| In 2002, Swedish researchers first reported the large amounts of acrylamide are widely existed in carbohydrate-rich food prepared by high temperature. Acrylamide has neurotoxicity, genotoxicity, reproductive and developmental toxicity, and potent ial carcinogenic, therefore taking measures to reduce the acrylamide content in food is of great significance. The results from the previous studies confirmed that asparagine and reducing sugars by the Maillard reaction is the main way to generate acrylamide. Most methods tried to reduce acrylamide levels to eliminate precursors of acrylamide formation（reducing sugars and asparagine） or to inhibit the intensity of the Maillard reaction. L-Asparaginase can catalyze the hydrolysis of asparagine and eliminate the most important precursor（asparagine） of acrylamide formation. By this way, L-asparaginase can control the formation of acrylamide in food without affecting the color, flavor and nutritional characteristics of the final product. Control the formation of acrylamide in foods using L-asparaginase is a method worthy of popularization.The present subject demonstrates cloning, expression, purification, and characterization of three different thermostable L-asparaginases from Thermococcus gammatolerans EJ3, Thermococcus zilligii AN1 Tzi AN11, and Pyrococcus yayanosii CH1 and also evaluates the potential for enzymatic control of acrylamide in French fries using those thermostable enzymes. The recombinant expression of those L-sparaginase genes were performed in E. coli BL21（DE3）. After induction, the products of overexpression were purified by nickel-affinity chromatography. The purified enzymes were analyzed by SDS-PAGE, and the results indicated that the molecular weight of the purified T. gammatolerans L-ASNase, T. zilligii L-ASNase, and P. yayanosii L-ASNase were 36.5, 35.5 and 37.0 k Da, respectively, and which were consistent with their theoretical molecular weight. The recombinant enzymes showed that the optimum temperatures at 85, 90 and 85 °C, and the optimum p Hs at 8.5, 8.5 and 8.0, respectively. The purified L-ASNase all exhibited promising thermostability at high temperatures. The kinetic constants of those three L-ASNase on the substrate L-asparagine were determined, the Km values were 10.0, 6.02 and 12.3 m M,the kcat values were 5721,3358 and 1989 s-1,and the kcat/Km values were 572.1,557.8 and 162.7 m M-1 s-1, respectively.The effects of blanching time and T. zilligii L-ASNase and P. yayanosii L-ASNase treatment on the formation of acrylamide in French fries were studied. It was found that with the extension of blanching time the content of acrylamide was slowly decreased. When potato strips were treated with hot water at 80 °C for 10 min, the acrylamide content was reduced by 39.3%. When potato strips were treated with 10 U/m L T. zilligii L-ASNase solution at 80 °C for 4 min and P. yayanosii L-ASNase solution at 80 °C for 5 min, the acrylamide levels were reduced by 80.5% and 79.0%, respectively. Compared with other non-thermostable enzyme treatment, this subject combined blanching and enzyme treatment into one step and this method effectively inhibited the formation of acrylamide in a very short period of time. |
