Expression of Bacillus amyloliquefaciens γ-Glutamyltransferase in Lactococcus lactis and Immobilization on Magnetic Nanoparticles

Abstract

γ-Glutamyltransferase (GGT) can catalyze the transfer of a γ-glutamyl group to amino acids or peptides. Bacterial GGTs have a wide substrate specificity, which means that a variety of γ-glutamyl amino acid derivatives or peptides can be synthesized under GGT catalysis. In this work, in order to enhance the expression and stability of GGT, a recombinant plasmid harboring Bacillus amyloliquefaciens ggt gene with a Usp45 signal peptide gene fused upstream was constructed and transferred into Lactococcus lactis NZ9000 to express BaGGT. After on-column hydrolysis and purification by immobilized metal affinity chromatography, 90.2 ± 0.7 mg/L of BaGGT-6His with an enzymatic activity of 57.3 ± 0.9 U/mg was purified. Subsequently, BaGGT-6His was immobilized onto magnetic mesoporous nanoparticles (Fe 3 O 4 NPs) by covalent binding. The immobilized BaGGT-6His largely retained the activity and, importantly, could be recovered and reused. After optimization of the production and immobilization conditions, the biotechnologically relevant product L-theanine was synthesized with free and immobilized BaGGT-6His.

Secretion of Bacillus amyloliquefaciens Levansucrase and Its Mutants from L. lactis NZ9000 and Their Applications in the Synthesis of Levan

“…Figure 5B shows that the immobilization efficiency decreases when the pH rises from 4 to 10, which may be due to the protonation of the surface of Fe 3 O 4 @NPs in acidic environment. 21 At the same time, AR reached the maximum value of 89.33% at pH 6. Thus, although the acidic environment facilitates the immobilization of the enzyme, considering the activity of the enzyme, pH 6 buffer was selected for the immobilization of the enzyme.…”

“…Additionally, the highest AR reached is 93.57%. Figure B shows that the immobilization efficiency decreases when the pH rises from 4 to 10, which may be due to the protonation of the surface of Fe 3 O 4 @NPs in acidic environment . At the same time, AR reached the maximum value of 89.33% at pH 6.…”

“…The possible reasons might be that the combination of the nanoparticles and the enzyme increases the stability of the structure. 21 At the same time, the stability makes the spatial structure of the E313F@NPs less prone to change under an extreme environment, thereby increasing the stability of Ba-SacB. 44 It is reported that the thermal stability of inulinase is increased by immobilizing inulinase on chitosan, and it has the ability of continuous hydrolysis of inulin.…”

“…Thus, we also observed that the prepared E313F @ NPs still had about 60% of the initial activity after 10 times of reuse, which is similar to previous reports. 21,46…”

“…During industrial applications, the enzymatic activity of soluble Ba-SacB may decrease or even disappear due to inappropriate reaction conditions, resulting in relatively high costs. The immobilization of enzymes has shown promising results in optimizing the performance of enzymes, such as thermal stability, storage stability, pH stability, easy separation of the product, reusability of the catalyst, catalysis of the unnatural substrates, novel reactions, and augmented enantioselectivity . Further, immobilized enzymes cannot easily penetrate the skin, so they exhibit low allergenicity .…”