Investigation of the Enzymatic Cleavage of Diastereomeric Oligo(3-hydroxybutanoates) Containing Two to Eight HB Units. A Model for the Stereoselectivity of PHB Depolymerase from Alcaligenes faecalis T1

Abstract

So far, it is known that various poly(3-hydroxybutanoate), PHB, depolymerases are able to degrade all-(R) chains, cyclic (R) oligomers, oligolides, and polymers composed of rac-hydroxybutanoate, HB, but not all-(S) or syndiotactic (R,S) chains. We have now studied the ability for configurational recognition (stereoselectivity) by the purified PHB depolymerase from Alcaligenes faecalis T1. To this end, a titristat/HPLC method has been developed for following the degradation of oligo(3-hydroxybutanoates), OHBs, providing baseline separation for OHB methyl esters up to at least the octamers (Figure 1). With this method, we have now investigated the degradation rates and cleavage patterns of OHBs (1−16) containing up to eight HB units with given sequences of (R) and (S) configurations along the chains (Figures 2−5 and 7). Analysis of the measurements now allows us to propose more detailed structural features at the binding site of the depolymerase (Figures 6, 8, 9): (i) the enzyme is an endo esterase; (ii) it recognizes the orientation of the chain relative to its active site; (iii) the binding site contains four subsites, three of which have to be occupied by HB units for cleavage to occur at all (rate vmaxβ) and all four for cleavage to take place at the maximum rate (vmaxα); and (iv) the central two subsites, between which cleavage occurs, must be occupied by (R) HB units, whereas the terminal subunits may also be occupied by (S) HB units.