Heterologous expression and characterization of exo-type alginate lyase from anaerobic bacterium Sunxiuqinia sp. SH-52

Abstract:

[Background] Most alginolytic bacteria currently reported are aerobic bacteria and studies on anaerobic ones have not been reported yet. Characterization of alginate lyases isolated from alginolytic bacteria are mainly focused on the endo-type alginate lyases and rarely on exo-type ones. [Objective] To investigate the genes encoding alginate lyases isolated from anaerobic alginolytic bacteria, to characterize the novel alginate lyase, and to elucidate its enzymatic properties, providing a theoretical basis for the diversity of alginate lyases and the mechanism of microbial degradation of alginate. [Methods] The gene encoding SHA-4, an alginate lyase isolated from an anaerobic alginolytic bacterium Sunxiuqinia sp. SH-52, was cloned and sequenced. Recombinant plasmid PGEX-4T-1-SHA-4 was constructed and heterologously expressed in E. coli. The expressed enzyme was purified and the enzymatic and degradation characteristics were analyzed. [Results] Maximum expression was achieved when induced by 0.1 mmol/L IPTG (Isopropyl-β-D-Thiogalactoside) at 28 °C for 6 hours and the specific activity of the purified enzyme was 21 U/mg. Characterization of the enzyme showed that the optimal condition for SHA-4 includes the temperature at 37 °C, pH at 7.5, and PolyMG (heteropolymeric MG blocks) as a preferred substrate. The activity of this enzyme was inhibited by Na+ and significantly elevated to about 168% by Cu2+. Km and Vmax of SHA-4 when used to catalyze alginate were 2.5 mg/mL and 8.7 mg/(mL·min), respectively. SHA-4 was an exo-type alginate lyase with monosaccharides as the final degradation products. [Conclusion] An alginate lyase SHA-4 isolated from an anaerobic alginolytic bacterium Sunxiuqinia sp. SH-52 is successfully expressed. Among other PL6 family members, SHA-4 is the first exo-type alginate lyase with PolyMG as a preferred substrate. Along with its relatively high enzymatic activity, SHA-4 may serve as a tool enzyme with promising application and provide new clues for further exploration of the mechanism of alginate degradation.