α-Chymotrypsin shows higher activity in water as well as organic solvents after three phase partitioning

Abstract

Alpha-Chymotrypsin was found to show a 119% increase in activity after three phase partitioning. The k cat /K m of the partitioned enzyme (TPP-C ) for hydrolysis of BzTyr-OEt in aqueous medium at 258C was found to be 48.3/10 4 mM 1 min 1 as compared to the corresponding value of 17.7 /10 4 mM 1 min 1 for the untreated control (C ). The l max of the fluorescence emission spectrum of TPP-C showed 178% increase in the quantum yield when compared to C. TPP-C showed a 2.94 and 3.58 fold increase (as compared to C ) in initial rates for formation of the ester Ac-Phe-OEt (from Ac-Phe and ethanol) in low water containing toluene and n-octane, respectively. It was found that TPP-C also showed the phenomenon of pH memory. At 5% (v v 1 ) water (in t-amyl alcohol), while no esterification was observed with C , TPP-C still showed significant level of esterification activity.

Enhancement of stability of a lipase by subjecting to three phase partitioning (TPP): structures of native and TPP-treated lipase from Thermomyces lanuginosa

“…TPP treatment has, in many cases, resulted in improvement of the catalytic rates of the various enzymes by the marginal to moderate degrees [23,24,[28][29][30][31]. As indicated in the introduction, TLL subjected to TPP under optimized conditions showed an increase in the initial rates of hydrolytic activity from 8.6 to 17.8 U/mg protein ( Table 1).…”

“…The complexity of the mechanism by which it works was discussed adequately by Lovrein et al [27], and more recently in the context of protein refolding [26]. There have been quite a few reports on the effect of TPP treatment on the structure and function of many enzymes and proteins [21][22][23][24][25][26][27][28][29][30][31]. In few cases, it has been clearly shown that the specific activity of the pure enzyme increased after being subjected to TPP [24,25,29].…”

“…However, unlike ammonium sulphate precipitation, this TPP often (though not always) results in subtle structural changes. This makes TPP valuable for wide range of applications such as protein purification, protein refolding and improving the catalytic efficiencies of the enzymes in both aqueous and non aqueous media [21][22][23][24][25][26][27][28][29][30][31]. The technique was introduced by Lovrein's group [21] who emphasized its importance in protein isolation and concentration [22].…”

“…The effect of TPP treatment on alpha chymotrypsin was even more dramatic. The enzyme subjected to TPP showed significantly high activity in both aqueous and non-aqueous media [23]. When attempts were made to crystallize the alpha chymotrypsin subjected to TPP, selective autolysis generated a 14 amino acid peptide which was found to bind to the active site of the enzyme [32].…”

“…TPP consists of precipitating a protein at the interface of two layers formed by the addition of tert-butanol and ammonium sulphate to a protein solution [21][22][23][24][25][26] (Fig. 1).…”