Partial purification and characterization of lipase from Geobacillus stearothermophilus AH22

Figures & data

Figure 1. Staining SDS–PAGE (12%) gel with Coomassie brilliant blue R-250 after activity staining. The lanes are as follows: M, marker proteins with relative molecular masses indicated on the left; Lane 1, crude extract of G. stearothermophilus AH22 lipase.

Figure 2. Nondenaturing PAGE pattern of partial purified lipase. The partial purified protein was electrophoresed on 10% (w/v) polyacrylamide gel under nonreducing conditions. (A) Activity staining gel. (B) Staining gel with Coomassie brilliant blue R-250 after activity staining. Lane 1: crude extract of G. stearothermophilus AH22 lipase; Lane 2: heat treatment (30 min at 70 °C); Lane 3: pooled fractions from DEAE-Cellulose chromatography; Lane 4: pooled fractions from Sephadex G-150 chromatography; Lane 5: pooled fractions from 2nd Sephadex G-150 chromatography; Lane 6: pooled fractions from Sephadex G-25 chromatography.

Table 1. Flowsheet of procedure used to partial purification of lipase from G. stearothermophilus AH22.

	Volume (mL)	Protein (mg/mL)	Total protein (mg)	Activity (U/mL)	Total activity (U)	Specific activity (U/mg protein)	Purification fold	Yield (%)
Crude extract	100	9.32	932	2.01	201	0.216	1	100
Heat treatment (30 min at 70 °C)	95	6.50	617.5	1.8	171	0.28	1.3	85
DEAE-Cellulose	30	2.60	78	3.15	94.5	1.21	5.6	47
Sephadex G-150	13	2.32	30.2	5.38	69.9	2.31	10.7	34.7
Sephadex G-150 2nd loading	12.5	1.37	17.1	4.1	51.3	2.99	13.9	25.5
Sephadex G-25	11	0.91	10	3.60	39.6	3.96	18.3	19.7

Figure 3. Optimum pH for activity of AH22 lipase.

Figure 4. Effect of pH on thermostability of AH22 lipase.

Figure 5. Optimal temperature for AH22 lipase.

Figure 6. Effect of temperature on thermostability of AH22 lipase at room temperature, 40 and 50 °C.

Figure 7. Effect of temperature on thermostability of AH22 lipase at 60 °C.

Figure 8. Effect of temperature on thermostability of AH22 lipase at 70, 80 and 90 °C.

Table 2. Thermodynamic parameters for thermal inactivation of G. stearothermophilus AH22 lipase.

Temparature (K)	k (s^−1)	ΔG# (J mol^−1)	ΔH# (J mol^−1)	ΔS# (J mol^−1 K^−1)
298	2.00 × 10^−7	295 589	127 043	−566
313	2.00 × 10^−7	310 595	126 918	−587
323	5.00 × 10^−7	323 063	126 835	−608
333	2.00 × 10^−6	336 988	126 752	−631
343	5.00 × 10^−4	362 937	126 669	−689
353	2.00 × 10^−4	370 914	126 586	−692
363	4.00 × 10^−4	383 597	126 502	−708

#Standard condition is 25 °C and 1 atm for physicochemical parameters.

Table 3. Effect of various metal ions and EDTA on the lipase activity.

	Relative activity (%)
Metal ions	0.1 mM	0.5 mM	1 mM
Control	100	100	100
Mn^2+	135	136	98
Ca^2+	105	119	91,5
Cu^2+	158	121	125
Ba^2+	112	124	153
Co^2+	145	127	152
Zn^2+	72.7	62.2	99
Fe^2+	122	103	96.8
Ni^2+	105	124	83.5
Mg^2+	104.5	120.7	93.8
Hg^2+	56	27.8	29.1
EDTA	105	119	106

Table 4. Effect of some surfactants and inhibitors on the lipase activity.

Surfactants and inhibitors	Concentration	Relative activity (%)	IC50	Surfactants and inhibitors	Concentration	Relative activity (%)	IC50
Control Triton X-114 (%)	–	100		Control PMSF (mM)	–	100
	0.1	67			0.5	70
	0.5	47	0.42		1.0	63	1.6
	1.0	23			2.0	43
Triton X-100 (%)	0.1	85		Catechol (mM)	1.0	80
	0.5	57	0.62		5.0	62	8.5
	1.0	37			10.0	48
Tween-80 (%)	0.1	65		Catechin (mM)	0.05	59
	0.5	46	0.40		0.1	31	0.06
	1.0	25			0.25	24
SDS (%)	0.1	31		Propyl paraben (mM)	0.5	52
	0.5	20	0.04		1.0	42	0.5
	1.0	12			2.0	29
β-Mercaptoethan ol (mM)	0.1	114		p-Coumaric acid (mM)	0.5	64
	0.5	107	–		1.0	53	1.4
	1.0	106			2.0	38
Orlistat (µg/mL)	2.0	68		3,4-Dihydroxy hydro-cinnamic acid (mM)	1.0	80
	4.0	51	4.2		2.5	43	1.7
	6.0	46			5.0	23