Influence of Water Miscible Organic Solvents on α-chymotrypsin in Solution and Immobilized on Eupergit CM

Abstract

The effects of the water-miscible organic solvents (methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, N,N'-dimethylformamide and tetrahydrofuran) on the stability and catalytic activity of alpha-chymotrypsin (CT) immobilized on Eupergit CM were studied. Enhanced stabilities and activities were observed both as a consequence of immobilization and the presence of organic solvent, which in combination provide long term (at least 24 h) retention of activity, and up to 50-fold increase in 50% (v/v) methanol in buffer. Low quantities (20%, v/v) of acetonitrile not only prevented CT inactivation by autolysis at 20 degrees C but also induced a significant increase in the activity of both free (six-fold) and immobilized (two-fold) CT.

Effects of water miscible organic solvents on the activity and conformation of the baeyer–villiger monooxygenases from Thermobifida fusca and Acinetobacter calcoaceticus: A comparative study

“…The increase in activity was only 1.2-fold for CHMO and maximal at 2% MeOH. It has to be noted that activation of enzymes at low co-solvent concentration, is a phenomenon already observed for other enzymes (Kudryashova et al, 2003;Ogino et al, 2000;Olofsson et al, 2006;Park et al, 2005;Tan and Lovrien, 1972). Interestingly, Tó th et al (2010) found a remarkable increase in activity of the flavoenzyme NADH oxidase from Thermus thermophilus in the presence of several alcohols (with MeOH the optimum was at about 40%).…”

Phage display screening in low dielectric media

“…Further, the marked difference in CT autolytic behaviour in buffer, relative to ACN, has been studied previously (Olofsson et al, 2006). The delicate balance between function and media dielectricity necessitated a series of optimization studies for the various steps of the biopanning protocol and the subsequent enzyme assay and inhibition analysis procedures, which resulted in those employed here.…”

“…To facilitate the biopanning process, the protein is ideally immobilized on a suitable matrix. We have recently reported (Olofsson et al, 2006) the immobilization of CT on porous oxirane functionalized polymer beads (Eupergit CM), and demonstrated that immobilization on this matrix not only enhances resistance to organic media, but also limits autolysis.…”

“…Analogy can here be drawn to the influence of immobilization of CT on a matrix. Similarly, immobilization has been shown to inhibit the detrimental effect of acetonitrile on chymotrypsin activity (Olofsson et al, 2006).…”

“…Pre-incubation (CT blank 10 min) results in lowering of CT activity from 100 to 70%. The inhibition caused by the pre-incubation step stems from the autolytic behaviour of CT (Kumar and Hein, 1970;Olofsson et al, 2006). The presence of each of the lysates, 1-4, during the 10 min of pre-incubation increased CT activity significantly ($60%) relative to CT blank 10 min.…”