase in the number of hy
14、drogen bonds, resulting in decreased thermal denaturationPost-production modification of industrial enzymes，Appl Microbiol Biotechnol ，2014PEG 修饰的胰蛋白酶PEG修饰可以降低酶分子的免疫原性，延长其在体内的半衰期，增加蛋白质的溶解性和热 力学稳定性以及抗蛋白酶降解的能力。综合修饰反应中酶活的损失及修饰后蛋白酶性质的改变，4000-6000分子量的PEG修饰效果 比较好；若考虑消除抗原性，40000-60000分子量的PEG效果好。THANK YOU FOR LISTENING!THANK YOU FOR LISTENING!THANK YOU FOR LISTENING!THANK YOU FOR LISTENING!
