Physical Adsorption Entrapment

Physical adsorption is the most convenient way to achieve enzyme immobilization, avoiding the tedious and time-consuming multistep procedure of covalent binding (Aleixo et al. 1985; Cleveland et al. 1981). Entrapment immobilizes enzymes by trapping them into porous materials (membranes). Because there is no covalent attachment, the activity of the enzymes immobilized by physical adsorption, and entrapment is easily maintained. However, loss of enzyme in applications is inevitable for the physical adsorption method, impeding the further utility of this method. At the same time, how substrates diffuse into the porous materials and how products diffuse out are the critical problems associated with the entrapment strategy.

Deng et al. (2004a,b,c) immobilized Candida rugosa lipase on a series of surface-modified PPMMs by adsorption and compared their effects on the enzyme activity. Three different kinds of modifiers, poly(a-allyl glu-coside) (PAG), two polypeptides with short and long hydrophobic side chains, poly(y-ethyl-L-glutamate) and poly(y-stearyl-L-glutamate), and phospholipid-analogous polymers (PAP) containing hydrophobic octyloxy, dodecyloxy, and octadecyloxy groups (8-PAP, 12-PAP, and 18-PAP respectively), were tethered onto PPMMs (see Figs. 1, 3, and 6). Then lipases from Candida rugosa were immobilized on these membranes by adsorption and their activity was examined. The specific activity and the activity retention of the lipase immobilized onto the hydrophobic polypeptide- and PAP-modified membranes were both higher than those of nascent and hy-drophilic PAG-modified membranes (Table 4). This can be ascribed to the large hydrophobic surface that surrounds the catalytic site of the lipase; lipase is thought to be activated in the presence of a hydrophobic interface, which can be helpful for the rearrangement of the protein conformation to yielding an "open state" of the lipase active site. There is a strong hydropho-bic interaction between the long alkyl chain of PAP and polypeptides and the hydrophobic domain around the active site of lipase, which stabilizes the "open state" conformation of lipases and favors the accessibility of the

Table 4. Comparing of lipase activity as function of different modifier. PAG Poly (a-allyl glucoside), PAP phospholipids analogous polymers, PELG poly(y-ethyl-L-glutamate), PSLG poly(y-stearyl-L-glutamate)

Membrane/Modifier

Grafting degree

Water contact Specific activity

Activity

(wt.%)

angle (°C)

(U/mg protein)

retention (%)

Nascent PPMM

-

118

69.9

57.5 ± 2.8

2.1

60

61.3

50.4 ± 2.7

PAG

2.8

48

62.3

51.2 ± 2.8

3.7

36

60.7

49.9 ± 2.9

8-PAP

10.6

53

90.1

74.1 ± 3.2

PAP

12-PAP

10.9

92

94.3

77.5 ± 3.7

18-PAP

10.8

96

101.2

83.2 ± 3.3

Polypeptide

PELG

3.5

113

76.4

62.8 ± 3.3

PSLG

3.6

122

88.1

72.4 ± 3.9

active site to substrates. On the other hand, after modification with PAG, the sharp increase in hydrophilicity induces the conformational equilibrium of lipase, to some extent shifting it toward the unfavorable "closed state".

Tanioka et al. (1998) entrapped invertase in the pores of PPMMs, and these membranes were used to hydrolyze sucrose. As show in Fig. 15, they embedded invertase into the pores of PPMMs and then grafted poly(acrylic acid) (PAA) onto the membrane surface to entrap the enzyme. The in-vertase-entrapped PPMM was immersed in sucrose aqueous solution to hydrolyze the sucrose. The sucrose that permeated through the PAA layer on the membrane surface was hydrolyzed into fructose and glucose by in-vertase, and the decomposed products exited to the external solution. The hydrolyzation process of the sucrose caused a change in optical rotation, and inspection could be made using a polarimeter. Figure 16a,b shows the hydrolysis process of the invertase-immobilized PPMMs with a high (Fig. 16a) and low (Fig. 16b) PAA grafting degree, respectively, as a function of time. Arrows indicate that the membranes were removed from the sucrose solution. After this removal, hydrolysis cannot be observed at all in the case with the high grafting degree (Fig. 16a), but can still be observed in that of the low grafting degree (Fig. 16b). This indicates that invertase leakage occurred in the case of a low grafting degree, in which the PAA layer was not thick enough to prevent the invertase from spilling. However, with higher PAA grafting degree invertase could be entrapped stably in the PPMM pores and the hydrolysis processed very well.

Schematic Protein Adsorption

Fig. 15. Schematic representation of enzyme entrapment

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