Characterization of two β-galactosidases LacZ and WspA1 from Nostoc flagelliforme with focus on the latter's central active region

Abstract

The identification and characterization of new β-galactosidases will provide diverse candidate enzymes for use in food processing industry. In this study, two β-galactosidases, Nf-LacZ and WspA1, from the terrestrial cyanobacterium Nostoc flagelliforme were heterologously expressed in Escherichia coli, followed by purification and biochemical characterization. Nf-LacZ was characterized to have an optimum activity at 40 °C and pH 6.5, different from that (45 °C and pH 8.0) of WspA1. Two enzymes had a similar Michaelis constant (Km = 0.5 mmol/liter) against the substrate o-nitrophenyl-β-D-galactopyranoside. Their activities could be inhibited by galactostatin bisulfite, with IC50 values of 0.59 µM for Nf-LacZ and 1.18 µM for WspA1, respectively. Gel filtration analysis suggested that the active form of WspA1 was a dimer, while Nf-LacZ was functional as a larger multimer. WspA1 was further characterized by the truncation test, and its minimum central region was found to be from residues 188 to 301, having both the glycosyl hydrolytic and transgalactosylation activities. Finally, transgenic analysis with the GFP reporter protein found that the N-terminus of WspA1 (35 aa) might play a special role in the export of WspA1 from cells. In summary, this study characterized two cyanobacterial β-galactosidases for potential applications in food industry.

Figure 1

Phylogenetic analysis of Nf-LacZ (COO91_07519) with its top 50 similar homologs from the KEGG database. The nodes with bootstrap values higher than 70% were highlighted with blue dots. The lacZ homologs from Cyanobacteria (green) form a distinct clade. The homologs from Betaproteobacteria were highlighted in blue, the homologs from Deltaproteobacteria were highlighted in orange, the homologs from Acidobacteria were highlighted in red, and the homologs from Deinococcus-Thermus group were highlighted in purple. The species names for these proteins were included in supplemental table S1.

Figure 2

In vitro expression and enzymatic analysis of Nf-LacZ protein. (A) In vitro expression of Nf-LacZ by the E. coli BL21/pET28a protein expression system. M marker protein, P total proteins before IPTG induction, IP total proteins after IPTG induction, W1 and W2 washed fractions, E eluted fraction. Blue arrow points to the Nf-LacZ protein. (B) The protein polymerization state analyzed by FPLC. Molecular weight markers: beta-amylase, 200 kDa; alcohol dehydrogenase, 150 kDa; albumin, 66 kDa; carbonic anhydrase, 29 kDa; cytochrome c, 12.4 kDa. (C) Michaelis kinetic analysis. V_max and K_m values were calculated. ONPG serves as the substrate. Reaction was conducted at 45 °C and pH 8.0.

Figure 3

In vitro expression of Wsp_A and analysis of the protein polymerization state. (A) In vitro expression of Wsp_A by the E. coli BL21/pET28a protein expression system. M marker protein, E eluted fraction. Blue arrow points to the target protein. (B) The protein polymerization state of native Wsp_A analyzed by FPLC at pH 7.5. (C) The effect of unfavorable acidic condition (pH 5.5) on the dimer of Wsp_A. (D) The effect of unfavorable alkaline condition (pH 8.5) on the dimer of Wsp_A.

Figure 4

The inhibitory effects of GBS on the activities of Nf-LacZ and Wsp_A. (A,B), the galactosyl hydrolytic reactions of Nf-LacZ and Wsp_A in presence of different concentrations of GBS, respectively. GBS concentration: 0 ~ 20 µM. The absorbance of the reaction product ONP at 405 nm (OD₄₀₅) was detected. Data shown are means ± SD (n = 3). (C,D), determination of the IC50 values of GBS inhibition on the activities of Nf-LacZ and Wsp_A, respectively. GBS concentration ranged from 0.01 µM to 50 µM in the tests.

Figure 5

Biochemical analysis of the truncated proteins of WspA1. (A) An illustration of the truncated protein variants. (B) Protein profiles of the in vitro expressed and purified target proteins in SDS-PAGE gels. M marker protein, E eluted fraction. No. 1–6, the purified fractions by FPLC. The red arrows indicate the target proteins. C1, C2, C3 and C4 represent Wsp_C1, Wsp_C2, Wsp_C3, and Wsp_C4, respectively. (C) Comparative analysis of hydrolytic activities in 0.1 M PBS buffer (pH 7.5) with ONPG as the substrate. Data shown are means ± SD (n = 3). Protein concentrations, 10 μg/ml. ONPG, 3 mM. Control, no addition of any protein in the reaction buffer. (D) In vitro activity analysis of the four proteins in X-Gal buffer (pH 7.5). Similar conditions were used as in (C). Reactions were performed for 6 h. (E) TLC analysis of the transgalactosylation activity of the truncated proteins. Glucose was used as the acceptor. Blue arrows indicate the generated products. The pink arrow indicates the solvent diffusion direction.

Figure 6

Examination of the potential export role of Wsp_N in transgenic Nostoc sp. PCC 7120 cells. Confocal microscopy observation of the GFP-fused proteins, WspA1::GFP (A), Wsp_B::GFP (B), and Wsp_N::GFP (C) in transgenic cells. For panel (A,B), a 40 × objective lens was used; for panel (C), a 60 × objective lens was used. Red arrows point to the secreted fluorescent foci; Red circles indicate the fluorescent foci that are seemingly in the process of secretion. (D) Western blotting analysis of WspA1::GFP and Wsp_B::GFP proteins in transgenic cells and culture solutions. An anti-WspA1 antibody was used. Blue arrows point to the target proteins. WT, wild-type cells.

Figure 7

An illustration of the two β-galactosidases LacZ and WspA in a cell of N. flagelliforme. LacZ is located intracellularly. WspA is stored intracellularly, but can be secreted into the glycan sheath upon rehydration. The activities of both enzymes are promoted by Mg²⁺, while Ca²⁺ is inhibitory for WspA. In the glycan sheath, the activity of WspA may also be affected by the extracellular pigment scytonemin and its own hydrolysis. scy scytonemin.