Glycosidation Of Trypsin With Galactose

Trypsin (EC 3.4.21.4) is a kind of alkaline protease which is widely applicated in food, feedstuff, medicine and other fields, and it's often used as model in the researches of protein engineering. In this research, a monoaminated galactose derivative was attached to trypsin to modify it via a carbodiimide-catalyzed reaction. A ricinus agglutinin affinity chromatography column was prepared and used to separate the modified and non-modified enzyme. The results showed that this affinity chromatography column had a good binding with the modified enzyme, and can purify it from reaction mixture. The modified trypsin was confirmed by absorption and fluorescence spectroscopy. The absorption peak at 280nm became weaker and wider. The result of fluorescence spectroscopy showed that the character emission peak of tryptophane residue at 340nm had a obvious decrease and blueshift. The thermostability of modified trypsin was enhanced, based on the examination of enzymatic activity. The result showed that galactose modified enzyme was more stable at temperatures higher than 40℃, in comparison with its native counterpart, and the thermostability was enhanced from 49.3 to 62.1℃for modified trypsin. This study will lay a foundation for widening application of trypsin.