| L-asparaginase can specifically catalyze the hydrolysis of L-asparagine into L-aspartic acid and ammonia.It has been widely used as an anti-tumor drug in the clinical treatment of acute lymphoblastic leukemia,non-Hodgkin’s lymphoma and other hematopoietic malignancies.In addition,L-asparaginase has the ability to inhibit the formation of acrylamide in high-temperature processed food products.Acrylamide is classified as“probably carcinogenic to humans”,which could form in a diversity of food products via Maillard reaction.L-asparaginase pre-treatment of the food products could inhibit the synthesis of acrylamide,by hydrolyzing the L-asparagine precursor of acrylamide,without affecting sensory or nutritional properties of products.However,there are many shortcomings with the existing L-asparaginases,such as low catalytic activity,poor thermal stability,poor substrate specificity and low catalytic efficiency,which limit the use of L-asparaginase in food processing.Meanwhile,the high immunogenicity and high metabolic rate of L-asparaginase in human body make it difficult to apply in the clinical treatment.To meet the demands of industrial application,it is significant to improve the catalytic and applicable performance of L-asparaginase so that it can adapt to the specific functionalities in food processing and clinical application by using protein engineering modification.In this study,a novel L-asparaginase producing strain Acinetobacter soli Y-3 was screened;and its L-asparaginase coding gene was cloned and expressed in E.coli.The recombined enzyme（As A）was characterized with high catalytic activity,high substrate specificity and unsatisfactory thermal stability.Therefore,the protein engineering methods were implemented to improve the thermal stability of As A,and the thermal stability improved molecular mechanism of mutant enzymes were analyzed through molecular dynamics simulation and homology modeling structure comparison.Finally,the mutant enzymes with improved thermal stability were applied to the acrylamide mitigation in fried potato chips.The main findings are as follows:1.Screening of novel L-asparaginase producing strain and cloning and expression of its L-asparaginase coding gene21 strains detected with L-asparaginase activity were isolated from 11 soil samples,and strain Y-3 with L-asparaginase activity of 0.14 IU/m L was identified as Acinetobacter soli.The A.soli L-asparaginase coding gene was cloned and efficiently expressed in E.coli.After the optimization of the induction conditions,the activity of the recombined enzyme As A reached 42.01 IU/m L,which was 300.07 times of the activity in wild A.soli.As A was characterized with the dedicated catalytic activity toward L-asparagine.Additionally,the optimal reaction conditions of As A were 40°C and p H8.0,respectively.However,the half-life of As A at 40°C was shorter than 10 min.Therefore,the molecular modification was performed to improve the thermal stability of As A.2.Improvement of the thermal stability of As A via directed evolutionEfficient high-throughput screening method for the thermal stability determine of L-asparaginase was established with the aid of microbial screening system and automatic pipetting workstation.After three rounds of error-prone PCR and one round of DNA shuffling,four thermal stability improved mutant enzymes were screened from 22000clones,the half-life of mutant enzymes F18-11（Q148E）and YC-5（Q148E/H292Y/D326V）at 45°C were 24.76 min and 19.92 min,which were 6.08 times and 4.85 times the wild-type half-life of 4.07 min,respectively.Therefore,the 3 mutation sites Gln148,His292 and Asp326,which could be the key amino acids related to the thermal stability of As A,were further saturated mutated and screened,and 7 thermal stability enhanced mutant enzymes Q148V,Q148L,Q148M,Q148I,Q148F,Q148E and Q148S were obtained.Among them,the thermal stability of mutant enzyme Q148V was greatly improved,its half-life at 40°C raised up to 16 h,which was 104.52 times that of the wild-type,the half-life at 45°C was330.07 min,which was 81.10 times that of the wild-type,the half-life at 50°C was 128.36min,and its Tm value increased by 4.38°C,additionally,its optimal reaction temperature increased from 40°C to 50°C.Through molecular dynamics simulation and structure analysis,the thermal stabilities of mutant enzymes Q148V,Q148L,Q148M and Q148I were mainly improved by introducing of hydrophobic amino acids in the inner of As A;the Ser148 in the mutant enzyme Q148S formed new hydrogen bonds to stabilized the enzyme conformation;the charged residue glutamate introduced in mutant enzyme Q148E was more conducive to structural stability than glutamine;and the aromatic group of phenylalanine in mutant enzyme Q148F formed new aromatic interaction with the nearby residues Phe150 and Phe171,which increased the thermal stability of As A.3.Improvement of the thermal stability of As A via semi-rational designTwo non-conserved cysteine residues Cys8 and Cys283 were selected out through Consensus design.Through saturation mutations,four mutant enzymes C8Y,C8F,C8W and C283Q with improved thermal stability were obtained.The half-life of mutant enzyme C8Y at 45°C was 151.23 min,which was 16.67 times that of the wild-type,the half-life at 45°C was 57.28 min,which was 14.07 times that of the wild-type,and the half-life at 50°C was16.84 min.Furthermore,three double-point mutants of C8Y/C283Q,C8F/C283Q and C8W/C283Q were constructed,which showed higher thermal stability than their corresponding single-point mutant enzymes.Among them,the half-life of mutant enzyme C8Y/C283Q at 40°C was 361.64 min,which was 39.87 times that of the wild-type,the half-life at 45°C was 67.96 min,which was 16.09 times that of the wild-type,the half-life at50°C was 25.21 min,and its Tm value increased by 7.07°C.Through molecular dynamics simulation and structure analysis,the mutations of C8Y,C8F,and C8W formed aromatic interactions with Tyr25 and Phe28 to stabilize the conformation of As A,and the mutation of C283Q could form additional hydrogen bonds through the ketone and amino groups on its side chain and reduce the flexibility of its neighboring conformation.Meanwhile,on the basis of Consensus design,secondary structure analysis,location and amino acid characteristics change of mutation sites,five non-conserved residues were selected as Asn2,Ala118,Thr146,Pro179 and Asn185 for mutation.6 thermal stability enhanced mutant enzymes were screened through site-directed saturation mutagenesis,N2K,P179T,P179A,P179I,P179E and N185T.Mutant enzyme P179A had a half-life of34.31 min at 45°C,which was 8.43 times that of the wild-type,and its Tm value increased by 2.64°C.Through molecular dynamics simulation and structure analysis,the mutant enzyme P179A introduced new hydrogen bonds to improve the stability of As A,which also retained the original hydrophobic interactions.4.Improvement of the thermal stability of As A via rational design and the application of thermal stability enhanced mutant enzymesA rational design strategy was implemented to improve the thermal stability of As A.The structure model of As A was compared with the structures of Bl A,Pf A,Ph A and Tk A that have been characterized with high thermal stabilities,and 3 regions with distinguished secondary structures were found.Combined with molecular dynamics simulation,sequence alignment and unfolding free energy calculation,3 potential thermal stability enhancing mutation sites Q29K,N116E and N129P were screened and constructed by site-directed mutagenesis.Among them,the thermal stabilities of mutant enzymes Q29K and N129P were improved.The half-life of mutant enzyme Q29K at 40°C was 39.84 min,which was4.39 times that of wild-type,and the half-life at 45°C was 13.84 min,which was 3.40 times that of wild-type,and its Tm value increased by 4.35°C.Mutant enzyme N129P had a half-life of 77.88 min at 40°C,which was 8.59 times that of the wild-type,and a half-life of25.86 min at 45°C,which was 6.35 times that of the mutant,and its Tm value was also increased by 4.97°C.Besides,the substrate affinity of mutant enzymes Q29K and N129P were both improved,and the Km value of them reduced from 3.81 m M to 1.24 m M and2.45 m M,respectively;their catalytic efficiency kcat/Km values increased from 51.97s-1·m M-1to 201.54 s-1·m M-1and 403.15 s-1·m M-1,respectively.Analyzed the thermal stability enhance mechanism,it was found that residue Gln29 located on the surface of the enzyme which was easy to deaminate,after mutated into positive charged residue lysine,a ionic bond was formed between residue Lys29 and Glu33,which greatly improved the thermal stability of As A.In mutant enzyme N129P,when temperature increased,the introduction of proline could prevent theα-helix from unfolding,and thus stabilize the As A.The thermal stability enhanced mutant enzymes Q148V,C8Y/C283Q and N129P were applied to inhibit the formation of acrylamide in fried potato chips.The results showed that the acrylamide content was reduced from 1.6559 mg/kg in the control group to 0.6781mg/kg in As A wild-type treated group,which was reduced by 59.05%;after the treatment with mutant enzyme Q148V,the acrylamide content in potato chips was reduced to 0.0928mg/kg,which was decreased by 94.40%;after treatment with mutant enzymes C8Y/C283Q and N129P,the acrylamide content in potato chips were reduced to 0.2235 mg/kg and0.3113 mg/kg,respectively,and the decreasing rate were 86.50%and 81.20%,respectively.The inhibition of acrylamide in fried potato chips treated with the mutant enzymes is positively correlated with their thermal stabilities. |
PDF Full Text Request