Expression Of Heterologous GGT By Lactococcus Lactis And Its Immobilization Of Food Enzymes

Lactococcus lactis is a Gram-positive bacterium that complies with the GRAS standard.Due to its fast growth,simple metabolism,and clear genetic background,it has received great attention in the food industry and clinical medicine area.There are many expression systems of L.lactis,among them,Nisin-induced NICE expression system is the most widely used.With the help of the L.lactis expression systems,more and more heterologous proteins have been produced in L.lactis.?-glutamyltranspeptidase?GGT?is widely used in the food industry and pharmaceutical fields due to its ability to catalyze the hydrolysis of glutathione or its derivatives/transfer?-glutamyl to various acceptors.In order to increase the yield of GGT and simplify the purification process,in this work,the ggt gene from Bacillus amyloliquefaciens BH072 and the gene of Usp45 signal peptide were fused together,and a recombinant strain which could secret and express GGT was constructed.Because of the instability and non-reusability of the free enzyme used in catalyze reactions,we immobilized the heterologous Ba GGT and MTG which were expressed in L.lactis on magnetic nanoparticles by covalent binding,high specific activity,high stability and reusable immobilized enzyme was obtained.Finally,the characteristics of the immobilized enzymes were explored under the optimal immobilization conditions.The main results are as follows:?1?Extract the genomic DNA of B.amyloliquefaciens BH072.Used this genomic DNA as a template to amplify the gene of ggt_?sp and fused with the signal peptide(sp_usp45).The fusion fragment was introduced into L.lactis to construct inducible and constitutive expression vectors,respectively,and the optimal expression conditions of these two expression systems were explored.Under the optimal expression conditions,Ba GGT was purified by immobilized metal affinity chromatography.The concentration and specific activity of Ba GGT from constitutive expression system were 95.0±0.4mg/L and 58.9±1.3 U/mg;The concentration and specific activity of Ba GGT from inducible expression system were 90.2±0.7 mg/L and 57.3±0.9 U/mg.The mature Ba GGT-6His could catalyze L-Gln and ethylamine hydrochloride to synthesize L-theanine.?2?Fe₃O₄MNPs with core-shell structure are prepared.These nanoparticles have magnetic properties and good dispersibility,Fe₃O₄MNPs could be covalent bonding with enzymes better after being modified by the coupling agent EDC-NHS.We immobilized the Ba GGT and MTG secreted from L.lactis on Fe₃O₄MNPs and optimized the immobilization conditions.The specific activity of the immobilized enzymes was higher than that of free enzymes.The specific activity of Ba GGT@Fe₃O₄MNPs was 77.8±1.7 U/mg,and the specific activity of MTG@Fe₃O₄MNPs was29.1±0.4 U/mg.Comparing the properties of the immobilized enzyme with free enzyme,respectively,we found that the catalytic efficiency,stability and reusability of the immobilized enzymes were significantly improved.However,the affinity?K_m?with the substrate was decreased.?3?The magnetic nanoparticles coated with chitosan were prepared,the properties and micromorphology of the nanoparticles were studied.The surface of the CS-Fe₃O₄MNPs is smooth,it has magnetic and better biocompatibility.The functional groups of magnetic nanoparticles were modified by 3-APTES,which made the magnetic nanoparticles with rich amino groups.Ba GGT was immobilized on CS-Fe₃O₄MNPs via covalent bonding,the specific activity of immobilized enzyme was68.2±1.2 U/mg under the optimal immobilization conditions.The catalytic efficiency was higher than Ba GGT@Fe₃O₄MNPs.