Immobilization of cells of the recombinant strain of Escherichiacoli LGE 36 with aminoacylase activity on an inorganic carrier

Background: Optically active amino acids obtained using native and immobilized aminoacylase enzyme are widely demanded for use in food, pharmaceutical, and biotechnology applications. In this study, cells of the recombinant strain of Escherichia coli LGE 36 with aminoacylase activity were immobilized on silica gel 5/40 using glutaraldehyde, following our developed method. The dependence of the properties of immobilized cells with aminoacylase activity on the degree of carrier activation by γ-aminopropyl-3-ethoxy silane was studied. The thermal stability of native and immobilized cells was compared. Objective: The objective of the work is to immobilize cells of the recombinant strain of Escherichia coli LGE 36 with aminoacylase activity, study the dependence of the properties of the immobilized cells on the degree of carrier activation with γ-aminopropyl-3-ethoxy silane and study the thermal stability of native and immobilized cells. Methods: A recombinant aminoacylase-producing strain of Escherichia coli LGE 36 was used in this study. E.coli cells were grown in supplemented M9 minimal medium at 37 oC. Aminoacylase activity of native and immobilized cells was measured at 37 oC in a 0.2 mL reaction mixture containing Buffer A (100 mM Na/K-phosphate buffer, pH 7.0), 0.2 mM CoCI2, 40 mM N-acetyl-D,L-methionine, and the required amount of cells or enzymes. One unit of acylase activity was defined as the amount of biocatalyst producing the formation of 1µmol of L-methionine per minute at 37 oC and pH 7.0. The protein concentration was determined by the method of Groves and Davis [22]. Relative activity was expressed as theFunctional Foods in Health and Disease 2026; 16(10): 971–981 FFHD Page 972 of 981 percentage of the corresponding initial activity measured under the same assay conditions. Results: Under the conditions examined, silica gel 5/40 activated for 5 h at 55 oC in an aqueous medium containing no more than 1% concentration of γ-aminopropyl-3-ethoxysilane produced immobilized cells with the highest observed activity and thermal stability limit. Immobilized cells retained activity at a temperature 13oC higher than native cells under the specified 20-min treatment conditions. Conclusion: Cell immobilization of the recombinant strain of Escherichia coli LGE36 cells on silica gel 5/40 with approximately 90 µmol/g amino groups produced a biocatalyst that retained up to 65–75% of its initial relative activity under the reported conditions. The preparation may be useful for further development of biocatalytic processes for producing optically active amino acids. It has potential to serve as a basis for creating new pharmaceutical drugs and biologically active supplements, but its suitability for these functional-food applications remains prospective, as food- grade status, product composition, biological effects, safety, and efficacy have not yet been evaluated and verified. Novelty: This study optimizes a carrier-based immobilization procedure specifically for whole cells of recombinant E. coli LGE36 with aminoacylase activity. The results indicate improved thermal performance relative to native cells under the reported assay conditions. Environmental and economic advantages of the process were not directly quantified and should be evaluated in future studies.