Production and Bioconversion of 3,6-Anhydro-D-galactose Derived from Red Macroalgae

Abstract

Red macroalgae are possible renewable sources for bio-product production because of their high polysaccharide content and small number of sugar types. The main components of red macroalgae are galactans (agaroses, porphyrans, and carrageenans), which are comprised of D-galactose (D-Gal) and modified D-Gal or L-galactose (L-Gal) units. Among red macroalgae, Kappaphycus is one of the most abundant macroalgae produced by aquaculture. This macroalgae contains kappa-carrageenans, which are sulfated polysaccharides that consist of backbone of alternating D-galactose-4-sulfate (D-Gal4S) and 3,6-anhydro-Dgalactose (D-AnG) with a similar proportion of D-Gal and D-AnG. D-Gal is a fermentable sugar that can be used by bioethanol-producing strains; but the utilization of red macroalgae derived D-AnG has not yet been studied. This study investigates the production and bioconversion of D-AnG. First, we described a new enzymatic method to quantify D-AnG and D-Gal, which are produced during acid-catalyzed hydrolysis of κ-carrageenan. The D-AnG and D-Gal contents in acid hydrolysates could be precisely determined by using D-AnG dehydrogenase (D-AnGDH) and D-Gal dehydrogenase (D-GalDH), respectively. Both D-AnGDH and D-GalDH use NAD+ as a cofactor, so the activity of both enzymes could be determined spectrophotometically by measuring an increase in the concentration of NADH. D-AnG content determined by enzymatic method was much lower than that determined by conventional colorimetric methods, such as the resorcinol method and the thymol method; the disparity occurs because the enzymatic method measures the amount of monomeric D-AnG, whereas the chemical method measures the total amount of D-AnG in the hydrolysates. We also found that the enzymatic determination of D-Gal using D-GalDH is not influenced by the presence of L-Gal, which is a minor sugar component of carrageenan. The enzymatic method presented in this study quantifies only fermentable or bioconvertable D-AnG and D-Gal molecules, and therefore would be useful when the acid hydrolysates are used as the substrate of fermentation or bioconversion processes. Second, we described acid-catalyzed production of D-AnG from κ-carrageenan. We analyzed four hydrolysis products (D-AnG, 5-hydroxymethyl-furfural (HMF), levulinic acid (LA), and D-Gal) and reducing sugar contents during acid hydrolysis. Acid screening was conducted using seven acid catalysts that have distinct acidities. Catalysts that showed high D-AnG production and high selectivity were chosen for subsequent experiments. We selected four acid catalysts (HCOOH, CF3COOH, HNO3, and HCl), and studied the effects of catalyst acidity, hydrolysis temperature T, and reaction time t on the production of D-AnG and other hydrolysis products. The optimal condition for maximum production of D-AnG by κ-carrageenan hydrolysis was T = 100 °C and t = 30 min using 0.2 M HCl. Under this condition, 2.81 g/L D-AnG (33.5% of theoretical maximum) could be obtained from 2% (w/v) κ-carrageenan. In general, the maximum values of D-AnG, D-Gal, and the sum of two by-products (HMF and LA) increased with the acidity of catalysts. However, HNO3 was an exception in that the maximum production levels of HMF and LA were unusually low compared with other acid catalysts. Silica gel chromatography successfully purified D-AnG from acid hydrolysates; the product was nearly 100% pure. This effective D-AnG production could facilitate studies on the conversion of D-AnG to biofuels and biochemicals. Third, we investigated the metabolic pathway of D-AnG degradation and the novel conversion method of D-AnG. In bioinformatic analysis, we found four genes that were believed to be involved in D-AnG metabolism. We then experimentally confirmed the enzymatic function of each gene product. D-AnG metabolizing genes were clustered and organized in operon-like arrangements, which we named the dan operon (3,6-d-anhydro-galactose). Combining bioinformatic analysis and experimental data, we showed that D-AnG is metabolized to pyruvate and D-glyceraldehyde-3-phosphate via four enzyme-catalyzed reactions: 3,6-anhydro-D-galactose -> 3,6-anhydro-D-galactonic acid -> 2-keto-3-deoxy-D-galactonate (D-KDGal) -> 2-keto-3-deoxy-6-phospho-D-galactonate -> pyruvate + D-glyceraldehyde-3-phosphate. The first two enzyme reactions are specific to the D-AnG pathway; the other two are identical to those in the DeLey–Doudoroff pathway. Finally, we suggested a novel conversion method of D-AnG by combining chemical and enzymatic reactions. In alkaline conditions, D-AnG was converted to 3-deoxygalactonic acid (3-dGA). Then we identified a novel enzyme 3-dGA dehydrogenase, that can convert 3-dGA to D-KDGal, which enters to the DeLey-Doudoroff pathway in E. coli. The end products of the DeLey-Doudoroff pathway are pyruvate and D-glyceraldehyde-3-phosphate; they can be readily converted to bioethanol in ethanologenic microorganisms such as E. coli KO11 and Saccharomyces cerevisiae. This disclosure of the metabolic pathway of D-AnG, a compound previously regarded as a useless, non-fermentable sugar, may enable construction of recombinant microorganisms that can produce bioethanol from D-AnG.