Immobilization of β-Galactosidases from Lactobacillus on Chitin Using a Chitin-Binding Domain

Abstract

Two β-galactosidases from Lactobacillus, including a heterodimeric LacLM type enzyme from Lactobacillus reuteri L103 and a homodimeric LacZ type β-galactosidase from Lactobacillus bulgaricus DSM 20081, were studied for immobilization on chitin using a carbohydrate-binding domain (chitin-binding domain, ChBD) from a chitinolytic enzyme. Three recombinant enzymes, namely, LacLM-ChBD, ChBD-LacLM, and LacZ-ChBD, were constructed and successfully expressed in Lactobacillus plantarum WCFS1. Depending on the structure of the enzymes, either homodimeric or heterodimeric, as well as the positioning of the chitin-binding domain in relation to the catalytic domains, that is, upstream or downstream of the main protein, the expression in the host strain and the immobilization on chitin beads were different. Most constructs showed a high specificity for the chitin in immobilization studies; thus, a one-step immobilizing procedure could be performed to achieve up to 100% yield of immobilization without the requirement of prior purification of the enzyme. The immobilized-on-chitin enzymes were shown to be more stable than the corresponding native enzymes; especially the immobilized LacZ from L. bulgaricus DSM20081 could retain 50% of its activity when incubated at 37 °C for 48 days. Furthermore, the immobilized enzymes could be recycled for conversion up to eight times with the converting ability maintained at 80%. These results show the high potential for application of these immobilized enzymes in lactose conversion on an industrial scale.

Keywords: Lactobacillus; chitin-binding domain; immobilization; β-galactosidase.

Figure 1

Construction of expression plasmids: pSCBDlac1 (for LacLM-ChBD) and pSCBDlac3 (for LacZ-ChBD) (A) and pSCBDlac2 (for ChBD-LacLM) (B).

Figure 2

SDS-PAGE analysis of expression and immobilization of recombinant enzymes. (A) Cell-free extracts of noninduced cells (lanes 1, 3) and induced cells (lanes 2, 4) of L. plantarum WCFS1 harboring pSCBlac1 (containing LacLM-ChBD) and pSCBlac2 (containing ChBD-LacLM), respectively. (B) Cell-free crude extracts (lanes 1, 4), flow through during immobilization (lanes 2, 5), and chitin-bound β-gals (lanes 3, 6) of L. plantarum harboring plasmids pSCBDlac1 (containing LacLM-ChBD) and pSCBDlac2 (containing ChBD-LacLM), respectively. (C) Cell-free crude extracts of L. plantarum WCFS1 harboring pSCBDlac3 (containing LacZ-ChBD) at OD ~ 0.3 (before induction) (lane 1) and at 16 h of induction (lane 2); flow through during immobilization (lane 3) and rinsing fractions (lanes 4, 5), chitin-bound LacZ (lane 6); and chitin beads (lane 7). Lane M shows the Precision Plus Protein standard (Bio-Rad). The arrows indicate subunits of the recombinant β-galactosidases.

Figure 3

Temperature optimum of the immobilized enzymes. I-LacLM-ChBD (●) and I-ChBD-LacLM (▽) indicate the recombinant enzymes with the ChBD fused to the C-terminal of LacM and the N-terminal of LacL, respectively. I-LacZ-ChBD (◆) indicates the immobilized LacZ-ChBD. The solid and dotted line indicate the substrate oNPG and lactose, respectively.

Figure 4

Optimum pH values of β-galactosidase activity of the immobilized enzymes. I-LacLM-ChBD (●) and I-ChBD-LacLM (▽) indicate the recombinant enzymes with the ChBD fused to C-terminal of the LacM and the N-terminal of the LacL, respectively. I-LacZ-ChBD (◆) indicates the immobilized LacZ-ChBD. The solid and dotted lines indicate substrates oNPG and lactose, respectively.

Figure 5

Reusability of the chitin-immobilized enzymes I-LacLM-ChBD (●), I-ChBD-LacLM (○), and I-LacZ-ChBD (▲). The converting ability (%) was calculated from the ratio of the product released (glucose or oNP released from lactose or oNPG, respectively) in different reaction cycles to the product obtained at the first batch (assumed as 100%).

Figure 6

TLC analysis of different batches of lactose conversion by the immobilized β-galactosidase from L. bulgaricus, I-LacZ-ChBD. The batch conversions were carried out in 50 mM sodium phosphate buffer, pH 6.5, 10 mM MgCl₂ with various initial lactose concentrations, activities, and temperatures: 50 g/L initial lactose at 55 °C using 3.2 U_lactose/mL (A); 205 g/L initial lactose concentration at 55 °C using 1.7 U_lactose/mL (B); 50 g/L initial lactose concentration at 60 °C using 9.7 U_lactose/mL (C); commercial whole milk, 1 h at 60 °C using 9.7 U_lactose/mL (D).

Figure 7

TLC of products from the batch lactose conversions by the immobilized enzymes I-LacLM-ChBD, I-ChBD-LacLM, and LacLM-ChBD (free soluble enzyme). The conversions were performed in 50 mM sodium phosphate buffer, pH 6.5, 10 mM MgCl₂, with an initial concentration of 205 g/L lactose at 30 °C, using 1.5 U_lactose/ml. Glc, glucose; Gal, galactose; Lac, lactose; GOS, galacto-oligosaccharides. The standards include a mixture of glucose, galactose, and lactose (1); Vivinal GOS (2); and GOS from conversion of free LacLM from L. reuteri L103 (3).

Figure 8

Carbohydrate composition during lactose transformation by the immobilized β-galactosidase from L. bulgaricus (I-LacZ-ChBD) determined by HPLC. The batch conversions were carried out in sodium phosphate buffer 50 mM, pH 6.5, in the presence of 10 mM MgCl₂ at various temperatures, enzyme activities, and initial lactose concentrations: (A) 50 g/L initial lactose concentration, at 55 °C using 3.2 U_lactose/mL; (B) 50 g/L initial lactose concentration at 37 °C using 3.2 U_lactose/mL; (C), 205 g/L initial lactose concentration at 37 °C using 1.7 U_lactose/mL; (D) 205 g/L initial lactose concentration at 55 °C using 1.7 U_lactose/mL. The concentrations of lactose (◆), glucose (□), galactose (▲), and total galacto-oligosaccharides (○) were analyzed during the conversion.

Figure 9

Carbohydrate composition during lactose transformation by the recombinant β-galactosidase LacLM-ChBD from L. reuteri L103 as determined by HPLC. The batch conversions were carried out in 50 mM sodium phosphate buffer, pH 6.5, in the presence of 10 mM MgCl₂ at 30 °C; 1.5 U_lactose/mL, and an initial concentration of lactose of 205 g/L with the free soluble LacLM-ChBD (A) or with the immobilized enzyme I-LacLM-ChBD (B). The concentrations of lactose (◆), glucose (□), galactose (▲), and total galacto-oligosaccharides (○) were analyzed during the conversion.

Figure 10

Predicted 3D structures of LacM-ChBD (A), ChBD-LacL (B), LacZ-ChBD (C), and ChBD (D). The 3D structures were predicted by the RaptorX tool (http://raptorx.uchicago.edu/) on the basis of the deduced amino acid sequences.