Application Of Transglutaminase In The Enzyme Modified

In recent years,the methods that can accomplish the fixation of multiple enzymes include co-immobilization,co-embedding,and co-crosslinking.However,these methods have some problems.For example,glutaraldehyde cross-linking will cause loss of enzyme activity.Larger,genipin cross-linking will make the product appear blue and expensive.Layer-by-layer self-assembly,electrospinning,and other methods cannot achieve both the precise control of the enzyme molecule ratio and the substrate transfer.Specific recognition and cross-linking of enzyme molecules can achieve a controlled and orderly arrangement of enzyme molecules,shorten the distance between enzyme molecules,and complete the cascade catalysis of multiple enzymes or the synergistic effect of multiple enzymes.Therefore,specific cross-linking of different enzyme molecules according to requirements is one of the current research directions.Transglutaminase(TGase)can catalyze the acyl transfer reaction between the amide group of glutamine(Gln)and the primary amino group of lysine(Lys)of proteins or peptides,generating isopeptide bonds,so that proteins Covalent cross-linking occurred.However,natural globular proteins and columnar proteins cannot be used as substrates for glutamine transaminase.The existing method is to connect the N-terminal peptide tag of the protein,so that the protein can be recognized and crosslinked by glutamine transaminase.In this paper,a peptide chain tag sequence was inserted into the gene fragment of a protein to obtain a protein with a peptide chain tag at the N-terminus.Transglutaminaseis used to catalyze the cross-linking of peptide tags to achieve specific cross-linking of proteins.Different cross-linking results can be obtained by changing the peptide chain tag.In this study,the feasibility of peptide tagging was first verified in enhanced green fluorescent protein(EGFP).By expressing EGFP with S peptide,F peptide,or M peptide and measuring the fluorescence,verify that the peptide chain tag will not affect the protein itself.At the same time,the peptide chain tag can be used to achieve the specific cross-linked protein of glutamine transaminase.purpose.The peptide tag was then applied to the cross-linking of carbonic anhydrase and formate dehydrogenase.Cross-linking the double enzyme can increase the conversion efficiency of carbon dioxide to formic acid and shorten the transmission distance of the intermediate product bicarbonate ion.The F peptide,K2 peptide,and K3 peptide are cross-linked with the M peptide,respectively,and can realize "one-to-one","one-to-two",and "one-to-three"cross-linking of the double enzymes.According to experimental results such as enzyme activity retention,thermal stability,and catalytic efficiency,the"one-to-two" cross-linking method has the best effect,and there is almost no loss of double-enzyme enzyme activity after cross-linking.The thermal stability of formate dehydrogenase is improved after cross-linking,and it can exist stably at 35?.At this temperature,the yield of formic acid was the highest,and the catalytic efficiency was the highest,which was 5.8 times that of free enzyme.The last part of the research is to prepare a water-soluble polymer with a structure similar to glutamine.Glutamine transaminase can catalyze the cross-linking of the polymer with the enzyme to form a "polymer-protein"form.Conjugate.Immobilizing the double enzyme on the polymer can increase the thermal stability of the enzyme to 45?.At the same time,at 45?,the catalytic efficiency of the immobilized dual enzyme is 4.4 times that of the free enzyme,and it can also greatly increase the service life of the dual enzyme.In addition,with the intervention of thrombin,the protein can be separated from the conjugate to achieve protein separation.