The marine bacteria V. splendidus 12B01, V. splendidus 13B01, and V. breoganii1C10 metabolize brown seaweeds. Brown seaweeds have many advantages overterrestrial feedstocks, including fast growth and non-recalcitrant carbon feedstocks, sobrown seaweeds are an attractive target for industrial fermentation. Alginate andlaminarin are two major components of brown seaweeds, comprising up to 60% of thedry weight. Alginate is a polysaccharide consisting of the 1,4-linked epimers α- L -guluronate and β- D -mannuronate. Laminarin is a storage glucan consisting of β-1,3- andβ-1,6-linked glucose monomers. In order to utilize these carbon sources, manyorganisms express enzymes that cleave the bonds linking the constituent monomerswithin alginate and laminarin. These enzymes are called alginate lyases andlaminarinases.V. splendidus 12B01, V. splendidus 13B01, and V. breoganii 1C10 each containbetween four and twelve putative alginate lyases. We have over-expressed and purified21 alginate lyases from these organisms and determined under what conditions theseenzymes are most active. We found these enzymes are optimally active between pH 6.5and 10 and between 20 to 30 °C. Additionally, these enzymes were broadly salt tolerantbetween 50 mM and 1 M NaCl. We also determined the enzyme kinetics for theseenzymes and found K m parameters towards alginate between 22 and 300 µM alginate.The computed turnover numbers range from 0.6 to 18 s -1 . Alginate lyases havepreferential specificity toward specific dyads within alginate. We found alginate lyaseswith all potential dyad specificities: G-G, G-M, M-G, and M-M specific alginate lyases.Having characterized the alginate lyases in 12B01, 13B01, and 1C10, we canbegin to understand the metabolism of alginate by these organisms. 12B01 was found topoorly degraded and metabolize alginate, and we found 12B01 to express and secrete itsenzymes at low levels. In addition we found the 12B01 alginate lyases have lowenzymatic activity and narrow dyad specificity. 13B01 was found to degrade andmetabolize alginate at high levels. We identified the presence of a unique enzyme to13B01, which upon knockout, resulted in eight-fold less secreted alginate lyase activity.We found that this high activity enzyme allows 13B01 to degrade alginate efficiently andiiithen metabolize the liberated monomers of alginate. 1C10 contains eleven alginatelyases within its genome. While this organism has 70% of the 13B01 secreted alginatelyase activity, we found that the 1C10 lyases do not have large enzymatic activity.Rather, the concerted action of enzymes with broad dyad specificity allow 1C10 toefficiently degrade alginate. Overall, we identified several attractive alginate lyases forfuture metabolic engineering to produce biofuels from alginate. While this would requireexpression of additional metabolic pathways, we present the first step to the industrialutilization of alginate.V. breoganii 1C10 contains four laminarinases which we over-expressed andpurified. These enzymes had optimal enzymatic activity between pH 6.5 and 8.0 andbetween 25 and 40 °C. These enzymes were shown to have especially broad tolerance tosalt between 50 mM and 1 M NaCl. The 1C10 laminarinases had K m parameters towardslaminarin between 3.4 and 6 mM laminarin. These enzymes also had computed turnovernumbers ranging from 0.69 to 6.1 s -1 . As the degraded monomer of laminarin is glucose,these enzymes can be expressed in fermentative hosts with no additional metabolicpathways, so laminarin utilization is an attractive target for biofuel production.
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Alginate and laminarin degrading enzymes from Vibrio splendidus and Vibrio breoganii
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