| L-alanine has been widely used in food,medicine,chemical synthesis and other fields because it has a variety of physiological and biochemical functions.L-aspartate beta decarboxylase is the only enzyme known at present,which can catalyze the decarboxylation of L-aspartate to L-alanine.The pH of the reaction solution after directly injecting L-aspartic acid rapidly decreases,and the enzyme activity is low or even inactivated under acidic conditions.In view of this,site-directed mutagenesis was used to modify the acidity of L-aspartate?-decarboxylase in order to increase its enzyme activity in acidic environment.The main contents are summarized as follows:?1?L-aspartate?-decarboxylase gene aspD was cloned into E.coli BL21?DE3?to construct the recombinant strain E.coli BL21/pET28a-aspD.After inducing expression with 1mmol·L-1 IPTG,the SDS-PAGE results showed that the target protein was mostly in the form of inclusion bodies.The strategy of eliminating inclusion bodies by removing N-terminal His6 sequences of vector pET28a or replacing vectors successfully solved the problem of inclusion bodies.The crude enzyme activity of the strain after deleting N-terminal His6sequences was up 72.47 U·m L-1,and the specific activity reached 11.56 U·mg-1.However,the enzyme activity of L-aspartic acid?-decarboxylase was not detected in the original strain of E.coli BL21.?2?Recombinant bacteria were induced to express the enzyme and the crude enzyme supernatant was recovered by cell disruption by sonification prior to centrifugation at 10000rpm for 10 minutes.The purified L-aspartate?-decarboxylase had an enzyme activity of 12.2U·m L-1,the specific activity reached 65.95 U·mg-1.Further,we studied the enzymatic properties of the enzyme,and results showed that the optimal reaction temperature of L-aspartate?-decarboxylase was 45?and its optimum pH was 5.5,and the recombinant L-aspartate?-decarboxylase had good stability in the range of 35?-45?and pH 5.5-7.0.Metal ions had no obvious promotion effect on L-aspartate?-decarboxylase enzyme activity,while Cu2+,Co2+,Fe3+had obvious inhibitory effect on enzyme activity.?3?The acidity modification of L-aspartate?-decarboxylase was performed,and the protein sequence of L-aspartate?-decarboxylase from different sources were selected for comparison,and the three-dimensional structure of the protein was analyzed and selected for detailed study.Six amino acid residues near the active site were chosen and a total of 7mutants were obtained.The mutant enzymes N34D and L484M had 8.67%and 10.36%higher specific activity than the original enzyme,respectively.The results of the enzymatic properties of the mutant enzymes showed that the optimum reaction temperature for N34D was 50?,and the optimal reaction temperature for L484M and the original enzyme was the same at 45?;the optimal reaction pH for both enzymes was the same as that of the original enzyme.The relative enzymatic activities of the mutants N34D and L484M were increased by14%and 40%,respectively,compared to the original enzyme at pH 5.5 and pH 4.5.The relative enzymatic activities of the mutants N34D and L484M were lower than those of the original enzyme at pH 5.0.The enzyme is 30%higher and 36%.Amino acid residues at the34th and 484th positions with increased enzyme activity and acid resistance were selected for mutation,and a combination mutant was constructed.The results of the biochemical properties showed that the specific enzymatic activity of N34D/L484M was 116.27 U·mg-1,which was 76.30%higher than that of the original enzyme.The optimum reaction temperature was 45?,which was the same as that of the original enzyme.The in-range half-lives were all>48 h;the relative enzyme activity residual was 93%at p H 5.0,while the original enzyme was only 51%,and the optimum pH and pH stability were comparable to those of the original enzyme.?4?Using the mutant strain N34D/L484M as a catalyst,the conditions for the conversion of L-aspartic acid to L-alanine from the whole cell were optimized in terms of transformation temperature,conversion pH and buffer concentration.The results showed that the optimal conversion temperature was obtained.At 40?,the optimal pH was 5.5,and the optimal buffer was 0.2 mol·L-1 acetate buffer.After whole cell transformation under optimized conditions,the results showed that the mutant N34D/L484M could transform 75g·L-11 L-aspartic acid to generate 50.05 g·L-1 L-alanine within 12 h.The molar conversion rate was 99.6%,and the L-alanine production rate was 4.17 g·L-1·h-1.Under the same conditions,only 33.26 g·L-1 L-alanine could be transformed by E.coli BL21/pET28a-aspD in 20 h,and the transformation rate was 2.15 g·L-1·h-1,The aspartic acid molar conversion was 66.2%.The mutant strain N34D/L484M was transformed into a 5 L bioreactor for whole-cell transformation.When transformed for 27 h,371.05 g·L-1 L-alanine was formed,and the molar conversion rate reached 92.3%. |
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