Study on oligomerization of glutamate decarboxylase from Lactobacillus brevis using asymmetrical flow field-flow fractionation (AF4) with light scattering techniques

Abstract

In this work, asymmetrical flow field-flow fractionation (AF4) coupled with UV/Vis, multi-angle light scattering (MALS), and differential refractive index (dRI) detectors (AF4-UV-MALS-dRI) was employed for analysis of glutamate decarboxylase (LbGadB) from Lactobacillus brevis (L. brevis). AF4 provided molecular weight (MW) (or size)-based separation of dimer, hexamer, and aggregates of LbGadB. The effect of pH on oligomerization of LbGadB was investigated, and then AF4 results were compared to those from molecular modeling. The MWs measured by AF4-UV-MALS-dRI for dimeric and hexameric forms of LbGadB were 110 and 350 kDa, respectively, which are in good agreements with those theoretically calculated (110 and 330 kDa). The molecular sizes determined by AF4-UV-MALS-dRI were also in good agreement with those obtained from molecular modeling (6 and 10 nm, respectively, for dimeric and hexameric from AF4-UV-MALS-dRI and 6.4 × 7.6 and 7.6 × 13.1 nm from molecular modeling). The effects of temperature, salt type, and salt concentration on oligomerization of LbGadB were also investigated using dynamic light scattering (DLS). It was found that the hexameric form of LbGadB was most stable at pH 6 and in presence of NaCl or KCl. The results indicate that AF4, in combination of various online detectors mentioned above, provides an effective tool for monitoring of oligomerization of LbGadB under different conditions, such as temperature, pH, type of salts, and salt concentrations.Electronic supplementary materialThe online version of this article (10.1007/s00216-017-0735-6) contains supplementary material, which is available to authorized users.

Proteins and antibodies in serum, plasma, and whole blood—size characterization using asymmetrical flow field-flow fractionation (AF4)

“…Therefore, AF4 is a highly powerful technique that is increasingly being used for the separation and characterization of biomacromolecules and pharmaceutical molecules [14–16]. It has been proven to be a potential tool for studying biological structures such as proteins, antigens, and antibodies [17–21]. In previous studies, field-flow fractionation (FFF) has been utilized for the separation and characterization of blood plasma and lipoproteins [22–24].…”

First Evidence of Acyl-Hydrolase/Lipase Activity From Human Probiotic Bacteria: Lactobacillus rhamnosus GG and Bifidobacterium longum NCC 2705

“…Thermal stability of the enzyme in different pH values was determined using the Prometheus NT 48 nanoDSF (NanoTemper Technologies, GmbH, Munich Germany) (Choi et al, 2018). Enzyme samples at 0.5 g/L were prepared in pH 4.3, 5.3, 6.2, 7, 8 (McIlvaine buffer system, 100 mM), 9 and 9.6 (glycin-NaOH buffer, 100 mM).…”

“…The biophysical characterization was focused on the determination of: (1) optimal temperature; (2) protein stability, in terms of melting temperature determined by differential scanning fluorimetry (DSF) in a range of pH from 4 to 10; and (3) multimerization, studied by asymmetrical flow field-flow fractionation (AF4) coupled to multiangle light scattering (MALS). AF4 separates multimeric proteins and aggregates based on their diffusion coefficient and, compared to size exclusion chromatography (SEC), does not have a stationary phase which reduce the potential loss of analytes from adsorption or shear-induced degradation ( Wahlund and Nilsson, 2012 ; Choi et al, 2018 ).…”