Immobilized l-aspartate ammonia-lyase from Bacillus sp. YM55-1 as biocatalyst for highly concentrated l-aspartate synthesis

Abstract

L-aspartate ammonia-lyase from Bacillus sp. YM55-1 (AspB, EC 4.3.1.1) catalyzes the reversible conversion of L-aspartate (Asp) into fumarate and ammonia with a high specific activity toward the substrate. AspB was expressed in Escherichia coli and partially purified by heat precipitation and saturation with ammonium sulfate reaching purification factor of 7.7 and specific activity of 334 U/mg of protein. AspB was immobilized by covalent attachment on Eupergit® C (epoxy support) and MANA-agarose (amino support), and entrapment in LentiKats® (polyvinyl alcohol) with retained activities of 24, 85 and 63 %, respectively. Diffusional limitations were only observed for the enzyme immobilized in LentiKats® and were overcome by increasing substrate concentration. Free and immobilized AspB were used for the synthesis of aspartate achieving high product concentration (≥450 mM) after 24 h of reaction. Immobilized biocatalysts were efficiently reused in 5 cycles of Asp synthesis, keeping over 90 % of activity and reaching over 90 % of conversion in all the cases.

Efficient aspartic acid production by a psychrophile-based simple biocatalyst

“…In previous studies, bacterial strains have been applied for the industrial production of aspartic acid [2,4,12,15,17]; however, the preparation of biocatalysts for efficient bioconversion was laborious and time consuming because fermentation had to be regulated to suppress fumarase activity [15]. In addition, recombinant enzyme extraction and purification were required to eliminate the influence of metabolic enzymes [2,4,12,17]. Compared with other methods to prevent byproduct formation, heat treatment of recombinant bacteria is a simple and easy way to obtain an efficient biocatalyst, such as PSCat.…”

“…To repress host basic metabolism, psychrophilic bacteria expressing a mesophilic enzyme (aspartase) were heat treated at 50 °C for 15 min, which effectively blocked the activity of psychrophilic fumarase to produce L-malic acid, a major by-product of aspartate biosynthesis. In previous studies, bacterial strains have been applied for the industrial production of aspartic acid [2,4,12,15,17]; however, the preparation of biocatalysts for efficient bioconversion was laborious and time consuming because fermentation had to be regulated to suppress fumarase activity [15]. In addition, recombinant enzyme extraction and purification were required to eliminate the influence of metabolic enzymes [2,4,12,17].…”

“…The products were eluted at 60 °C using 0.1 % (v/v) phosphoric acid as a mobile phase at a flow rate of 0.7 mL/ min. Aspartic acid was measured by the derivatization with 2,4-dinitrofluorobenzene (DNFB) as previously described with some modifications [2]. Briefly, 10 μL of 1 M NaHCO 3 was added to 25 μL of the sample and reacted with 40 μL of 37.6 mM DNFB in acetone at 40 °C for 90 min.…”

“…Thus, free and immobilized thermostable aspartase of Bacillus sp. YM55-1 expressed in E. coli could yield over 430 mM aspartic acid after a 24-h fermentation [2]. However, enzyme purification involves steps such as mechanical cell disruption, removal of cell debris by prolonged centrifugation, and fractionation by ammonium sulfate, which are major bottlenecks for the industrialization of the process [14].…”

Co-immobilised aspartase and transaminase for high-yield synthesis of l-phenylalanine

“…Aspartase from Bacillus sp. YM55-1 (AspB) is a thermostable enzyme which was used in the synthesis of high concentrations of l-aspartate with reaction yields over 90% [18]. Hence, by coupling the reactions of aspartase and transaminase ( Fig.…”