Cloning,Expression And Molecule Reforming Of L-asparaginase From Bacillus Licheniformis

L-asparaginase is an amide hydrolase that hydrolyzes amide groups of L-asparagine side chains into aspartic acid and ammoni—L-asparaginase widely exists in microorganisms.A series of studies have reported that L-asparaginase can inhibit the formation of carcinogens-acrylamide during thermal food processing,while it has no influences on food appearance,flavor and nutritional values.In addition,L-asparaginase can also be used to treat children with acute lymphoblastic leukemia,Hodgkin's lymphoma and other diseases.However,L-asparaginase has low enzymatic activity and thermal stability,which limit its application in food and medical industries.Therefore,it is necessary to develop high-quality L-asparaginase.In this thesis,the L-asparaginase gene derived from Bacillus licheniformis was cloned and heterologously expressed.Meanwhile,the enzymatic properties of the recombinant enzyme were studied.The L-asparaginase gene was modified by molecular directional evolution and semi-rational design.Then,the enzymatic properties of the resulting mutant enzyme were studied.All the studies can provide guidance about scaling up the production of L-asparaginase.The results of this study are shown as follows.1.Cloning and expression of L-asparaginase from Bacillus licheniformis.The ansZ(1131)sequence of L-asparaginase gene was successfully cloned according to the whole genome sequence of Bacillus licheniformis in Genbank,which encodes 376 amino acids with a theoretical molecular weight of about 40 kDa.The recombinant expression vector pET-30a-Bli-ansZ was constructed and the ansZ gene was expressed in Escherichia coli.The recombinant enzyme activity was 3.97±0.16 IU/mL.The recombinant enzyme specific activity was 148.83 IU/mg after purification.The optimum reaction conditions for this recombinant enzyme were pH 8.0,40 ?.More than 70%of the relative activity can be retained in the pH range between 5.0 to 10.0.After treatment at pH 8.0 and 50? for 8 h,this recombinant enzyme still had 80%of the relative activity.The Km values of the recombinant enzyme were 0.692 mM and the maximum reaction rate Vmax was 1.68 IU/?g.These results indicated that this recombinant enzyme had a considerably high pH adaptability and thermal stability,which can been potentially used in food industry.2.Directed evolution of Bacillus licheniformis L-asparaginase.The optimal error-prone PCR reaction conditions were of dTTP 0.2 mM,dCTP 0.2 mM,Mg2+concentration 3/5/7 mM,and Mn2+concentration 0.05mM.The highest activity of mutant F5A11 screened from more than 12,600 mutant strains had the highest activity of 8.961 ±0.225 IU/mL,which was 2.26 fold higher than that of the wild strain.Next,the first four highest mutants were selected as parent genes,and three mutants S10,S16 and S21 were screened from 6500 DNA shuffling mutant libraries.The mutant S10 had the highest activity of 8.961 ±0.225 IU/mL,which was 2.26 fold higher than that of the wild strain and the specific activity of the purified mutant S10 was 306.589 IU/mg,which was 2.06 fold higher than that of the wild strain.At the same time,the Km value of the mutant enzyme S10 was lower than that of wild type enzyme,and the Kcat/Km value was higher than that of wild type enzyme,indicating that the affinity of mutant enzyme was enhanced and the catalytic efficiency was improved.Three-dimensional modeling analysis and circular dichroism results showed that 43-terminal mutant amino acids could enhance the interactions between substrates and the catalytic hydrogen bonds 67 amino acids could increase the mutagenicity,thus favoring enzyme binding with the substrate and the release of the products.As a result,the substrate affinity and conversion efficiency of this enzyme was improved.This study indicated that the directional evolutionary strategy can effectively improve the activity of Bacillus licheniformis L-asparaginase.3.Semi-rational design of Bacillus licheniformis L-asparaginase.The mutant S10 plasmids was used as a template,and the mutations were established at the points of 43 and 67,respectively.The mutants K43Y/N67S and K43E/N67A with enhanced hige enzyme activity were selected.Meanwhile,the mutant K43Y/N67A was obtained by two-point combination mutagenesis from K43Y/N67S and K43E/N67A mutants.The studies about enzymatic properties of the mutants K43Y/N67S,K43Y/N67A and K43Y/N67A showed that the enzyme activity of K43Y/N67S was 14.45±0.37 IU/ml,which was 3.64 fold higher than that of the wild enzyme.After thermal treatment at 50 C for 8 h,the half-life of the mutant enzyme K43Y/N67S was extended by 3 h compared with that of the wild enzyme.The results of three-dimensional modeling and circular dichroism exhibited that the number of a-helix in the secondary structure of the mutant K43Y/N67S increased,and the increase of the benzene ring structure and the enhancement of the hydrophobicity could change the protein conformation.The increase in the flexibility of the monomeric interface may be beneficial to the enhancement of the catalytic activity.Semi-rational design improved the Bacillus licheniformis L-asparaginase enzyme activity,and its thermal stability was also enhanced.