The Improved Cross-linked Enzyme Aggregates And Its Application In Proteomics And Biocatalysis

In order to overcome the problems in preparation and application of cross-linked enzyme aggregates (CLEAs), two kinds of improved strategy have been developed in this thesis. The conventionally prepared CLEAs of trypsin, the sugar-assisted CLEAs of penicillin G acylase and the porous-CLEAs (p-CLEAs) of papain have been applied in the digestion process of proteomics, synthesis of amoxicillin and degradation of chitosan, respectively.1. Conventional CLEAs of trypsin has been prepared with 90% ethanol as precipitant and 0.075% glutaraldehyde as cross-linker with the activity yield of 44%. The optimum temperature (70oC), optimum pH (9.0), thermal stability and storage stability were all increased compared with free trypsin. The excellent operation stability and activity depended on its unique“scaffold-like”structure.2. Applied the conventional CLEAs of trypsin in the protein digestion of proteomics. CLEAs showed excellent performance of anti-autolysis and rapid digestion rate under high enzyme concentration. With the assistant of high temperature and ultrasound, high-through analysis has been realized. The digestion time has been sharply shortened to 60min and 60s, respectively, as well as with the higher sequence coverage.3. In order to increase the activity yield during the preparating step in preparing CLEAs of penicillin G acylase, an improved strategy has been developed through addition of sugar in the precipitation step to prepare sugar-assisted CLEAs. The activity yield of sugar-assisted CLEAs increased 30% by this improved preparation method compared with conventional CLEAs. The“water-substitute mechanism”can be inducted to explain the protection of sugar on penicillin G acylase molecules during the precipitating step.4. Applied the sugar-assisted CLEAs of penicillin G acylase in the synthesis of amoxicillin. The results showed that the yield and selectivity of penicillin G acylase CLEAs increased obviously when CLEAs were prepared by the improved strategy. The amoxicillin yield can reached 57% when using 30% methanol/buffer as reaction medium and with the high substrate radio. 5. In order to overcome the defect of low catalytic efficiency of conventional CLEAs in macromolecular substrate reaction, an improved method has been developed through using starch as pore-making agent to prepare porous-CLEAs. (p-CLEAs). Compared with conventional CLEAs, p-CLEAs of papain increased the hydrolysis efficiency on bovine serum albumin and ovalbumin by 95.9% and 90.4%, respectively. p-CLEAs were characterized by SEM, which showed that the higher catalysis performance of p-CLEAs resulted from its porous structure and higher specific surface area.6. Applied the p-CLEAs of papain in the degradation of chitosan to produce bioactive chitosan oligomers. The reducing sugar concentration was 85% of that catalyzed by free papain, which is 4 times higher than conventional CLEAs under the same condition. With ultrafiltration membrane separation, it obtained 67.5% weight of the antibacterial oligomers which molecular weight was in the range of 5~10kD.