The active sites of cellulases are involved in chiral recognition: a comparison of cellobiohydrolase 1 and endoglucanase 1

Abstract

The cellulases ceHobiohydrolase 1 (CBH 1) and endoglucanase 1 (EG 1) from the fungus Trichoderma reesei are closely related with 40% sequence identity and very similar in structure. In CBH 1 the active site is enclosed by long loops and some antiparallel ~strands forming a 40 A long tunnel, whereas in EG 1 part of those loops are missing so that the enzyme has a more common active site groove. Both enzymes were immobilized on silica and these materials were used as chiral stationary phases for chromatographic separation of the enantiomers of two chiral drugs, propranolol and alprenolol. The CBH 1 phase showed much better resolution than did the EG 1 phase, suggesting that the tunnel structure of the protein may play an important role in the chiral separation. The chiral compounds were found to be competitive inhibitors of both enzymes when p-nitrophenyl lactoside (pNPL) was used as substrate. (S)-enantiomers showed stronger inhibitory effects and also longer retention time on the stationary phases than the (R)-enantiomers. The consistency between kinetic data and retention on the stationary phases clearly shows that the enzymatically active sites of CBH 1 and EG 1 are involved in chiral recognition.

A statistical experimental design to study factors affecting enantioseparation of propranolol by capillary electrophoresis with cellobiohydrolase (Cel7A) as chiral selector

“…Previous studies using LC with Cel7A as a chiral selector have shown that resolution, selectivity and retention times increase with increasing pH [1,2,6,[30][31][32][33][34][35][36] due to stronger electrostatic binding between the analyte and Cel7A. Similar pH dependence for the interaction between lipophilic amino alcohols and cellulases has been observed in CE [19].…”

Apparent and true enantioselectivity in enantioseparations

“…16 If alprenolol is less hydrophobic than propranolol, why is the apparent enantioselectivity of the former always larger than that of the latter? 16,24 Our study shows that this is explained by the chiral equilibrium constant of the first enantiomer (a 1,II ) and the equilibrium constant of the nonselective interactions (a I ) being both relatively small compared to the chiral equilibrium constant of the second enantiomer (a 2,II ) in the case of alprenolol, while this is not so for propranolol. In fact, the relative values of the three constants is such that both the apparent and the true enantioselectives of alprenolol are much larger than those of propranolol, although S-propranolol has a larger enantioselective binding constant than S-alprenolol, in agreement with its higher hydrophobicity.…”

Toward an aggregated understanding of enzymatic hydrolysis of cellulose: Noncomplexed cellulase systems

“…The active site of EG1 is a groove rather than a tunnel (Henriksson et al, 1996), allowing glucan chains to be cleaved randomly to two shorter chains resulting in a rapid decrease in DP (KlemanLeyer et al, 1992(KlemanLeyer et al, , 1994Srisodsuk et al, 1998;Whitaker, 1957;Selby, 1961;Wood and McCrae, 1978). Endoglucanase activity is most often measured based on the rate of change of the viscosity of a soluble cellulose derivative such as carboxymethylcellulose (CMC) (Miller et al, 1960;Wood and McCrae, 1972).…”