Biochemical and molecular characterization of poly(aspartic acid) hydrolase-2 from sphingomonas sp. KT-1

Abstract

Poly(aspartic acid) (PAA) hydrolase-2 was purified from crude soluble cellular extracts of Sphingomonas sp. KT-1 (JCM10459) and characterized to elucidate the mechanism of alpha,beta-poly(d,l-aspartic acid) (tPAA) biodegradation. The molecular mass of PAA hydrolase-2 was 42 kDa, and the isoelectric point was 9.6. The optimum values of pH and temperature for the hydrolysis of alpha-di(l-aspartic acid) by PAA hydrolase-2 were 7.0 and 55 degrees C, respectively. The effect of inhibitors on the hydrolysis of alpha-di(l-aspartic acid) showed that the activity of PAA hydrolase-2 was significantly inhibited by EDTA. Thermally synthesized tPAA was hydrolyzed in the presence of two enzymes, PAA hydrolase-1 and PAA hydrolase-2, to generate aspartic acid. The PAA hydrolase-2 was capable of hydrolyzing alpha-poly(l-aspartic acid) of high molecular weights but had limited activity for tPAA. These results lead us to propose the following mechanism. First, PAA hydrolase-1 hydrolyzes tPAA to yield oligo(aspartic acid) via an endo-mode cleavage, and subsequently, PAA hydrolase-2 hydrolyzes the resultant oligo(aspartic acid) to yield aspartic acid. Analysis of hydrolyzed products from alpha- and beta-penta(l-aspartic acid) revealed that PAA hydrolase-2 catalyzed the exo-mode hydrolysis of alpha- and beta-penta (l-aspartic acid). The gene encoding PAA hydrolase-2 from Sphingomonas sp. KT-1 was cloned, and genetic analysis showed that the deduced amino acid sequence of PAA hydrolase-2 is similar to a putative peptidase, which belongs to the M20/M25/M40 family of proteins, from Caulobacter crescentus CB15.