| Lipoxygenases catalyze oxidative coupling reactions of 1-cis,4-cis-pentadiene structure of polyunsaturated fatty acids such as linoleic acid and linolenic acid. This oxidizing reaction will result in the formation of unstable hydroperoxides which is an important chemical intermediate with broad application prospects in food, medicine and chemical industry. The important application value in food processing is improving flour and its products quality, preparation of flavor compounds and tea processing. The application of lipoxygenase is limited in food processing due to its poor enzyme activity and thermal stability. So far, the lipoxygenase molecular modification has been the attention of the researchers at home and abroad.In order to improve the characteristics of LOX and lay the foundation of new type of enzyme, we need to improve the thermal stability of lipoxygenase. This paper will study the lipoxygenase gene by directed evolution and site directed mutagenesis. And the mutants picked out are to be simulated of 3d structure and their enzyme characteristics will be studied too. Mechanism of improving enzyme activity and thermal stability was parsed. The detail results are described as following.1. A sensitive and reliable high-throughput approach to screen lipoxygenase (LOX) mutants was developed. The recombmant E.coli cell was disrupted with the chemical penetration method, freezing and thawing method and enzymatic lysis method to get lipoxygenase. A L25(56) orthogonal array design generated based on single-factor experiments was employed to optimize conditions for E.coli cell disruption. The results showed that the optimal cell disruption conditions were lysozyme dose of 1.5mg/mL, lysozyme treatment time of 40 min, EDTA-2Na dose of 2.0mmol/L, Tween-60 dose of 2%, freezing and thawing temperature of -70-37℃ and freezing and thawing times of 3. We evaluated whether this assay was reliable or not for high-throughput screening of LOX mutant libraries in 96-well plate format and the "Z-factor" was calculated during in the range of 0.5-1.0 which indicating an excellent assay suitable for the screening of LOX mutant libraries.2. The technology of error-prone PCR was used to improve the activity and thermal stability lipoxygenase. Different concentration of Mg2+, Mn2+, dNTPs and Taq DNA polymerase were studied in the error-prone PCR reaction. The optimal condition is Mg2+ with 2 mM, Mn2+ with 0.1 mM, dNTPs with 0.2 mM and TaqDNA polymerase with 0.05 U/uL in the error-prone PCR reactions. Four mutants from two rounds of epPCR around 8,000 colonies were selected due to their improved thermostability and similar or low initial activity compared to the wild-type ana-LOX. Half-lives of all of the mutant enzymes at 50℃ increaseed from 8 min to 11.9min,14.6 min,13.3 min, and 11 min, respectively. Total of seven amino acid substitutions from two round of screening, T94A, V328A, E330G, N305D, L358I, E126G and T171A, were found to be of possible benefit for enhancing the thermal tolerance of ana-LOX. Thermal stability was improved because of the incorporation of hydrophobic interaction, hydrophobic forces and hydrogen bond.3. The technology of DNA Shuffling was used to improve the activity and thermal stability lipoxygenase. Mutants from error-prone PCR were chosen as templates to carry out DNA shuffling. Subsequently, three mutants were identified that had significantly improved thermostability and activity after heat treatment from 6,000 clones. Mutant D22 had a higher LOX activity which is 2.2-fold greater than the wild-type and its t1/2 at 50℃ was 15.2 min. Compared to the wild-type enzyme, the thermal inactivation t1/2 values at 50℃ of mutants D111 and D2221 were increased to 16.8 min and 18 min. The amino acid sequences of wild-type and mutant LOX enzymes were inputted into the SWISS-MODEL database and a template was selected that generated a homologous mimic diagram. Locus mutation effects were assessed using forecasts from the protein three-dimensional structure analysis software. The enzyme activity and thermal stability were improved because of side chain groups of chargeability, lower fold entropy and the electron density. The mutants D22, D111 and D2221 displayed over 50% residual activity at 50℃, while wild-type ana-LOX quickly lost 80% activity. The optimum temperatures of D22, D111 and D2221 were 50℃, 50℃ and 55℃, respectively. And the optimum pH of D22,D11 and D2221 were 10.0,9.0, 9.0. Protein secondary structures were determined by circular dichroism spectrum, the results showed that the increase of number of turns and folding of mutant D22, D111 and D2221, which is beneficial to maintain the stability of the protein. Analysis of the thermal denaturation temperatures of mutants showed introduction of hydrogen bonding and hydrophobic forces made thermal denaturation temperature increase 1℃,1.2℃ and 2℃, respectively.4. The technology of saturation mutagenesis to study the relationship between the structure and function of the recombinant LOXs. To confirm that the amino acid substitution at position 305 contributed to enhanced thermostability and activity, saturation mutagenesis was employed with eighteen pairs of mutation primers. As a result of these substitutions, the amino acid change at position 305 (N305D) demonstrates that stabilization of the α-helix is a key factor for stability. It can be concluded that the stability of the α-helix reduces according to the amino acid at position 305 following the order: Asp> Gin> Phe> Tyr>Arg> Lys> Cys> Asn. Mutants N305W, N305C, N305R and N305D from saturation site-directed mutagenesis were chosen as templates to carry out site-directed mutagenesis. Mutants L160Y/N305D, Y184L/N305D, Y184L/N305C, Y184L/N305W and L160Y/N305R obtained higher thermal stability and their half-lives reached 30 min at 50℃. The amino acid sequences mutant L160Y/N305D, Y184L/N305D, Y184L/N305C, Y184L/N305W and L160Y/N305R were inputted into the SWISS-MODEL database to get three-dimensional structure. It turned out that mutants got high thermal ability due to increasing the number of charged residues, the number of hydrophobic amino acids and turns, the number of hydrogen bonds and helix formation and folding sheets.5. Study on the rheological properties of mutant enzyme which added in dough. The determination of rheological properties of mutant enzyme under 30℃,40℃ and 50℃ showed that the mutant enzyme N305D had a larger elastic modulus G’. The elastic modulus of mutant enzyme Y184/N305D had increasd 104% and 108% Compar ed to the wild enzyme and the elastic modulus between 40-50℃ had stabled over 60000 Pa. And the elastic modulus G" of mutant enzyme N305D had increased 16.84% and 8.53% compared to controled group and wild enzyme. The elastic modulus G" of mutant enzyme Y184L/N305D had increasd 91.8% and 78.2% compared to controled group and wild enzyme. |
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