Bioorthogonal Immobilization Of Enzyme Depending On The Orientation Of Unnatural Amino Acids

Enzyme immobilization technology has many advantages, such as maintaining the stability of enzyme, adjusting the total enzyme activity and easy recycling, which have great broad application prospects and market potential. The covalently immobilized enzyme prepared using conventional method is often achieved by glutaraldehyde or benzoquinone crosslinker. In addtion, the enzyme used in the immobilization needs purification, which polluting the environment seriously and taking a long time. It is the most serious that a covalent bond connection relies mostly on the side chain amino of natural amino acid and occurred random. The active site of enzyme was usually buried and destroyed from the random chemical linkage, which resulted in great decrease or complete loss of the enzyme catalytic performance and stability. With the rise of the one step purification and immobilization technology, pepole could purify and immobilize enzymes by one-step reaction from the crude enzyme solution, which simplifies the experimental procedures and reduces the production costs. However, there is no more than ten research articles in the existent method now, heavy metal ions such as Co2+、Cu2+、Ni2+are often used and the only method is by means of His-Tag labeling. In this paper, on the base of the oriented modification of the natural enzyme using unnatural amino acid (UAA), we achieved the site specificity covalent immobilization of enzyme, and achieved the purification of enzymes in the process at the same time using the orientation function and the connection reaction of orthogonality of UAA,to open up new methods to overcome the problems in traditional method.In our work, We described the site-specific introduction of aldehyde groups into lipase and the6-amino-acid consensus sequence (LCTPSR, aldehyde tag, ald6) was recognized by the formylglycine-generating enzyme(FGE). We used the site-directed mutagenesis primer that is aldehyde tag-encoding bases amplify the gene of lipase, and inserted it into the expression vector pET28a(+). The recombinant plasmid of pBAD/myc-his A-FGE and pET28a-lipald6were transfected into the expression host E. coli BL21(DE3). The FGE oxidizes the cysteine of LCTPSR to formylglycine by post-translational modification and the aldehyde tag was formed. The fluorescence experiments was performed to prove that the formylglycine lipase containing aldehyde groups. The recombinatant lipase keeps the same activity as the natural lipase. We use the schiff base reaction betweeen the aldehyde group of lipald6and MCFS-NH2to achieve the one-step purification and immobilization of enzyme from the crude lipald6. The activity of the immobilized formylglycine lipase was0.5times of free lipase and2.5times of the immobilized lipase using1.5mM glutaraldehyde. The catalytic efficiency (Kcat/Km) of the immobilized formylglycine lipase was about3.0times of the immobilized lipase using1.5mM glutaraldehyde. In the case of thermal stability at50℃, it still retained58.7%of its activity after heating for19h, which was1.5-fold and1.7-fold greater than those of the immobilized lipase when1.5mM and15mM of glutaraldehyde were used, respectively. Because formylglycine modified lipase fixed on N or C terminal, in order to further develop the point fixed enzyme method, we use the biological orthogonal method to fixed-point modification of lipase, and using the azide group of azide phenylalanine lipase to fixed point by click on the chemical reaction happen without copper. By analysing the three-dimensional structure of lipase, we chose and determined the non-active site of the lipase, mutated the lipase using the overlap extension PCR technology. The gene was amplified and inserted into the expression vector pET24a(+), the recombinant plasmids of pACYC184-2MjtRNACUATyr, pBR322-MjTyrRS10and pET24a-lipTAG were transfected into the expression host E.coli BL21(DE3). Five recombinant enzymes protein (AzPhe-lip50、AzPhe-lipl37、AzPhe-lip243、AzPhe-lip274、AzPhe-lip355) were produced with0.5mM AzPhe and100μ.M IPTG induction, respectively. By Western Blot and ESI-MS test, all of these recombinant enzymes protein were confirmed to have azide group which indicated that the AzPhe were inserted into the lipase protein stucture to complete the oriented modification. Among the five recombinatant enzyem, the AzPhe-lip137has a maximum enzyme activity and its value is213.3U/mg. The AzPhe-lip355has a better protein expression than others, its value is5.7mg/L. The activity of the immobilized AzPhe-lipl37is175.8U/mg, it is106.9%of the free enzyme. In the case of thermal stability at50℃, it still retained70.9%of its activity after heating for29h, which was1.5-fold greater than the immobilized lipase when1.5mM of glutaraldehyde were used.The aim of this research is to specific modify the lipase by the H-4-Azido-phenylalanine and formylglycine. Using this method, a variety of functional groups were introduced into enzyme to create a new technology for one-step purification and oriented immobilization enzyme from the crude enzyme. This research is expected to enrich the one-step purification and oriented immobilization and provide a new way of enzyme immobilization study to achieve low cost and no heavy metal pollution. In our method, random covalent linkage that occurs usually in the traditional covalent immobilization could be avoided, which would enrich and promotethe development of the enzyme immobilization in theory and application research.