Expression of β-Glucosidases from the Yak Rumen in Lactic Acid Bacteria: A Genetic Engineering Approach

Abstract

β-glucosidase derived from microorganisms has wide industrial applications. In order to generate genetically engineered bacteria with high-efficiency β-glucosidase, in this study two subunits (bglA and bglB) of β-glucosidase obtained from the yak rumen were expressed as independent proteins and fused proteins in lactic acid bacteria (Lactobacillus lactis NZ9000). The engineered strains L. lactis NZ9000/pMG36e-usp45-bglA, L. lactis NZ9000/pMG36e-usp45-bglB, and L. lactis NZ9000/pMG36e-usp45-bglA-usp45-bglB were successfully constructed. These bacteria showed the secretory expression of BglA, BglB, and Bgl, respectively. The molecular weights of BglA, BglB, and Bgl were about 55 kDa, 55 kDa, and 75 kDa, respectively. The enzyme activity of Bgl was significantly higher (p < 0.05) than that of BglA and BglB for substrates such as regenerated amorphous cellulose (RAC), sodium carboxymethyl cellulose (CMC-Na), desiccated cotton, microcrystalline cellulose, filter paper, and 1% salicin. Moreover, 1% salicin appeared to be the most suitable substrate for these three recombinant proteins. The optimum reaction temperatures and pH values for these three recombinant enzymes were 50 °C and 7.0, respectively. In subsequent studies using 1% salicin as the substrate, the enzymatic activities of BglA, BglB, and Bgl were found to be 2.09 U/mL, 2.36 U/mL, and 9.4 U/mL, respectively. The enzyme kinetic parameters (Vmax, Km, Kcat, and Kcat/Km) of the three recombinant strains were analyzed using 1% salicin as the substrate at 50 °C and pH 7.0, respectively. Under conditions of increased K⁺ and Fe²⁺ concentrations, the Bgl enzyme activity was significantly higher (p < 0.05) than the BglA and BglB enzyme activity. However, under conditions of increased Zn²⁺, Hg²⁺, and Tween20 concentrations, the Bgl enzyme activity was significantly lower (p < 0.05) than the BglA and BglB enzyme activity. Overall, the engineered lactic acid bacteria strains generated in this study could efficiently hydrolyze cellulose, laying the foundation for the industrial application of β-glucosidase.

Keywords: L. lactis NZ9000; enzyme activity; fusion expression; β-glucosidase.

Figure 1

Maps of the three recombinant plasmid constructs and their electropherogram. (a) pMG36e-usp45-bglA recombinant plasmid; (b) pMG36e-usp45-bglB recombinant plasmid; (c) pMG36e-usp45-bglA-usp45-bglB recombinant plasmid; (d) M: DNA molecular weight marker; 1: PCR product of bglA; 2: PCR product of bglB; 3: PCR product of bglA-bglB; 4: pMG36e plasmid after Pst I digestion; 5: pMG36e-bglA plasmid after Pst I and Hind III digestion; 6: pMG36e-bglB plasmid after Pst I and Hind III digestion; 7: pMG36e-bglA-bglB plasmid after Pst I digestion; 8: pMG36e-bglA-bglB plasmid after Pst I and Hind III digestion.

Figure 2

The analysis of recombinant enzymes activity using agar plates with 0.1% sodium carboxymethyl cellulose CMC-Na-stained Congo red. The halos dimeter is proportional to enzyme’s activity. To each well 20 μL of the enzyme with concentration 0.26–0.27 mg/mL of recombinant enzymes was transferred; 1: The inoculum of L. lactis NZ9000/pMG36e-usp45-bglA; 2: The inoculum of L. lactis NZ9000/pMG36e; 3: The inoculum of L. lactis NZ9000/pMG36e-usp45-bglA-usp45-bglB; 4: The inoculum of L. lactis NZ9000/pMG36e-usp45-bglB; 5: Negative control ddH₂O; 6: The inoculum of L. lactis NZ9000/pMG36e; 7: β-glucosidase standard.

Figure 3

SDS-PAGE of supernatant from recombinant strains. M: Protein molecular weight marker, 1: Bacterial supernatant precipitate of L. lactis NZ9000/pMG36e; 2: Bacterial supernatant precipitate of L. lactis NZ9000/pMG36e-usp45-bglA; 3: Bacterial supernatant precipitate of L. lactis NZ9000/pMG36e-usp45-bglB; 4: Bacterial supernatant precipitate of L. lactis NZ9000/pMG36e-usp45-bglA-usp45-bglB; 5: TCA/acetone precipitated protein of pMG36e from the L. lactis NZ9000/pMG36e bacterial supernatant; 6: TCA/acetone-precipitated BglA protein from L. lactis NZ9000/pMG36e-usp45-bglA bacterial supernatant; 7: TCA/acetone-precipitated BglB protein from L. lactis NZ9000/pMG36e-usp45-bglB bacterial supernatant; 8: TCA/acetone-precipitated Bgl protein from L. lactis NZ9000/pMG36e-usp45-bglA-usp45-bglB bacterial supernatant.

Figure 4

Effect of temperature, pH, and substrate on recombinant enzyme activity. (a) Substrate specificity of recombinant proteins; (b) Effect of temperature on recombinant enzyme activity; (c) Effect of pH on recombinant enzyme activity; (*: Represents the difference within experimental groups; *: p < 0.05; **: p < 0.01; error bars in graphs represent standard deviations).

Figure 5

Effect of different concentrations of metal ions and chemical reagents on recombinant enzyme activity. (a) Effect of 1 mmol/mL ions and 1% chemical solutions on enzyme activity; (b) Effect of 5 mmol/mL ions and 10% chemical solutions on enzyme activity (#: Represents the difference between experimental groups and the control group; #: p < 0.05; ##: p < 0.01; ###: p < 0.001 *: Represents the difference within experimental groups; *: p < 0.05; **: p < 0.01; Error bars in graphs represent standard deviations).