Bryjak Jolanta, Liesiene Jolanta, Kolarz Bozena N
Faculty of Chemistry, Department of Bioorganic Chemistry, Wrocław University of Technology, 50-370 Wrocław, Wybrzeze Wyspiańskiego 27, Poland.
Colloids Surf B Biointerfaces. 2008 Jan 15;61(1):66-74. doi: 10.1016/j.colsurfb.2007.07.006. Epub 2007 Jul 21.
The main point was the search for a proper carrier and the kind of carrier activation for trypsin (EC 3.4.21.4) immobilization. The acrylic and cellulose-based carriers were specially prepared in that they possessed the most often used anchor groups: -OH, -NH(2), DEAE and/or -COOH. The immobilization procedures were selected to apply mainly to protein amine groups and appropriate anchor groups on the carrier. As activity tests low (N-benzoyl-dl-arginine-p-nitroanilide, BAPNA) and high (casein) molecular weight substrates were used. It was found, as a rule, that trypsin bound to -COOH groups with the help of carbodiimide was less active and that the amount of bound protein and measured activity (BAPNA) are considerably higher when protein is immobilized via divinyl sulfone. Both rules were observed irrespective of the nature of the polymer matrix. Both types of carriers were found suitable for trypsin immobilization and they were far better than the corresponding Eupergit C-bound enzyme preparations. Taking into account storage stability and activity for both substrates, the divinylsulfone linkage formed between unmodified Granocel and trypsin was the most effective method for the enzyme immobilization. For this preparation, BAPNA and casein conversion, thermal stability at 60 degrees C and estimated kinetic parameters were compared with those obtained for the native enzyme. It was shown that mass transport limitations could be effectively eliminated by suitable conditions and immobilized trypsin was considerably more stable. The values k(cat)/K(m) indicated that the immobilized enzyme was even better as amidase activity was regarded and its potential for protein hydrolysis was only less than twice.
主要要点是寻找合适的载体以及用于固定化胰蛋白酶（EC 3.4.21.4）的载体活化方式。基于丙烯酸和纤维素的载体经过特殊制备，因为它们具有最常用的锚定基团：-OH、-NH₂、二乙氨基乙基（DEAE）和/或-COOH。选择的固定化程序主要适用于蛋白质胺基和载体上合适的锚定基团。作为活性测试，使用了低分子量（N-苯甲酰基-dl-精氨酸对硝基苯胺，BAPNA）和高分子量（酪蛋白）底物。通常发现，借助碳二亚胺与-COOH基团结合的胰蛋白酶活性较低，而当通过二乙烯基砜固定蛋白质时，结合的蛋白质量和测得的活性（BAPNA）要高得多。无论聚合物基质的性质如何，这两条规律都成立。发现这两种类型的载体都适用于胰蛋白酶固定化，并且它们比相应的与Eupergit C结合的酶制剂要好得多。考虑到两种底物的储存稳定性和活性，未改性的Granocel与胰蛋白酶之间形成的二乙烯基砜键合是酶固定化的最有效方法。对于这种制剂，将BAPNA和酪蛋白转化率、60℃下的热稳定性以及估算的动力学参数与天然酶获得的参数进行了比较。结果表明，通过合适的条件可以有效消除传质限制，并且固定化胰蛋白酶的稳定性大大提高。k(cat)/K(m)值表明，就酰胺酶活性而言，固定化酶甚至更好，其蛋白质水解潜力仅略低于天然酶的两倍。
