Hee Taek Kim

The complete enzymatic saccharification of agarose and its application to simultaneous saccharification and fermentation of agarose for ethanol production.

A sugar platform equipped with acetic acid, multiple agarases and neoagarobiose hydrolase (NABH) converted recalcitrant agar polysaccharide into monosugars, which was evaluated by simultaneous saccharification and fermentation (SSF). The sugar platform was divided into chemical liquefaction and enzymatic saccharification. The chemical liquefaction was carried out in mild conditions (using a dilute acetic acid at 80°C for 1-6h) to avoid the production of fermentation inhibitors and hence the highest degree of liquefaction of 95.6% (w/w) was obtained. We mimicked the natural agarolytic pathway using three microbial agarases (Aga16B, Aga50D and DagA) and NABH, and the enzyme system converted 79.1% of agarose to monosugars. The chemical liquefaction and SSF of 30 g/l agarose resulted in 4.4 g/l ethanol concentration and 49.3% of the theoretical ethanol yield to d-galactose. This is the first report on the complete enzymatic conversion of agarose into its monosugars and the SSF of agarose into ethanol.

Crystal structure of a key enzyme in the agarolytic pathway,α-neoagarobiose hydrolase from Saccharophagus degradans 2–40

In agarolytic microorganisms, α-neoagarobiose hydrolase (NABH) is an essential enzyme to metabolize agar because it converts α-neoagarobiose (O-3,6-anhydro-alpha-l-galactopyranosyl-(1,3)-d-galactose) into fermentable monosaccharides (d-galactose and 3,6-anhydro-l-galactose) in the agarolytic pathway. NABH can be divided into two biological classes by its cellular location. Here, we describe a structure and function of cytosolic NABH from Saccharophagus degradans 2-40 in a native protein and d-galactose complex determined at 2.0 and 1.55 Å, respectively. The overall fold is organized in an N-terminal helical extension and a C-terminal five-bladed β-propeller catalytic domain. The structure of the enzyme-ligand (d-galactose) complex predicts a +1 subsite in the substrate binding pocket. The structural features may provide insights for the evolution and classification of NABH in agarolytic pathways.

The novel catabolic pathway of 3,6‐anhydro‐L‐galactose, the main component of red macroalgae, in a marine bacterium

The catabolic fate of the major monomeric sugar of red macroalgae, 3,6-anhydro-L-galactose (AHG), is completely unknown in any organisms. AHG is not catabolized by ordinary fermentative microorganisms, and it hampers the utilization of red macroalgae as renewable biomass for biofuel and chemical production. In this study, metabolite and transcriptomic analyses of Vibrio sp., a marine bacterium capable of catabolizing AHG as a sole carbon source, revealed two key metabolic intermediates of AHG, 3,6-anhydrogalactonate (AHGA) and 2-keto-3-deoxy-galactonate; the corresponding genes were verified in vitro enzymatic reactions using their recombinant proteins. Oxidation by an NADP(+) -dependent AHG dehydrogenase and isomerization by an AHGA cycloisomerase are the two key AHG metabolic processes. This newly discovered metabolic route was verified in vivo by demonstrating the growth of Escherichia coli harbouring the genes of these two enzymes on AHG as a sole carbon source. Also, the introduction of only these two enzymes into an ethanologenic E. coli strain increased the ethanol production in E. coli by fermenting both AHG and galactose in an agarose hydrolysate. These findings provide not only insights for the evolutionary adaptation of a central metabolic pathway to utilize uncommon substrates in microbes, but also a metabolic design principle for bioconversion of red macroalgal biomass into biofuels or industrial chemicals.

Pretreatment and saccharification of red macroalgae to produce fermentable sugars

Red macroalgae are currently considered as renewable resources owing to their high carbohydrate and low lignin and hemicellulose contents. However, utilization of red macroalgae has been limited owing to the lack of established methods for pretreatment and an effective saccharification system. Furthermore, marine red macroalgae consist of the non-favorable mixed sugars for industrial microorganisms. In this review, we suggest strategies for converting red macroalgae to bio-based products, focusing on the pretreatment and saccharification of red macroalgae to produce fermentable sugars and the microbial fermentation of these sugars by industrial microorganisms. In particular, some recent breakthroughs for the efficient utilization of red macroalgae include the discovery of key enzymes for the complete monomerization of red macroalgal carbohydrate and the catabolic pathway of 3,6-anhydro-l-galactose, the most abundant sugar in red macroalgae. This review provides a comprehensive perspective for the efficient utilization of red macroalgae as sustainable resources to produce bio-based products.

A novel agarolytic β-galactosidase acts on agarooligosaccharides for complete hydrolysis of agarose into monomers.

ABSTRACT Marine red macroalgae have emerged to be renewable biomass for the production of chemicals and biofuels, because carbohydrates that form the major component of red macroalgae can be hydrolyzed into fermentable sugars. The main carbohydrate in red algae is agarose, and it is composed of d-galactose and 3,6-anhydro-l-galactose (AHG), which are alternately bonded by β1-4 and α1-3 linkages. In this study, a novel β-galactosidase that can act on agarooligosaccharides (AOSs) to release galactose was discovered in a marine bacterium (Vibrio sp. strain EJY3); the enzyme is annotated as Vibrio sp. EJY3 agarolytic β-galactosidase (VejABG). Unlike the lacZ-encoded β-galactosidase from Escherichia coli, VejABG does not hydrolyze common substrates like lactose and can act only on the galactose moiety at the nonreducing end of AOS. The optimum pH and temperature of VejABG on an agarotriose substrate were 7 and 35°C, respectively. Its catalytic efficiency with agarotriose was also similar to that with agaropentaose or agaroheptaose. Since agarotriose lingers as the unreacted residual oligomer in the currently available saccharification system using β-agarases and acid prehydrolysis, the agarotriose-hydrolyzing capability of this novel β-galactosidase offers an enormous advantage in the saccharification of agarose or agar in red macroalgae for its use as a biomass feedstock for fermentable sugar production.

High temperature and low acid pretreatment and agarase treatment of agarose for the production of sugar and ethanol from red seaweed biomass

To obtain fermentable sugar from agarose, pretreatment of agarose by using acetic acid was conducted for short durations (10-30 min) at low acid concentrations (1-5% (w/v)) and high temperatures (110-130 °C). On testing the pretreated agarose by using an endo-β-agarase I (DagA), an exo-β-agarase II (Aga50D), and neoagarobiose hydrolase (NABH), we observed that the addition of the endo-type agarase did not increase the sugar yield. Use of the crude enzyme of Vibrio sp. EJY3 in combination with Aga50D and NABH including acetic acid pretreatment resulted in a 1.3-fold increase in the final reducing sugar yield (62.8% of theoretical maximum based on galactose and 3,6-anhydrogalactose in the initial agarose), compared to those obtained using Aga50D and NABH only after acetic acid pretreatment. The simultaneous saccharification and fermentation of pretreated agarose yielded ethanol of 37.1% theoretical maximum yield from galactose contained in the pretreated agarose.

Fatty acid profiling and proteomic analysis of Salmonella enterica serotype Typhimurium inactivated with supercritical carbon dioxide

Non-thermal sterilization and microbial inactivation processes are currently receiving much attention in food and pharmaceutical industries. In particular, since supercritical carbon dioxide (SC-CO2) treatment, which is conducted at relatively low temperatures, is considered to be a promising alternative method to replace thermal sterilization processes that cannot be safely used in foods and bioactive materials. Although SC-CO2 has been applied to many microorganisms, the inactivation of microbial cells by SC-CO2 has only been evaluated by using a conventional viable cell count such as a plating method, by which it is not possible to systematically elucidate the microbial cell inactivation process. Therefore, in this study the physiological status of SC-CO2 treated Salmonella enterica serotype Typhimurium was analyzed by using GC-MS analysis of fatty acids with principal component analysis and two-dimensional electrophoresis for protein profiling. From the results of these systemic analyses, it was revealed that SC-CO2 caused significant alterations to the profiles of fatty acids and proteins of the cells.

Flow cytometric analysis of Salmonella enterica serotype Typhimurium inactivated with supercritical carbon dioxide

Non-thermal processes for the effective sterilization and inactivation of microorganisms are currently receiving a great deal of attention in food, pharmaceutical and other relevant industries. Supercritical carbon dioxide (SC-CO(2)) treatment is an alternative method of microbial inactivation that can be safely used in foods and bioactive materials at relatively low temperatures. However, to date, the inactivation of microbial cells by treatment with SC-CO(2) has only been evaluated using a conventional plating method. Therefore, it is difficult to quantitatively determine the damage to cells other than counting colony forming units and also to find the possible inactivation mechanism by SC-CO(2) treatment. For the first time in the area of SC-CO(2) inactivation of microorganisms, we analyzed the physiological status of SC-CO(2) treated Salmonella enterica serotype Typhimurium using flow cytometry and then compared the flow cytometric data to the survival rate obtained by the plating method. The results of these systemic analyses revealed that SC-CO(2) caused damage to various aspects of the cells, including the efflux pump and membrane integrity.