| L-asparaginase amidohydrolase（EC 3.5.1.1）can hydrolyze L-asparagine to produce L-aspartic acid and ammonia,which is widely present in the serum of microorganisms,plants and some rodents.In the pharmaceutical industry,it can be used to treat Hawkinson’s disease,acute lymphoblastic leukemia and malignant lymphoma.In the food industry,it can be used in high-temperature fried foods to reduce the production of carcinogen acrylamide.In this study,L-asparaginase mutants with significantly improved enzyme activity and thermal stability were constructed,and 5’untranslated region（UTR）transformation and medium optimization were used to achieve high-yield fermentation of L-asparaginase.The main findings are as follows:（1）The L-asparaginase encoding gene derived from Rhizomucor miehei was ligated into the vector p ET28a and introduced into Escherichia coli,then successfully expressed in E.coli.After fragmentation and purification,its specific enzyme activity was determined to be 509.1U·mg-1.The results of the enzymatic properties showed that the optimal temperature was45°C,the optimal p H was 7.0.It was relatively stable at 30°C and poor at 45°C,and it had good stability in the range of p H 6.0-7.0.Among the investigated metal ions,there was no ion that had a significant activation effect on the enzyme.（2）To address the shortcomings of low enzyme activity of Rm Asnase and poor thermal stability at 45°C,molecular modifications were made.Based on sequence alignment and Fold X algorithm,15 sites were selected for site-directed mutation,and one mutant with significantly improved enzyme activity and two mutants with significantly improved thermal stability were obtained.The specific enzyme activities of mutants A344E,S302I,and S302M were increased by 54.5%,32.0%,and 27.7%,respectively.The difference in enzymatic properties between the wild type and the mutants were studied.The optimal temperature of mutant A344E was 40°C,and the optimal temperature of S302I and S302M was 50°C.After placing the mutant enzyme and the original enzyme at 45°C for 35 h,the residual enzyme activity of the wild enzyme was only about 10%,while the residual enzyme activity of S302I and S302M still retained more than 50%.（3）According to the results of single point mutation,forward mutants A344E,S302I,and S302M were selected for combined mutation,thus two combined mutants called A344E/S302I and A344E/S302M were obtained.The specific enzyme activities of mutants A344E/S302I and A344E/S302M were increased by 43.8%and 39.2%,respectively,compared with the original enzyme.The enzymatic properties of the combined mutants were studied.The results showed that the optimum temperature of A344E/S302I and A344E/S302M was 50°C,and the thermal stability at 45°C was better than that of the original enzyme.After being placed at 45°C for 35 hours,the combined mutant strain still retained more than 60%of the enzyme activity,and the wild enzyme only retained about 10%.The optimum p H and p H stability were not much different than the original enzyme.（4）Using the food-safe strain Bacillus subtilis 168 as the host,construct B.subtilis168/p MA5-A344E/S302I.The 5’untranslated region（UTR）transformation strategy was used to increase the expression level of L-asparaginase derived from Rhizomucor miehei in Bacillus subtilis.After UTR modification,the crude enzyme activity of L-asparaginase increased from the original 2.1 U·m L-1to 13.3 U·m L-1.Through the optimization of fermentation medium and the amplification experiment of 5 L fermenter,the enzyme activity output of recombinant bacteria B.subtilis 168/p MA5 UTR-A344E/S302I reached 521.9U·m L-1,which provided a new strategy for the safe industrial production of L-asparaginase. |
