Table 5.

Kinetic Parameters of the Immobilized β-d-Galactosidases for the Hydrolysis of Lactose and o-Nitrophenyl-β-d-galactopyranoside (oNPG) in Comparison to the Respective Free Enzymes without ChBD^a

substrate	method for determination of enzyme activity	kinetic parameter	LacLM-ChBDb	I-LacLM-ChBD	I-ChBD-LacLM	L103c	EcoL103d	LpL103e	I-LacZ-ChBD	LacZf
lactose	release of d-glucose	vmax,Glu (μmol min−1 mg−1)	17.83 ± 0.14	1.74 ± 0.07	0.72 ± 0.01	34	38	43	39.9 ± 0.9	123 ± 5
		Km,Lac (mM)	51.74 ± 1.80	36.82 ± 0.33	12.25 ± 0.39	13	12	12	45.5 ± 4.2	19.2 ± 3.8
		Kcat (s−1)	9.00 ± 0.07	0.88 ± 0.03	0.36 ± 0.01	18	20	23	79.8 ± 1.8	234 ± 13
		Kcat/Km (s−1 M−1)	174	24	29	1389	1681	1902	1754	12300
oNPG	release of oNP	vmax,oNP (μmol min−1 mg−1)	103.9 ± 1.82	65.40 ± 1.78	32.09 ± 0.14	nd	nd	nd	87.1 ± 2.8	317 ± 6
		Km,oNPG (mM)	0.91 ± 0.05	2.18 ± 0.45	1.34 ± 0.03	nd	nd	nd	13.7 ± 1.1	0.92 ± 0.09
		Kcat (s−1)	52.39 ± 0.92	32.98 ± 0.90	16.18 ± 0.07	nd	nd	nd	174.2 ± 5.6	603 ± 15
		Kcat/Km (s−1 M−1)	54970	15096	12032	nd	nd	nd	12715	655000

^aThe three chitin-immobilized enzymes of LacLM-ChBD, ChBD-LacLM, and LacZ-ChBD are designated I-LacLM-ChBD, I-ChBD-LacLM, and I-LacZ-ChBD, respectively. The bound amount of protein on the chitin beads was calculated by subtraction of the protein content in the cell crude extract (before immobilization) and in the liquid phase (after immobilization). The difference of v_max and K_m values among the immobilized enzymes and the free-soluble enzyme is statistically significant (p < 0.05). nd, not determined.

^bFree soluble LacLM-ChBD.

^cNative enzyme from L. reuteri L103.¹⁴

^dRecombinant enzyme expressed and purified from E. coli.¹⁵

^eRecombinant enzyme expressed and purified from L. plantarum WCFS1.¹⁷

^fPurified recombinant enzyme LacZ from L. bulgaricus expressed in L. plantarum.¹⁶