Cloning,Expression And Directed Evolution Of L-Asparaginase From Bacillus Megaterium

L-asparaginase(L-asparagine amidohydrolase;EC 3.5.1.1)can catalyze the hydrolysis and deamidation of L-asparagine to form L-aspartic acid and ammonia.L-asparaginases have received widespread attention in the treatment of acute lymphoblastic leukemia,lymphoid system malignancies and Hodgkin's lymphoma.Meanwhile,L-asparaginase can inhibit the generation of acrylamide in fried and baked foods without affecting food appearance and taste.However,most L-asparaginases have poor stability and low catalytic activity.Their activity can only stay stable at a narrow pH range.Considering the great theoretical and practical values of L-asparaginases,it is necessary to search for novel L-asparaginases.In this thesis,Bacillus megaterium H-1 was first isolated from soil.The ansA,ansZ gene encoding the L-asparaginase was cloned and expressed.The resulting products were then purified and the characteristics of enzyme were studied.Moreover,the directed evolution of this enzyme were studied.In addition,the modified L-asparaginase was used in fried potato chips,so as to prevent acrylamide formation.The detailed results are described as follows:1.Isolation and identification of bacteria producing L-asparaginase.Over 100 strains that produced L-asparaginase were screened using modified Czapek Dox medium.A strain(H-1)with a high L-asparaginase production was then selected,which was originally isolated from the soil containing wastes from a factory(Suzhou,China)specializing in asparagine production.The activity of enzyme produced by this strain reached 0.086±0.0023 IU/mL.Through microscope observation,biophysical and biochemical analysis,and molecular identification,it was found that the isolated strain H-1 was most closely related to Bacillus megaterium.2.Cloning and expression of L-Asparaginase from Bacillus megaterium H-1.The ansA(1050bp)and ansZ(1113 bp)genes were cloned on the basis of B.megaterium WSH-002 whole genome sequence from Genbank.These two genes encoded 349 and 370 amino acids,respectively.These genes were then connected to the expression vectors pET-30a and pET32a,separately,thus building four plasmids.The ansA and ansZ genes were expressed in Escherichia coli.The host E.coli named pET-30a-BM-ansZ produced enzyme with the highest catalytic activity,being 4.26±0.22 IU/mL after optimization.The activity was 50 fold higher than that of wild strain.In the end,the recombinant enzyme was named BmAase.3.Separation,purification and Biochemical Characterization of BmAase.The recombinant enzyme showed high activity(146.48 IU/mg).The molecular weight of BmAase was 39.63 kDa according to the results of SDS-PAGE and MALDI-TOF-MS.The optimum pH and temperature for BmAase activity were 7.0 and 40?,respectively.The enzyme was stable in the pH ranging 5.0-8.0.BmAase had satisfactory stability.After thermal treatment for 12 h,70%of activity was retained at 60 ? and 50%activity was retained at 70 ?.The Km and Vmax values of BmAase were 0.80 mM and 1.58IU/?g respectively.4.Directed Evolution of BmAase.The optimal conditions were 2 mM Mg2+ and 0.06mM Mn2+ in the error-prone PCR reactions.Mutants 2MH6 with the highest enzyme activity was selected from 8000 colonies through two rounds of error-prone PCR.The resulting enzyme activity was enhanced by 30%compared to that of BmAase.The mutants from error-prone PCR were further chosen as templates for DNA shuffling.Subsequently,the mutant S-CA3 was obtained from 5,000 mutants,the enzyme of which reached 249.01 IU/mg.The activity was 70%higher than that of BmAase.Moreover,this enzyme stayed stable at pH between 5.0 to 9.0,thus broadening the pH range in practical application.5.Application of L-Asparaginase in fried potato chips.Before frying,potato chips were treated by L-Asparaginase and the content of acrylamide was then measured using HPLC-MS.It was found that after frying,acrylamide content in control samples was 0.738± 0.0163 mg/kg,while the corresponding content in chips treated by L-Asparaginase was 0.056 ± 0.0056 mg/kg.Therefore,L-Asparaginase from B.megaterium can inhibit the formation of acrylamide during potato chip processing.