Heterologous Expression Of Glutamine Transaminase And Its Application In Preparation Of Antimiciobial Emulsifier

Transglutaminase（TGase）has excellent protein cross-linking ability,so it is widely used in food,medicine,textile,leather and other industries.TGase are widely present in nature,and microbial derived TGase also known as mTGase,it has excellent enzymatic properties that other sources of TGase do not have.Currently,all of commercial TGase are mTGase.The industrial production of mTGase is mainly achieved through the fermentation of Streptomyces mobaraensis.Although the yield is considerable,but the fermentation cycle is long,the fermentation process is difficult to control,and the later purification is difficult.Therefore,expressing the gene fragment encoding mTGase to obtain recombinant strains with short fermentation cycles and single fermentation broth composition is the future trend of mTGase production.The S.mobaraensis CICC 11019 strain is a wild strain obtained through the CICC,and its mTGase can be safely used in various areas such as food and medicine.Bacillus subtilis is a certified food grade safe（GRAS）strain.In this thesis,we will take mTGase gene fragment from S.mobaraensis CICC 11019,and the mTGase successfully expressed in B.subtilis.In order to improve the enzyme activity of the recombinant strain,the mTGase from the recombinant strain was modified by site directed mutagenesis,and recombinant strains with significantly improved enzyme activity were obtained.The mTGase in the fermentation broth was purified by nickel column.The chitosan andε-Poly-L-lysine was successfully crosslinked to prepare an antimiciobial emulsifier,and the emulsifier was characterized.The main research results are as follows:（1）The pro-mTGase and mTGase gene obtained from S.mobaraensis CICC 11019 was successfully heterologously expressed in B.subtilis.The gene sequence of pro-mTGase and mTGase was found in NCBI.According the sequence,we designed two pairs of primers for amplifying the sequence（pro-mTGase and mTGase）by PCR.Then this two target fragments were connected with p MA5 shuttle plasmid separately,and the recombinant plasmids were transformed into Escherichia coli DH5αreceptive cells for obtaining the positive transformants.The recombinant plasmids were extracted from the positive transformants and transformed into B.subtilis 168 and B.subtilis WB600 receptive cells,and the recombinant strains（B.subtilis 168/p MA5-pro-mTG,B.subtilis 168/p MA5-mTG and B.subtilis WB600/p MA5-mTG）were constructed.The final mTGase productions were 0.0837 U/m L,0.1134 U/m L and 0.1297 U/m L.（2）In order to improve the enzyme activity of the recombinant strain,recombinant strain B.subtilis 168/p MA5-mTG was selected using computer aided rational design to transform it through site directed mutagenesis.After sequencing the mTGase,three-dimensional modeling is carried out,and dock with the substrate,key amino acids affecting the activity of mTGase are analyzed to determine the site for site directed mutagenesis.The amino acid sequence of mTGase is blast compared in NCBI,and the sequences with a similarity of 70%-90%are selected.The key amino acids was replaced with amino acids that are less conserved and frequently presented in homologous sequences.Four mutation sites were identified:Arg121-Glu,Glu239-Leu,Glu374-Pro and Tyr376-Gly.Using reverse PCR to mutate the identified amino acid sites,the plasmid p MA5-mTG was used as a template to amplify the recombinant plasmid with the mutation site.The recombinant plasmid with the mutation site was transferred to DH5αreceptive cells.After amplification,it was transformed into B.subtilis 168 receptive cells,This four recombinant strains(B.subtilis 168/p MA5-mTG（R121E）,B.subtilis 168/p MA5-mTG（E239L）,B.subtilis 168/p MA5-mTG（E374P）and B.subtilis168/p MA5-mTG（Y376G）were constructed.The final mTGase productions were 0.509 U/m L,0.505 U/m L,0.469 U/m L and 0.377 U/m L,respectively,which were 4.49,4.45,4.14 and 3.32times higher than before.And the mTGase from the fermentation supernatant of the recombinant strain B.subtilis 168/p MA5-mTG（R121E）was purified using a nickel column,and the high-purity mTGase with no obvious impurities was successfully obtained.（3）The mTGase was used to crosslink the tchitosan andε-PL to prepara nanoparticle emulsifier and to optimizate the preparation conditions of chitosan-ε-PL nanoparticles.Determine the optimal conditions for chitosan andε-PL is 3:1,reaction at 50℃for 3 hours.The prepared nanoparticle emulsifier was characterized,and the results of size,FT-IR and DSC showed successful crosslinking.The lotion Em and Em_mTG were prepared,and the salt and heat resistance of the prepared lotion were tested.The results proved that the lotion Em_mTG had better heat resistance and higher concentration of salt resistance.The antimiciobial activity of the prepared nanoparticles was tested,and the test results showed that they were effective against Staphylococcus aureus and E.coli has good inhibitory effects.