Kinetics of enzyme attack on substrates covalently attached to solid surfaces: influence of spacer chain length, immobilized substrate surface concentration and surface charge.
The use of alpha-chymotrypsin to cleave covalently bound N-acetyl- l-tryptophan (Ac-Trp-OH) from the surfaces of aminopropylated controlled pore glass (CPG) and the polymer PEGA 1,900 was investigated. Oligoglycine spacer chains were used to present the covalently attached Ac-Trp-OH substrate to the aqueous enzyme. In the absence of the oligoglycine spacer chain, the rate of release was relatively slow, especially from the PEGA 1,900. These slow rates reflect the position of the amino group to which Ac-Trp-OH is covalently attached. On the glass there was a clear optimum with a chain of four glycine residues. For PEGA 1,900 there is no real apparent change beyond two glycine residues. The decline in rate beyond these optima are a possible result of changes in oligoglycine structure. Comparing different surface loadings of bound substrate the rate of release of Ac-Trp-OH from CPG with a pore diameter of 1,200 A was optimal when using 83% of the maximum that can be coupled, then fell again at higher loading. The rate of Ac-Trp-OH release from CPG was the same for surface coverages of 0.4 and 1.0. The introduction of permanent surface charges on CPG 1,200 exhibits a distinct influence on enzymatic cleavage with an increase in the rate of biocatalysis at the surface. Optimal presentation of covalently immobilized substrate on different supports by use of appropriate linkers leads to favorable biocatalysis from the support.
Deere
Joseph
J
Department of Pure and Applied Chemistry, Thomas Graham Building, 295 Cathedral Street, University of Strathclyde, Glasgow, G1 1XL, U.K. joseph.deere@strath.ac.uk
De Oliveira
Rui F
RF
Tomaszewski
Bartłomiej
B
Millar
Sarah
S
Lalaouni
Antonia
A
Solares
Laura F
LF
Flitsch
Sabine L
SL
Halling
Peter J
PJ
D000818:Animals; D002384:Catalysis; D002417:Cattle; D002627:Chemistry, Physical / Q000379:methods; D002918:Chymotrypsin / Q000737:chemistry; D005898:Glass; D005998:Glycine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D008970:Molecular Weight; D011092:Polyethylene Glycols / Q000737:chemistry*; D011108:Polymers / Q000737:chemistry; D013499:Surface Properties; D013997:Time Factors
D011108:Polymers; C109511:bis(polyoxyethylene bis(amine)); D011092:Polyethylene Glycols; D002918:Chymotrypsin; D005998:Glycine