Characterization And Application Of L-asparaginase

As a potential carcinogen,acrylamide（AA）is widely found in carbohydrate-rich foods that are processed at high temperatures,raising international health alerts.Currently,many strategies have been developed to control the level of AA in food.L-asparaginase（L-ASNase,E.C.3.5.1.1）can effectively inhibit the formation of the AA by hydrolyzing the AA precursor L-Asparagine,and this mild and controlled biological control of AA has received widespread attention.Therefore,it is crucial to explore L-ASNase with excellent properties,especially good thermostability.Firstly,to meet the need for thermostability in the food industry,the L-ASNase gene from thermophilic microorganism Palaeococcus ferrophilus was discovered through the NCBI database and heterologously expressed in E.coli BL21.The pure enzyme Pafe-L-ASNase was obtained by Ni²⁺column,with the molecular weight of 36 k Da,and existed in dimeric forms.However,the expression level was not high.The identification results of Pafe-L-ASNase showed that:the optimum p H was 8.5 and the optimum temperature was 95℃.The optimum metal ion was Mg²⁺,and 1 m M Mg²⁺could significantly enhance the activity of Pafe-L-ASNase by about 4.08-fold.The results of the thermostability study showed that the residual enzyme activity was about 40%after holding at 95℃for 2 h,and the T_m value was 96.53℃,indicating that it has excellent thermostability.As shown by the substrate specificity and reaction kinetics study,the enzyme showed strict substrate specificity for L-asparagine without glutaminase activity.The K_m value and the k_cat value of Pafe-L-ASNase was 5.00 m M and 1340 s^-1,respectively.The storage stability was analyzed,and the enzyme activity remained more than98%after 3 months of storage at 4℃.Then,to enhance the application safety of Pafe-L-ASNase,the recombinant plasmid was constructed by“simple cloning”and heterologously expressed in B.subtilis 1A751（dal^-）to achieve the food-grade expression of Pafe-L-ASNase.After shake flask fermentation,the highest OD₆₀₀ value reached about 12.6 at 15 h and the highest fermentation enzyme activity reached about 29.9 U/m L at 31 h.The conventional french fries process was improved by combining enzyme treatment and blanching treatment as one step.Notably,the Pafe-L-ASNase pre-treatment（10 U/m L,85℃,15 min）could significantly reduce the production of 8469μg/kg AA in French fries,which has good prospects for food application.Afterward,due to the low level of expression of Pafe-L-ASNase,it is difficult to adapt to industrial needs.Therefore,the L-ASNase gene from medium temperature source Lactobacillus secaliphilus was selected and heterologously expressed in E.coli BL21.The pure enzyme Lase-L-ASNase was obtained by Ni²⁺column,with the molecular weight of 35 k Da,and existed in dimeric forms.The enzymatic properties of Lase-L-ASNase were identified as follows:the optimum p H was 8.0 and the optimum temperature was 60℃.The enzyme could still retain more than 70%of its initial activity after holding at 55°C for 10 min,and the T_m value was65.44℃,indicating that its thermostability still needs to be improved.The K_m value and the k_catvalue of Lase-L-ASNase was 4.78 m M and 887 s^-1,respectively.The enzyme could use asparagine and glutamine as substrates,which may have clinical toxicity.Finally,the thermostability of Lase-L-ASNase was further enhanced to meet the needs of the food industry.The homology and conservatism of Lase-L-ASNase and L-ASNase from thermophilic microorganisms were analyzed by amino acid sequence comparison to predict the key amino acid residue sites that may cause the difference in thermostability of L-ASNase from medium-temperature and thermophilic sources.Ten single-site mutants were constructed,among which L24Y,S55T,and V155S showed more than 1℃elevated T_m values.Notably,the residual enzyme activity of the mutant V155S was about 47.66-fold that of the Lase-L-ASNase after incubating at 65℃for 5 min.The half-life of mutant V155S at 65℃was 6.25-fold that of Lase-L-ASNase,indicating that the thermostability of mutant V155S was enhanced.