Cloning, expression and improvement of catalytic activity of alginate lyase by site-directed mutation

Abstract

Alginate lyase mainly produces active alginate oligosaccharides (AOS) by degrading alginate via β-elimination process. In this study, the Pseudoalteromonas sp. Alg6B alginate lyase-encoding gene alg6B-7 from polysaccharide lyase (PL)-7 family was successfully cloned, sequenced, expressed in Escherichia coli. Based on rational design and amino acid sequence alignment of the alginate lyase from various sources, four positive mutants were obtained. The specific enzyme activities of four mutants I62A, A99K, V132S, and L157T were 38.84%, 42.85%, 75.8% and 51.83% higher than that of the wild enzyme, respectively. The K cat /K m values of the four mutants were both increased, and the catalytic efficiency of V132S was 1.92-fold higher than that of the wild enzyme, especially. The rational design that was employed in this study achieved the dramatic improvement of catalytic activity, which may provide the application potential in industrial production.

Characterization of degradation patterns and enzymatic properties of a novel alkali-resistant alginate lyase AlyRm1 from Rubrivirga marina

“…AlyRm1 also performed the highest enzymatic activity at pH 10.0, and was stable within pH 4.0–11.0, indicating that AlyRm1 shows broad pH stability as an alkali-resistant alginate lyase. Alkali treatment usually is used to pretreat alginate for improving the saccharification biomass in enzymatic reaction ( Lu et al, 2022 ). Thus, AlyRm1 with extensive pH performance and alkali resistance could be suitable for industrial application.…”