An exotype alginate lyase in Sphingomonas sp Al:: overexpression in Escherichia coli, purification, and characterization of alginate lyase IV (A1-IV)

Sphingomonas sp. Al (strain Al) cells contain three kinds of endotype alginate lyases [Al-I, Al-II, and Al-III], all of which are formed from a common precursor through posttranslational processing. In addition to these lyases, another type of lyase (Al-IV) that acts on oligoalginates exists in the bacterium. Al-IV was overexpressed in Escherichia coli cells through control of its gene under the T7 promoter. The expression level of the enzyme in E coli cells was 8.6 U/L-culture, which was about 270-fold higher than that in strain A I cells. The enzyme was purified to homogeneity through three steps with an activity yield of 10.9%. The optimal pH and temperature, thermal stability, and mode of action of the purified enzyme were similar to those of the native enzyme from strain Al cells. Al-IV exolytically degraded oligoalginates, which were produced from alginate through the reaction of Al-I, Al-II, or Al-III, into monosaccharides, indicating that the cooperative actions of these four enzymes cause the complete depolymerization of alginate in strain Al cells. (C) 2003 Elsevier Science (USA). All rights reserved.