Immobilization Of Buckwheat Trypsin Inhibitor And Preparation Of Sustained-release Carrier

Buckwheat Trypsin Inhibitor?BTI?is a competitive inhibitor of trypsin.It has specific affinity with trypsin and can be used to prepare affinity resin to purify trypsin by one-step chromatography.Recent studies have shown that BTI can inhibit the proliferation of a variety of tumor cells in vitro,and can delay the healthy lifespan of C.elegans,indicating its good application prospect.Based on the analysis of the BTI primary sequence and three-dimensional structure,the mBTI mutant with improved inhibitory activity was prepared and optimized for its immobilization conditions,which provided theoretical basis for the further application of BTI.The main contents of this study are as follows:The first part: The mutant mBTI with enhanced activity was prepared by site-directed mutagenesis technique,and its inhibitory activity was studied.The p QE-30-mBTI plasmid was constructed by PCR using a site-directed mutagenesis kit.After induced expression by IPTG in E.coli M15,purification by Ni²⁺ affinity chromatography and desalination on a sephadex G25 gel column,mBTI was purified and identified by SDS-polyacrylamide gel electrophoresis.The inhibitory activities of mutant mBTI and BTI were compared,and the effects of three organic reagents on mBTI inhibitory activity were also investigated.The results showed that the p QE-30-mBTI plasmid was successfully constructed in this experiment.The electrophoresis-purified mBTI was obtained by affinity chromatography.The inhibitory activity of the mutant mBTI was significantly enhanced.Methanol reduced the inhibitory activity of BTI.Dimethylacetamide and acetonitrile had a certain enhancement effect on mBTI.When the concentration of acetonitrile was more than 40%,the enhancement effect was weakened.The second part:The immobilization of mBTI was carried out by using polyvinyl alcohol and sodium alginate as the carrier,and the immobilization conditions were optimized.According to the three single-factor results of CaCl₂ concentration,carrier-inhibitor volume ratio and immobilization time,the Box-Behnken center test was designed.The response surface method was used to optimize the immobilization conditions,and the optimal immobilization conditions and main influencing factors were obtained.The results showed that the optimal immobilization conditions were optimized by the response surface: CaCl₂ concentration was 5.5%,the ratio of carrier to inhibitor volume was 1.6:1?m L/m L?,and the immobilization time was 31 min.The inhibition rate of immobilized BTI is 72.4%,which is less than the predicted value of 74.3%.It is proved that the response surface method is optimized for the mBTI immobilization process parameters,which lays a theoretical foundation for the preparation of mBTI sustained-release carrier.The third part: The chitosan-alginate microspheres containing mBTI were prepared by three methods,and their sustained release properties were studied.Method 1: The mBTI-sodium alginate solution is directly dropped into a mixed solution of chitosan and CaCl₂ for fixation?one-step method?;the second method is to first drop mBTI-alginate into the CaCl₂ solution,and then transfer to 30 minutes later.Fixation in chitosan solution?two-step method?;method three is to transfer the microsphere prepared by the two-step method to sodium alginate solution for fixation?double cross-linking?.The results showed that the entrapment rate of mBTI-alginate-chitosan microspheres prepared by one-step method was higher than that of two-step method and double cross-linking method.The prepared microspheres can slowly release mBTI in both simulated gastric fluid and intestinal fluid,but the release rate in simulated intestinal fluid is faster than that of simulated gastric juice.In 0-10 min,the release rate of mBTI in the reeze-dried microspheres was faster than that in the un-lyophilized microspheresf.After 10 min,both forms of freeze-dried microspheres could achieve slow release of mBTI.