L-asparaginase catalyze the hydrolysis of L-asparagine to produce L-aspartic acid, which can effectively inhibit the formation of acrylamide (class 2A carcinogen) in high-temperature food processing.Although type I L-asparaginase has good thermal stability, its catalytic activity still needs to be improved.This study aimed to improve the catalytic activity of Thermococcus zilligii type I L-asparaginase (TzI) through molecular modification.First, 10 TzI mutants with potential increased activity were screened using molecular docking and virtual saturation mutation, and their expression, purification, and enzymatic properties were analyzed.The results showed that the specific enzyme activity of mutant enzyme L265W reached 2 751.13 U/mg, 29% higher than the original enzyme TzI.Although the optimal reaction temperature was still 90 ℃, the half-lives of mutant enzyme L265W at 80, 85, and 90 ℃ were increased by 1.5, 1.56, and 1.43 times compared to TzI, respectively.Second, the potato chips were treated with an equal amount of enzyme under 80 ℃ blanching conditions, and the acrylamide content after frying was measured.The results showed that L265W can reduce acrylamide in fried French fries by 65%, which is better than the treatment with commercial enzymes and original enzyme TzI.Therefore, the mutated enzyme L265W constructed in this study has good potential for application, which will help promote the industrial application of L-asparaginase.
[1] BATOOL T, MAKKY E A, JALAL M, et al.A comprehensive review on L-asparaginase and its applications[J].Applied Biochemistry and Biotechnology, 2016, 178(5):900-923.
[2] SHAKAMBARI G, ASHOKKUMAR B, VARALAKSHMI P.L-asparaginase:A promising biocatalyst for industrial and clinical applications[J].Biocatalysis and Agricultural Biotechnology, 2019, 17:213-224.
[3] WANG Y M, WU H, ZHANG W L, et al.Efficient control of acrylamide in french fries by an extraordinarily active and thermo-stable L-asparaginase:A lab-scale study[J].Food Chemistry, 2021, 360:130046.
[4] CASTRO D, MARQUES A S C, ALMEIDA M R, et al.L-asparaginase production review:Bioprocess design and biochemical characteristics[J].Applied Microbiology and Biotechnology, 2021, 105(11):4515-4534.
[5] KAUR N, HALFORD N G.Reducing the risk of acrylamide and other processing contaminant formation in wheat products[J].Foods, 2023, 12(17):3264.
[6] LI R, ZHANG Z H, PEI X M, et al.Covalent immobilization of L-asparaginase and optimization of its enzyme reactor for reducing acrylamide formation in a heated food model system[J].Frontiers in Bioengineering and Biotechnology, 2020, 8:584758.
[7] DA CUNHA M C, DOS SANTOS AGUILAR J G, DE MELO R R, et al.Fungal L-asparaginase:Strategies for production and food applications[J].Food Research International, 2019, 126:108658.
[8] ABEDI E, MOHAMMAD BAGHER HASHEMI S, GHIASI F.Effective mitigation in the amount of acrylamide through enzymatic approaches[J].Food Research International, 2023, 172:113177.
[9] JIA R Y, WAN X, GENG X, et al.Microbial L-asparaginase for application in acrylamide mitigation from food:Current research status and future perspectives[J].Microorganisms, 2021, 9(8):1659.
[10] ZUO S H, ZHANG T, JIANG B, et al.Reduction of acrylamide level through blanching with treatment by an extremely thermostable L-asparaginase during french fries processing[J].Extremophiles, 2015, 19(4):841-851.
[11] SELLÉS VIDAL L, ISALAN M, HEAP J T, et al.A primer to directed evolution:Current methodologies and future directions[J].RSC Chemical Biology, 2023, 4(4):271-291.
[12] BILAL M, ZHAO Y P, NOREEN S, et al.Modifying bio-catalytic properties of enzymes for efficient biocatalysis:A review from immobilization strategies viewpoint[J].Biocatalysis and Biotransformation, 2019, 37(3):159-182.
[13] XING H G, ZOU G, LIU C Y, et al.Improving the thermostability of a GH11 xylanase by directed evolution and rational design guided by B-factor analysis[J].Enzyme and Microbial Technology, 2021, 143:109720.
[14] ZHOU Y W, JIAO L S, SHEN J, et al.Enhancing the catalytic activity of type II L-asparaginase from Bacillus licheniformis through semi-rational design[J].International Journal of Molecular Sciences, 2022, 23(17):9663.
[15] PANG C P, LIU S, ZHANG G Q, et al.Improving the catalytic efficiency of Pseudomonas aeruginosa lipoxygenase by semi-rational design[J].Enzyme and Microbial Technology, 2023, 162:110120.
[16] ROBERT X, GOUET P.Deciphering key features in protein structures with the new ENDscript server[J].Nucleic Acids Research, 2014, 42(Web Server issue):W320-W324.
[17] RODRIGUES C H, PIRES D E, ASCHER D B.Dynamut:Predicting the impact of mutations on protein conformation, flexibility and stability[J].Nucleic Acids Research, 2018, 46(W1):W350-W355.
[18] ABRAHAM M J, MURTOLA T, SCHULZ R, et al.GROMACS:High performance molecular simulations through multi-level parallelism from laptops to supercomputers[J].SoftwareX, 2015, 1:19-25.
[19] GUO J X, COKER A R, WOOD S P, et al.Structure and function of the thermostable L-asparaginase from Thermococcus kodakarensis[J].Acta Crystallographica.Section D, Structural Biology, 2017, 73(Pt 11):889-895.
[20] LI X, ZHANG X, XU S Q, et al.Insight into the thermostability of thermophilic L-asparaginase and non-thermophilic L-asparaginase II through bioinformatics and structural analysis[J].Applied Microbiology and Biotechnology, 2019, 103(17):7055-7070.
[21] ANISHKIN A, VANEGAS J M, ROGERS D M, et al.Catalytic role of the substrate defines specificity of therapeutic L-asparaginase[J].Journal of Molecular Biology, 2015, 427(17):2867-2885.
[22] YAO M, YASUTAKE Y, MORITA H, et al.Structure of the type I L-asparaginase from the hyperthermophilic archaeon Pyrococcus horikoshii at 2.16 Å resolution[J].Acta Crystallographica.Section D, Biological Crystallography, 2005, 61(3):294-301.
[23] GRIBENKO A V, PATEL M M, LIU J J, et al.Rational stabilization of enzymes by computational redesign of surface charge-charge interactions[J].Proceedings of the National Academy of Sciences of the United States of America, 2009, 106(8):2601-2606.
[24] WANG X Y, ZHANG X B, PENG C, et al.D3DistalMutation:A database to explore the effect of distal mutations on enzyme activity[J].Journal of Chemical Information and Modeling, 2021, 61(5):2499-2508.
[25] BIGNARDI C, CAVAZZA A, GRIMALDI M, et al.Acrylamide determination in baked potatoes by HPLC-MS:Effect of steam and correlation with colour indices[J].European Food Research and Technology, 2019, 245(11):2393-2400.
