Abstract
Arginine metabolism in Enterococcus faecium sp. GR7 was enhanced via arginine deiminase pathway. Process parameters including fermentation media and environmental conditions were optimized using independent experiments and response surface methodology (central composite design). Fermentation media (EAPM) were optimized using independent experiments which resulted in 4-fold increase in arginine deiminase specific activity as compared to basal medium. To further enhance arginine deiminase activity in E. faecium sp. GR7 and biomass production including a five-level central composite design (CCD) was employed to study the interactive effect of three-process variables. Response surface methodology suggested a quadratic model which was further validated experimentally where it showed approximately 15-fold increase in arginine metabolism (in terms of arginine deiminase specific activity) over basal medium. By solving the regression equation and analyzing the response surface cartons, optimal concentrations of the media components (g/L) were determined as arginine 20.0; tryptone 15.0; lactose 10.0; K2HPO4 3.0; NaCl 1.0, MnSO4 0.6 mM; Tween 80 1%; pH 6.0 for achieving specific arginine deiminase activity of 4.6 IU/mG with concomitant biomass production of 12.1 mg/L. The model is significant as the coefficient of determination (R 2) was 0.87 to 0.90 for all responses. Enhanced arginine deiminase yield from E. faecium, a GRAS lactic acid bacterial strain, is desirable to explore in vitro therapeutic potential of the arginine metabolizing E. faecium sp. GR7.
Highlights
L-arginine is classified as conditionally essential amino acid for protein synthesis that is metabolized through citrulline, ornithine, creatine, proline, and polyamines in human body [1, 2]
The highest specific arginine deiminase (ADI) activity of 0.182 ± 0.001 international enzyme units present per mg (IU/mG) was observed in TGYE media when supplemented with 15 mM arginine, which was selected as a basal media for further study of process parameter variables on nutrients, inducers, salts, surfactants, and processing parameters on ADI production of E. faecium sp
Previous reports on Lactobacillus buchneri CUC-3 [32], Lactobacillus buchneri NCDO110 [29], Lactobacillus sanfranciscensis CB1 [12], Streptococcus lactis [9], Weissella confusa GR7 [27], Weissella koreensis MSI-3 [10], and Weissella koreensis MSI-14 [10] have suggested the role of arginine in enhancing ADI activity in various LAB strains
Summary
L-arginine is classified as conditionally essential amino acid for protein synthesis that is metabolized through citrulline, ornithine, creatine, proline, and polyamines in human body [1, 2]. It is used by a number of microorganisms to generate ATP fermentatively via arginine deiminase (ADI) pathway which is known as arginine dihydrolase (ADH) pathway [3]. The present study was aimed to screen and optimize media components that increase ADI activity of Enterococcus sp. RSM, formerly known as Box-Wilson methodology, is the most widely used statistical technique to evaluate relationship between a set of controllable experimental factors and observed results [11].
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