Identification and Characterization of the Biosynthetic Pathway of the Sulfonolipid Capnine.

Capnine (2-amino-3-hydroxy-15-methylhexadecane-1-sulfonate) and capnoids (N-fatty acylated capnine derivatives) are sulfonolipids present in the outer membrane of gliding bacteria in the phylum Bacteroidetes and play a role in their unique gliding motility. They are structurally similar to sphingolipids and are thought to be biosynthesized via a similar pathway. Here we report the identification and biochemical characterization of the capnine biosynthetic enzymes cysteate synthase (CapA) and cysteate-C-fatty acyltransferase (CapB) from the pathogenic gliding bacterium Capnocytophaga ochracea and NAD(P)H-dependent dehydrocapnine reductase CapC from the avian pathogen Ornithobacterium rhinotracheale. CapA catalyzes the formation of cysteate from O-phospho-l-serine and sulfite, and CapB catalyzes the formation of dehydrocapnine from cysteate and 13-methyl-myristoyl-CoA, followed by reduction by CapC. CapA is closely related to cystathionine-ß-synthase but distantly related to the archaeal cysteate synthase. Close homologues of CapA, CapB, and the CapA isozyme archaeal cysteate synthase are present in many Bacteroidetes bacteria, including environmental, pathogenic, and human oral and intestinal microbiome bacteria, suggesting the widespread ability of these bacteria to biosynthesize capnine and related sulfonolipids.

Expression of Aeromonas punctata ME-1 exo-xylanase X in E. coli for efficient hydrolysis of xylan to xylose.

exo-Xylanase X from Aeromonas punctata ME-1 was functionally expressed in Escherichia coli with a carboxy terminal His tag (6×) and a molecular mass of 39.42 kDa, which is in agreement with the prediction from its amino acid composition. The recombinant exo-xylanase reached 186 mg l(-1) after induction by isopropyl ß-D-1-thiogalactopyranoside. Its optimal temperature and pH were 50 °C and 6, respectively. The enzyme showed not only an exo-xylanase activity with K m of 3.90 mg ml(-1) and V max of 12.9 U µg(-1) for hydrolysis of Remazol Brilliant Blue-xylan but also a considerable exo-glucanase activity (27.9 U mg(-1)) on P-nitrophenyl ß-D-cellobioside. It hydrolyzed xylan predominantly to xylobiose, xylotriose, xylotetraose, and xylose. An enzyme mixture of exo-xylanase and endo-xylanase (50 µg ml(-1) each) yielded a larger amount (330 mg l(-1)) of xylose from beechwood xylan than the controls (270 and 150 mg l(-1)) using them alone at 100 µg ml(-1), indicating a synergistic action between the two xylanases favoring the hydrolysis of beechwood xylan to release more xylose.