Multiple components of endo-polyguluronide lyase of Pseudomonas sp.

A lyophilized alginate lyase preparation obtained from dialyzed extract of sonicated Pseudomonas sp. cells was fractionated by gell filtration on a Sephadex G-150 column, and three alginate lyase [EC4.2.2.3] fractions, peaks I, II, and III, were obtained. They were remarkably thermolabile. The lyase fractions degraded two kinds of alginate fragments, a polygluuronide (SG), and a polyuronide consisting of both mannuronic and guluronic acid residues (SMG), as well as commerical alginate, but were virtually inactive toward polymannuronide fragment (SM). The modes of degradation of these substrates by the lyase fractions were endowise with different degrees of randomness. Attack by the peak I fraction was more random than those by peaks II and III. The main lysis products formed from SG and SMG by these layses were identified as mixtures of unsaturated tri- and monouronides. The unsaturated triuronide from SG was deltatugg and SMG yielded a mixture of deltaUGG and a poorly characterized unsaturated trimer, possibly deltaUMG. However, the patterns of monomer and trimer production by theselyase fractions changed in different ways during incubation.

Fine structure of SMG alginate fragment in the light of its degradation by alginate lyases of Pseudomonas sp.

An alginate fragment named SMG, consisting of mannuronic (M) and guluronic acid residues (G)(DP=25), was prepared from the partial acid hydrolysate of a commercial alginate. Two subfractions, SMG-ppt (DP=52) and SMG-sup (DP=18) were obtained from SMG by fractionation with MgC12 and CaC12. The M/G ratios of these alginate fragment were 1.4-1.9. Their lysis products by a pseudomonad alginate lyase [EC 4.2.2.3] preparation were fractionated by gel filtration, giving similar patterns. The major products in their digests were unsaturated monouronides (53-50%) and triuronides (30-35%). The former was identified as a delta4,5-hexuronic acid (deltaU) and the latter was identified as a mixture of delta4,5-hexuronosyl-(1 leads to 4)-beta-D-mannuronosyl-(1 leads to 4)-L-guluronic acid (deltaUMG) and delta4,5-hexuronosyl-(1 leads to 4)-alpha-L-guluronosyl-(1 leads to 4))L-guluronic acid (deltaUGG). The two unsaturated triuronides were present in roughly equal amounts. The presence of 4-O-alpha-L-guluronosyl-L-guluronic acid (GG) and 4-O-beta-D-mannuronosyl-L-guluronic acid (MG) or 4-O-beta-L-guluronosyl-D-mannuronic acid (GM) was also demonstrated inthe digest. Moreover, indirect evidence suggested nonreducing terminal deltaU residue and free deltaU in the digest to be derived more from M than G of the original SMG. Thus, it was concluded that more than one-third of uronic acid residues of SMG molecules may be composed of almost equal amounts of MG and GG sequences, most of which may be connected by M to form MMG and MGG sequences, respectively.