Affinity purification lipase from wheat germ: comparison of hydrophobic and metal chelation effect

Figures & data

Figure 1. Images of cryogels before (a) and after (b) Cu(II) immobilization.

Table 1. Purification steps of lipase from wheat germ.

Purification steps	Volume (mL)	Protein amount (mg/mL)	Activity (U/mL)	Specific activity (U/mg)	Overall yield (%)	Purification factor
Crude extract	50.0	9.80 ± 0.5	345.7 ± 17.3	35.3	100.0	1.00
Ammonium sulfate	48.0	5.90 ± 0.3	317.4 ± 15.9	53.8	88.1	1.52
poly(HEMA-MATrp)	10.0	2.22 ± 0.1	214.5 ± 10.7	96.6	12.4	2.74
poly(HEMA-MATrp)-Cu(II)	10.0	2.30 ± 0.1	240.8 ± 12.0	104.7	13.9	2.97

Figure 2. SEM images of poly(HEMA-MATrp) (a) and poly(HEMA-MATrp)-Cu(II) (b) cryogels.

Figure 3. FTIR graphs of (a) poly(HEMA), (b) poly(HEMA-MATrp) and (c) poly(HEMA-MATrp)-Cu(II) cryogels.

Figure 4. Effect of pH on adsorption of lipase onto poly(HEMA-MATrp) (C_lipase: 1.5 mg/mL, interaction time: 40 min., temperature: 25 °C) and poly(HEMA-MATrp)-Cu(II) (C_lipase: 2.0 mg/mL, interaction time: 30 min., temperature: 25 °C) cryogels.

Figure 5. Effect of interaction time on adsorption of lipase onto poly(HEMA-MATrp) (C_lipase: 1.5 mg/mL, pH: 6.0, temperature: 25 °C) and poly(HEMA-MATrp)-Cu(II) (C_lipase: 2.0 mg/mL, pH: 7.0, temperature: 25 °C) cryogels.

Figure 6. Effect of temperature on adsorption of lipase onto poly(HEMA-MATrp) (pH: 6.0, interaction time: 40 min., C_Lipase: 1.5 mg/mL) and poly(HEMA-MATrp)-Cu(II) (pH: 7.0, interaction time: 30 min., C_Lipase: 2.0 mg/mL) cryogels.

Figure 7. Effect of ionic strength on adsorption of lipase onto poly(HEMA-MATrp) (pH: 6.0, interaction time: 40 min., C_Lipase: 1.5 mg/mL, temperature: 25 °C) and poly(HEMA-MATrp)-Cu(II) (pH: 7.0, interaction time: 30 min., C_Lipase: 2.0 mg/mL, temperature: 25 °C) cryogels.

Figure 8. SDS-PAGE of desorbed lipase obtained from Candida cylindracea. Lane 1. Lipase, lysozyme, and hemoglobin marker, Lane 2: Lipase marker, Lane 3: Initial [(Before adsorption for poly(HEMA-MATrp)], Lane 4: Final [(After adsorption for poly(HEMA-MATrp)], Lane 5: Desorbed sample [(After dialysis for poly(HEMA-MATrp)], Lane 6: Initial [(Before adsorption for poly(HEMA-MATrp)-Cu(II)], Lane 7: Final [(After adsorption for poly(HEMA-MATrp)-Cu(II)], Lane 8: Desorbed sample [(After dialysis for poly(HEMA-MATrp)-Cu(II)].

Figure 9. UV-VIS spectra of purification steps of lipase from wheat germ.

Table 2. Parameters for adsorption isotherms of poly(HEMA-MATrp) and poly(HEMA-MATrp)-Cu(II).

		Langmuir constants			Freundlich constants
	Qexp (mg/g)	Qmax (mg/g)	b (L/mg)	R^2	Kf	n	1/n	R^2
poly(HEMA-MATrp)	166.4	1666.67	0.000081	0.9565	0.5147	0.7887	1.27	0.8798
poly(HEMA-MATrp)-Cu(II)	196.4	35.71	0.0093	0.9897	2.39	0.6113	1.64	0.9267