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One-step biosynthesis of α-keto-γ-methylthiobutyric acid from L-methionine by an Escherichia coli whole-cell biocatalyst expressing an engineered L-amino acid deaminase from Proteus vulgaris.

Hossain GS, Li J, Shin HD, Du G, Wang M, Liu L, Chen J - PLoS ONE (2014)

Bottom Line: Then, error-prone polymerase chain reaction was used to construct P. vulgaris l-AAD mutant libraries.Among approximately 104 mutants, two mutants bearing lysine 104 to arginine and alanine 337 to serine substitutions showed 82.2% and 80.8% molar conversion ratios, respectively.Furthermore, the combination of these mutations enhanced the catalytic activity and molar conversion ratio by 1.3-fold and up to 91.4% with a KMTB concentration of 63.6 g/L.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, China; School of Food Science and Technology, Jiangnan University, Wuxi, China.

ABSTRACT
α-Keto-γ-methylthiobutyric acid (KMTB), a keto derivative of l-methionine, has great potential for use as an alternative to l-methionine in the poultry industry and as an anti-cancer drug. This study developed an environment friendly process for KMTB production from l-methionine by an Escherichia coli whole-cell biocatalyst expressing an engineered l-amino acid deaminase (l-AAD) from Proteus vulgaris. We first overexpressed the P. vulgaris l-AAD in E. coli BL21 (DE3) and further optimized the whole-cell transformation process. The maximal molar conversion ratio of l-methionine to KMTB was 71.2% (mol/mol) under the optimal conditions (70 g/L l-methionine, 20 g/L whole-cell biocatalyst, 5 mM CaCl2, 40°C, 50 mM Tris-HCl [pH 8.0]). Then, error-prone polymerase chain reaction was used to construct P. vulgaris l-AAD mutant libraries. Among approximately 104 mutants, two mutants bearing lysine 104 to arginine and alanine 337 to serine substitutions showed 82.2% and 80.8% molar conversion ratios, respectively. Furthermore, the combination of these mutations enhanced the catalytic activity and molar conversion ratio by 1.3-fold and up to 91.4% with a KMTB concentration of 63.6 g/L. Finally, the effect of immobilization on whole-cell transformation was examined, and the immobilized whole-cell biocatalyst with Ca2+ alginate increased reusability by 41.3% compared to that of free cell production. Compared with the traditional multi-step chemical synthesis, our one-step biocatalytic production of KMTB has an advantage in terms of environmental pollution and thus has great potential for industrial KMTB production.

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Result of mutagenesis on the molar conversion efficiency and time profile for product formation.(A) Conversion efficiency of different mutants from l-methionine to KMTB (mol/mol) and (B) Time profile for the production of KMTB (▪) from l-methionine by engineered double mutant l-AAD containing whole cell biocatalyst.
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pone-0114291-g004: Result of mutagenesis on the molar conversion efficiency and time profile for product formation.(A) Conversion efficiency of different mutants from l-methionine to KMTB (mol/mol) and (B) Time profile for the production of KMTB (▪) from l-methionine by engineered double mutant l-AAD containing whole cell biocatalyst.

Mentions: For industrial processes, directed evolution has become an influential tool in the production of variants that improve enzyme structure as well as function for specific purposes. Because the similarity of l-AAD to other l-AAOs for which crystal structures have been determined is very low (less than 20%), we could not predict the active site by developing homology modeling to improve biocatalytic efficiency. Therefore, random mutagenesis was used to improve the bioconversion efficiency of l-AAD in this study. Using er-PCR, we obtained two mutants with increased turnover of l-methionine to KMTB. The initial activities of these mutants were higher than that of l-AAD. Using sequencing analysis, we determined the mutations responsible for the increased activity and biotransformation efficiency. In the first point mutation, lysine 104 was replaced by arginine, which increased the bioconversion ratio from 71.2% to 82.2% (Fig. 4A). We found that the maximal reaction rate (Vmax) of the mutant l-AAD (Lys104Arg) was higher than that of the wild type (Table 2). Moreover, compared to that of the wild-type l-AAD, the Km of the mutant also decreased, and as a result, Vmax/Km increased (see Table 2, S2 Fig.).


One-step biosynthesis of α-keto-γ-methylthiobutyric acid from L-methionine by an Escherichia coli whole-cell biocatalyst expressing an engineered L-amino acid deaminase from Proteus vulgaris.

Hossain GS, Li J, Shin HD, Du G, Wang M, Liu L, Chen J - PLoS ONE (2014)

Result of mutagenesis on the molar conversion efficiency and time profile for product formation.(A) Conversion efficiency of different mutants from l-methionine to KMTB (mol/mol) and (B) Time profile for the production of KMTB (▪) from l-methionine by engineered double mutant l-AAD containing whole cell biocatalyst.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4273966&req=5

pone-0114291-g004: Result of mutagenesis on the molar conversion efficiency and time profile for product formation.(A) Conversion efficiency of different mutants from l-methionine to KMTB (mol/mol) and (B) Time profile for the production of KMTB (▪) from l-methionine by engineered double mutant l-AAD containing whole cell biocatalyst.
Mentions: For industrial processes, directed evolution has become an influential tool in the production of variants that improve enzyme structure as well as function for specific purposes. Because the similarity of l-AAD to other l-AAOs for which crystal structures have been determined is very low (less than 20%), we could not predict the active site by developing homology modeling to improve biocatalytic efficiency. Therefore, random mutagenesis was used to improve the bioconversion efficiency of l-AAD in this study. Using er-PCR, we obtained two mutants with increased turnover of l-methionine to KMTB. The initial activities of these mutants were higher than that of l-AAD. Using sequencing analysis, we determined the mutations responsible for the increased activity and biotransformation efficiency. In the first point mutation, lysine 104 was replaced by arginine, which increased the bioconversion ratio from 71.2% to 82.2% (Fig. 4A). We found that the maximal reaction rate (Vmax) of the mutant l-AAD (Lys104Arg) was higher than that of the wild type (Table 2). Moreover, compared to that of the wild-type l-AAD, the Km of the mutant also decreased, and as a result, Vmax/Km increased (see Table 2, S2 Fig.).

Bottom Line: Then, error-prone polymerase chain reaction was used to construct P. vulgaris l-AAD mutant libraries.Among approximately 104 mutants, two mutants bearing lysine 104 to arginine and alanine 337 to serine substitutions showed 82.2% and 80.8% molar conversion ratios, respectively.Furthermore, the combination of these mutations enhanced the catalytic activity and molar conversion ratio by 1.3-fold and up to 91.4% with a KMTB concentration of 63.6 g/L.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, China; School of Food Science and Technology, Jiangnan University, Wuxi, China.

ABSTRACT
α-Keto-γ-methylthiobutyric acid (KMTB), a keto derivative of l-methionine, has great potential for use as an alternative to l-methionine in the poultry industry and as an anti-cancer drug. This study developed an environment friendly process for KMTB production from l-methionine by an Escherichia coli whole-cell biocatalyst expressing an engineered l-amino acid deaminase (l-AAD) from Proteus vulgaris. We first overexpressed the P. vulgaris l-AAD in E. coli BL21 (DE3) and further optimized the whole-cell transformation process. The maximal molar conversion ratio of l-methionine to KMTB was 71.2% (mol/mol) under the optimal conditions (70 g/L l-methionine, 20 g/L whole-cell biocatalyst, 5 mM CaCl2, 40°C, 50 mM Tris-HCl [pH 8.0]). Then, error-prone polymerase chain reaction was used to construct P. vulgaris l-AAD mutant libraries. Among approximately 104 mutants, two mutants bearing lysine 104 to arginine and alanine 337 to serine substitutions showed 82.2% and 80.8% molar conversion ratios, respectively. Furthermore, the combination of these mutations enhanced the catalytic activity and molar conversion ratio by 1.3-fold and up to 91.4% with a KMTB concentration of 63.6 g/L. Finally, the effect of immobilization on whole-cell transformation was examined, and the immobilized whole-cell biocatalyst with Ca2+ alginate increased reusability by 41.3% compared to that of free cell production. Compared with the traditional multi-step chemical synthesis, our one-step biocatalytic production of KMTB has an advantage in terms of environmental pollution and thus has great potential for industrial KMTB production.

Show MeSH
Related in: MedlinePlus