Characterization and Engineering of a Novel Functional Algal Polysaccharide Degrading Enzymatic Machinery for Biofuels Production

Characterization and Engineering of a Novel Functional Algal Polysaccharide Degrading Enzymatic Machinery for Biofuels Production

Marine algae exhibit several key features of an ideal feedstock for bioconversion into biofuels. It is limited by the availability of tractable microorganisms that can metabolize alginate polysaccharides.  More efforts are required to harness the enzymatic machinery that bacteria use to convert marine algal polysaccharides into bioenergy substrate. Here, we present the characterization of three novel oligoalginate lyases (Oals) and four novel alginate lyases (Alys) from Vibrio splendidus that catalyze depolymerization of alginate into monomer substrates which were later converted to biofuels. The OalA protein was purified as an active soluble form using MBP-tag affinity chromatography. Protein refolding of OalB and OalC using the flash dilution method resulted in refolding of the protein into its native structure and regaining full biological activity. The PL-15 exotype OalA shows broad substrate specificity towards poly-M block, poly-G block, polyMG-block and sodium alginate. The turnover number was inversely proportional to degree of polymerization of substrates. The OalA showed highest activity towards PolyG substrate than previously reported recombinant exotype oligoalginates. The OalA utilized all alginate oligomers produced by Alys, while the OalB and OalC were specific to dimers and tetramers which were finally converted into monomer. The Alys belonging to family PL-5 and PL-7 endolytic AlyA, AlyB specifically depolymerize heteropolymer poly MG in alginate molecules into oligomers. AlyC, AlyD belongs to family PL-14, specific for poly α-L-guluronate (polyM) and poly β-D-mannuronate (poly G) respectively. The detection, quantification, and structure determination of the alginate products were carried out using TLC, HPLC, ESIMS or NMR. The Oals and Alys can be used as biocatalysts for saccharification of alginate since they can efficiently degrade alginate and alginate oligomers into alginate monosaccharides. The characterized algal polysaccharide degradation enzymatic machinery is being repackaged using the DNA assembler method to create recombinant yeast strains capable of producing biofuels and chemicals.