Optimization of covalent immobilization of Trichoderma reesei cellulase onto modified ReliZyme HA403 and Sepabeads EC-EP supports for cellulose hydrolysis, in buffer and ionic liquids/buffer media

Figures & data

Table I. The variables and their lowest, middle, and highest values.

			Variable level
			Lowest	Middle	Highest
Support	Variables	Symbol	− 1	0	+ 1
	Immobilization pH	X 1	5.0	6.5	8.0
Sepabeads EC-EP	Immobilization time (h)	X 2	6	15	24
	Initial protein concentration (mg/mL)	X 3	0.2	1.0	1.8
	Immobilization pH	X 1	5.0	6.5	8.0
ReliZyme HA403	Immobilization time (h)	X 2	2	5	8
	Initial protein concentration (mg/mL)	X 3	0.2	1.0	1.8

Figure 1. The procedure for immobilization of cellulase onto modified ReliZyme HA403 and Sepabeads EC-EP commercial supports.

Table II. The results of using 3-factor and 3-level BBD for the optimal conditions of cellulase immobilization.

Support	Run number	Immobilization pH	Immobilization time (h)	Initial protein concentration (mg/mL)	Immobilization yield (%)	Specific activity (U/mg protein) (Experimental)	Specific activity (U/mg protein) (Predicted)
	1	6.5	15	1.0	51	17.4	18.0
	2	8.0	15	1.8	62	38.4	36.8
	3	6.5	6	1.8	34	54.0	64.3
	4	5.0	24	1.0	65	14.4	23.0
	5	6.5	24	0.2	5	57.0	46.7
	6	5.0	15	0.2	32	19.2	20.9
	7	8.0	24	1.0	65	15.6	23.2
Sepabeads EC-EP	8	6.5	15	1.0	44	18.0	18.0
	9	5.0	6	1.0	62	32.4	24.8
	10	8.0	15	0.2	64	9.6	12.3
	11	6.5	24	1.8	36	45.6	39.7
	12	8.0	6	1.0	68	21.0	12.4
	13	6.5	6	0.2	50	7.2	13.1
	14	5.0	15	1.8	63	43.2	40.5
	15	6.5	15	1.0	53	18.6	18.0
	1	6.5	5	1.0	75	17.1	18.3
	2	8.0	5	1.8	88	35.3	16.6
	3	6.5	2	1.8	71	41.4	14.5
	4	5.0	8	1.0	66	21.6	25.7
	5	6.5	8	0.2	60	27.6	44.7
	6	5.0	5	0.2	74	12.6	24.5
	7	8.0	8	1.0	55	21.6	32.9
ReliZyme HA403	8	6.5	5	1.0	87	16.2	16.6
	9	5.0	2	1.0	76	25.8	23.0
	10	8.0	5	0.2	88	16.2	16.7
	11	6.5	8	1.8	51	32.2	33.4
	12	8.0	2	1.0	87	27.1	22.9
	13	6.5	2	0.2	67	17.4	36.0
	14	5.0	5	1.8	88	38.1	16.6
	15	6.5	5	1.0	55	16.5	24.3

Figure 2. (a) Response surface plot of the effect of immobilization pH and immobilization time on the specific activity of immobilized cellulase preparations at the fixed initial enzyme concentration of 1 mg mL^{− 1}. (b) Response surface plot of the effect of immobilization pH and initial protein concentration on the specific activity of the immobilized cellulase preparations at the fixed immobilization time of 5 h. (c) Response surface plot of the effect of immobilization time and initial protein concentration on the specific activity of the immobilized cellulase preparations at the fixed immobilization pH of 6.5.

Figure 3. The activities of free (♦) and immobilized cellulase preparations (■ for immobilization onto Sepabeads EC-EP, and ▲ for immobilization onto modified ReliZyme HA403), at the pH range of 3.0–6.5.

Figure 4. The activities of free (♦) and immobilized cellulase preparations (■ for immobilization onto Sepabeads EC-EP, and ▲ for immobilization onto modified ReliZyme HA403), at the temperature range of 25–65°C.

Figure 5. FE-SEM images of pure Sepabeads EC-EP (a) and modified ReliZyme HA403 (d), and cellulase immobilized onto Sepabeads EC-EP (b and c) and onto ReliZyme HA403 (e and f).

Figure 6. The hydrolysis of CMC in 25% (v/v) of ILs/buffer media. The pH/temperature values were 4.0/45°C, 4.0/50°C and 3.5/60°C, respectively, for the free cellulase, cellulase immobilized onto Sepabeads EC-EP, and onto modified ReliZyme. Values for 100% activities are 170, 66, and 60 U/mg protein, respectively for the free cellulase, cellulase immobilized onto Sepabeads EC-EP and onto ReliZyme HA403.

Figure 7. The hydrolysis of CMC in different volume ratios of [Bmim][PF₆]/buffer media. The pH/temperature values were 4.0/45°C, 4.0/50°C and 3.5/60°C, respectively, for the free cellulase, cellulase immobilized onto Sepabeads EC-EP, and onto modified ReliZyme. Values for 100% activities are 170, 66, and 60 U/mg protein, respectively, for the free cellulase, cellulase immobilized onto Sepabeads EC-EP, and onto ReliZyme HA403.

Figure 8. The thermal stability performance of free (♦) and immobilized cellulase preparations (■ for immobilization onto Sepabeads EC-EP, and ▲ for immobilization onto modified ReliZyme HA403) at 50°C (A) and at 70°C (B).

Figure 9. The reusability performance of immobilized cellulase preparations in a batch type reactor (♦for immobilization onto Sepabeads EC-EP, and ■ for immobilization onto modified ReliZyme HA403).