Sung Ok Han

Biodiesel production by a mixture of Candida rugosa and Rhizopus oryzae lipases using a supercritical carbon dioxide process

In this study, various factors, such as temperature, pressure, agitation speed, water content, and the concentration and ratio of immobilized ROL and CRL were investigated for the efficient enzymatic production of biodiesel using a supercritical carbon dioxide process. Furthermore, a stepwise reaction method for the maintenance of immobilized lipase activity was optimized. Optimal conditions for biodiesel production were determined to be as follows: 130 bar pressure, 45 °C temperature, 250 rpm agitation speed, 10% water content, and 20% immobilized ROL and CRL (1:1). When batch process was performed under optimal conditions, the biodiesel conversion yield was 99.13% at 3 h. Biodiesel conversion yield was 99.99% at 2 h when 90 mmol methanol was used in a stepwise reaction. Moreover, the conversion yield of biodiesel produced by the repeated recycling of immobilized lipase in the stepwise reactions was 85% after 20 reuses.

Synthesis of FAEEs from glycerol in engineered Saccharomyces cerevisiae using endogenously produced ethanol by heterologous expression of an unspecific bacterial acyltransferase.

The high price of petroleum‐based diesel fuel has led to the development of alternative fuels, such as ethanol. Saccharomyces cerevisiae was metabolically engineered to utilize glycerol as a substrate for ethanol production. For the synthesis of fatty acid ethyl esters (FAEEs) by engineered S. cerevisiae that utilize glycerol as substrate, heterologous expression of an unspecific acyltransferase from Acinetobacter baylyi with glycerol utilizing genes was established. As a result, the engineered YPH499 (pGcyaDak, pGupWs‐DgaTCas) strain produced 0.24 g/L FAEEs using endogenous ethanol produced from glycerol. And this study also demonstrated the possibility of increasing FAEE production by enhancing ethanol production by minimizing the synthesis of glycerol. The overall FAEE production in strain YPH499 fps1Δ gpd2Δ (pGcyaDak, pGupWs‐DgaTCas) was 2.1‐fold more than in YPH499 (pGcyaDak, pGupWs‐DgaTCas), with approximately 0.52 g/L FAEEs produced, while nearly 17 g/L of glycerol was consumed. These results clearly indicated that FAEEs were synthesized in engineered S. cerevisiae by esterifying exogenous fatty acids with endogenously produced ethanol from glycerol. This microbial system acts as a platform in applying metabolic engineering that allows the production of FAEEs from cheap and abundant substrates specifically glycerol through the use of endogenous bioethanol. Biotechnol. Bioeng. 2012;109: 110–115.

Enzymatic coproduction of biodiesel and glycerol carbonate from soybean oil and dimethyl carbonate.

The enzymatic coproduction of biodiesel and glycerol carbonate by the transesterification of soybean oil was studied using lipase as catalyst in organic solvent. To produce biodiesel and glycerol carbonate simultaneously, experiments were designed sequentially. Enzyme screening, the molar ratio of dimethyl carbonate (DMC) to soybean oil, reaction temperature and solvent effects were investigated. The results of enzyme screening, at 100 g/L Novozym 435 (immobilized Candida antarctica lipase B), biodiesel and glycerol carbonate showed conversions of 58.7% and 50.7%, respectively. The optimal conditions were 60 °C, 100 g/L Novozym 435, 6.0:1 molar ratio with tert-butanol as solvent: 84.9% biodiesel and 92.0% glycerol carbonate production was achieved.

Engineering of glycerol utilization pathway for ethanol production by Saccharomyces cerevisiae

Saccharomyces cerevisiae was metabolically engineered to improve ethanol production from glycerol. High rates of glycerol utilization were achieved by simultaneous overexpression of glycerol dehydrogenase (Gcy) and dihydroxyacetone kinase (Dak), which are the enzymes responsible for the conversion of glycerol to glycolytic intermediate dihydroxyacetone phosphate. As a result, ethanol production in YPH499 (pGcyaDak) was about 2.4-fold higher than wild strain. We have also successfully expressed a glycerol uptake protein (Gup1). The overall ethanol production in strain YPH499 (pGcyaDak, pGupCas) was 3.4-fold more than in wild strain, with about 2.4gL(-1) ethanol produced. These experimental results confirmed our metabolic pathway strategies which improve the production of ethanol.

Production of minicellulosomes for the enhanced hydrolysis of cellulosic substrates by recombinant Corynebacterium glutamicum

Although cellulosic materials of plant origin are the most abundant utilizable biomass resource, the amino acid-producing organism Corynebacterium glutamicum can not utilize these materials. Here we report the engineering of a C. glutamicum strain expressing functional minicellulosomes containing chimeric endoglucanase E bound to miniCbpA from Clostridium cellulovorans that can hydrolyze cellulosic materials. The chimeric endoglucanase E consists of the endoglucanase E catalytic backbone of Clostridium thermocellum fused with the endoglucanase B dockerin domain of C. cellulovorans. The resulting strain degraded cellulose efficiently by substrate targeting via the carbohydrate binding module. The assembly of minicellulosomes increased the activity against carboxymethyl cellulose approximately 2.8-fold compared with that for the corresponding enzymes alone. This is the first report of the formation of Clostridium minicellulosomes by C. glutamicum. The development of C. glutamicum strain that is capable of more effective cellulose hydrolysis brings about a realization of consolidated bioprocessing for the utilization of cellulosic biomass.

Cellulosic alcoholic fermentation using recombinant Saccharomyces cerevisiae engineered for the production of Clostridium cellulovorans endoglucanase and Saccharomycopsis fibuligeraβ‐glucosidase

In this study, Saccharomyces cerevisiae was engineered for simultaneous saccharification and fermentation of cellulose by the overexpression of the endoglucanase D (EngD) from Clostridium cellulovorans and the beta-glucosidase (Bgl1) from Saccharomycopsis fibuligera. To promote secretion of the two enzymes, the genes were fused to the secretion signal of the S. cerevisiaealpha mating factor gene. The recombinant developed yeast could produce ethanol through simultaneous production of sufficient extracellular endoglucanase and beta-glucosidase. When direct ethanol fermentation from 20 g L(-1)beta-glucan as a substrate was performed with our recombinant strains, the ethanol concentration reached 9.15 g L(-1) after 50 h of fermentation. The conversion ratio of ethanol from beta-glucan was 80.3% of the theoretical ethanol concentration produced from 20 g L(-1)beta-glucan. In conclusion, we have demonstrated the construction of a yeast strain capable of conversion of a cellulosic substrate to ethanol, representing significant progress towards the realization of processing of cellulosic biomass in a consolidated bioprocessing configuration.

Production of cellulosic ethanol in Saccharomyces cerevisiae heterologous expressing Clostridium thermocellum endoglucanase and Saccharomycopsis fibuligera β-glucosidase genes

Heterologous secretory expression of endoglucanase E (Clostridium thermocellum) and β-glucosidase 1 (Saccharomycopsis fibuligera) was achieved in Saccharomyces cerevisiae fermentation cultures as an α-mating factor signal peptide fusion, based on the native enzyme coding sequence. Ethanol production depends on simultaneous saccharification of cellulose to glucose and fermentation of glucose to ethanol by a recombinant yeast strain as a microbial biocatalyst. Recombinant yeast strain expressing endoglucanase and β-glucosidase was able to produce ethanol from β-glucan, CMC and acid swollen cellulose. This indicates that the resultant yeast strain of this study acts efficiently as a whole cell biocatalyst.

Cellulosome-based, Clostridium-derived multi-functional enzyme complexes for advanced biotechnology tool development: Advances and applications

The cellulosome is one of natures most elegant and elaborate nanomachines and a key biological and biotechnological macromolecule that can be used as a multi-functional protein complex tool. Each protein module in the cellulosome system is potentially useful in an advanced biotechnology application. The high-affinity interactions between the cohesin and dockerin domains can be used in protein-based biosensors to improve both sensitivity and selectivity. The scaffolding protein includes a carbohydrate-binding module (CBM) that attaches strongly to cellulose substrates and facilitates the purification of proteins fused with the dockerin module through a one-step CBM purification method. Although the surface layer homology (SLH) domain of CbpA is not present in other strains, replacement of the cell surface anchoring domain allows a foreign protein to be displayed on the surface of other strains. The development of a hydrolysis enzyme complex is a useful strategy for consolidated bioprocessing (CBP), enabling microorganisms with biomass hydrolysis activity. Thus, the development of various configurations of multi-functional protein complexes for use as tools in whole-cell biocatalyst systems has drawn considerable attention as an attractive strategy for bioprocess applications. This review provides a detailed summary of the current achievements in Clostridium-derived multi-functional complex development and the impact of these complexes in various areas of biotechnology.

Enzymatic production of glycerol carbonate from by-product after biodiesel manufacturing process.

Glycerol carbonate is one of the higher value-added products derived from glycerol. In this study, glycerol carbonate (GC) was synthesized by transesterification of glycerol and dimethyl carbonate (DMC) using Novozym 435 (Candida antarctica Lipase B) at various conditions. For the enzymatic production of GC, the optimum conditions were the amount of enzyme (75 g/L), DMC/glycerol molar ratio (2.00), reaction temperature (60°C) and organic solvent (acetonitrile). Experimental investigation of the effect of water content revealed that the conversion of GC was maximized with no added water. The addition of surfactant such as Tween 80 increased the GC conversion, which finally reached 96.25% under the optimum condition and with surfactant addition.

Antiplatelet activities of newly synthesized derivatives of piperlongumine

Piperlongumine, a pyridone alkaloid isolated from Piper longum L., exhibited a potential inhibitory effect on washed rabbit platelet aggregation induced by collagen, arachidonic acid (AA) and platelet activating factor (PAF), without any inhibitory effect on that induced by thrombin. Piperlongumine was used as a lead compound for the synthesis of new antiplatelet agents. Seven synthetic compounds were newly synthesized from 3,4,5‐trimethoxycinnamic acid (TMCA). They were 1‐piperidin‐1‐yl‐3‐(3,4,5‐trimethoxy‐phenyl)prop‐2‐en‐1‐one (1′), 1‐morpholin‐4‐yl‐3‐(3,4,5‐trimethoxyphenyl)prop‐2‐en‐1‐one (2′), 1‐(3,5‐dimethylpiperidin‐1‐yl)‐3‐(3,4,5‐trimethoxyphenyl)prop‐2‐en‐1‐one (3′), 1‐(2‐methylpiperidin‐1‐yl)‐3‐(3,4,5‐tri‐methoxyphenyl)prop‐2‐en‐1‐one (4′), 1‐(3‐hydroxypiperidin‐1‐yl)‐3‐(3,4,5‐trimethoxyphenyl)‐ prop‐2‐en‐1‐one (5′), 1‐[3‐(3,4,5‐tri‐methoxyphenyl) acryloyl]‐piperidin‐2‐one (6′) and ethyl 1‐[3‐(3,4,5‐trimethoxyphenyl)‐acryloyl]piperidine‐4‐carboxylate (7′). Among those seven synthetic derivatives, 1‐(3,5‐dimethylpiperidin‐1‐yl)‐3‐(3,4,5‐trimethoxyphenyl)prop‐2‐en‐1‐one (3′) had the most inhibitory effect on platelet aggregation induced by collagen, AA and PAF. Copyright