Optimization and purification of mannanase produced by an alkaliphilic-thermotolerant Bacillus cereus N1 isolated from Bani Salama Lake in Wadi El-Natron

Figures & data

Table 1. Factors examined as independent variables affecting mannanase enzyme activity and their levels in the Plackett–Burman experimental design.

		Levels
Factors	Symbols	Low level (−1)	Basal medium (0)	High level (+1)
MgSO4.7H2O	Mg	0.1	0.2	0.5
K2HPO4	K2	0.5	1	1.5
NaCl	Na	5	10	15
Yeast extract	YE	2	5	7
Peptone	P	2	5	7
Inoculum size	IS	0.5	1	1.5
Glucose	G	5	10	15

Table 2. The Plackett–Burman experimental design matrix for seven factors.

Trials	Mg	K2	Na	YE	P	IS	G	Mannanase activity (UmL^−1)	Mannanase specific activity Umg^−1 protein
1	−1	−1	−1	+1	+1	+1	−1	7	6
2	+1	−1	−1	−1	−1	+1	+1	32	29.5
3	−1	+1	−1	−1	+1	−1	+1	22	29
4	+1	+1	−1	+1	−1	−1	−1	8.8	15.7
5	−1	−1	+1	+1	−1	−1	+1	15	13
6	+1	−1	+1	−1	+1	−1	−1	0	0
7	−1	+1	+1	−1	−1	+1	−1	10	14
8	+1	+1	+1	+1	+1	+1	+1	29	25
9	0	0	0	0	0	0	0	6.5	10.5

Table 3. Statistical analysis of the Plackett–Burman experimental result for B. cereus N1.

	Mannanase activity
Factors	Main effect	t-value	P-value	Significance level
MgSO4.7H2O	3.95	0.468	0.66	–
K2HPO4	3.95	0.468	0.65	–
NaCl	−3.95	−0.468	0.65	–
Yeast extract	1.45	0.16	0.87	–
Peptone	−1.95	−0.228	0.83	–
Inoculum size	8.05	1.01	0.35	–
Glucose	18.05	4.1	0.0093	99%

Figure 1. Elucidation of cultivation factors affecting mannanase production by B. cereus N1 using Plackett–Burman experimental design.

Figure 2. Verification experiment for mannanase activity produced by B. cereus N1.

Table 4. Purification scheme of mannanase enzyme from the isolate B. cereus N1.

Purification step	Volume (mL)	Mannanase activity (UmL^−1)	Total protein (mgmL^−1)	Specific activity (Umg^−1)	Yield (%)	Purification (fold)
Culture filtrate	1000	0.9	0.22	4.22	100.00	1.00
Concentrating using ultrafiltration Amicon	100	8.8	1.88	4.68	97.78	1.11
DEAE-sepharose CL6B	10	60.2	0.71	84.79	88.89	20.09
32/60 Sephacryl S-100 HR	4	80.4	0.13	628.28	35.73	148.88

Figure 3. DEAE-sepharose CL6B column as anion exchange column chromatography of B. cereus N1 mannanase preparation. Buffer: 20 mmol/L tris-acetate, pH 7.

Figure 4. Sephacryl S-100 HR column as a gel filtration chromatography of B. cereus N1 mannanase preparation. Buffer: 50 mmol/L sodium acetate containing 0.2 mol/L NaCl, pH 6.

Figure 5. SDS-gel polyacrylamide electophoresis (PAGE), showing the purified mannanase from B. cereus N1. Lane 1: Showed mannanase extracted and purified from B. cereus N1. Lane 2: Showed the SDS-6H standard proteins (kDa).