-Han Chung

Characterization and temporal expression of a ω-6 fatty acid desaturase cDNA from sesame (Sesamum indicum L.) seeds☆

Abstract A cDNA (SeFAD2) encoding a microsomal ω-6 desaturase was characterized in association with its temporal expression and changes in linoleic acid levels in sesame (Sesamum indicum L.) oilseeds. Much higher sequence identities of the SeFAD2 protein were found with microsomal ω-6 (71–81%) than with plastidial ω-6 desaturases (22–24%) and the sequences within the conserved histidine boxes were some different from those of other plant histidine boxes examined. Phylogenetic analysis revealed that the SeFAD2 gene might have independently diverged as a different subfamily or family member. At least 1–2 copies of the SeFAD2 gene existed in sesame genome and its expression was seed-specific. The SeFAD2 gene vigorously expressed between 18 and 27 days after flowering (DAF). Changes in linoleic acid levels occurred coincidently with changes in the SeFAD2 transcript levels according to the development of sesame oilseeds. It was assumed that the SeFAD2 gene is responsible for production of storage linoleic acid in sesame seeds.

Isolation and characterization of a myo-inositol 1-phosphate synthase cDNA from developing sesame (Sesamum indicum L.) seeds: functional and differential expression, and salt-induced transcription during germination.

Abstract. A cDNA (SeMIPS1) encoding myo-inositol 1-phosphate synthase (EC 5.5.1.4) (MIPS) has been characterized from sesame (Sesamum indicum L. cv. Dan-Baek) seeds and its functional expression analyzed. The SeMIPS1 protein was highly homologous with those from other plant species (88–94%), while a much lower degree of sequence homology (53–62%) was found with other organisms such as humans, mouse, algae, yeast, Drosophila, bacteria and other prokaryotes. A yeast-based complementation assay in yeast mutants containing a disrupted INO1 gene for yeast MIPS confirmed that the SeMIPS1 gene encodes a functional MIPS. Phylogenetic analysis suggested that the SeMIPS1 gene diverged as a different subfamily or family member. Southern hybridization revealed several copies of the SeMIPS1 gene present in the sesame genome and northern blotting indicated that expression of the SeMIPS1 gene may be organ specific. Salt stress during sesame seed germination had an adverse influence on transcription of SeMIPS1 and greatly reduced transcript levels as the duration of exposure to a saline environment increased and NaCl concentration increased. Germination initiation of sesame seeds was severely delayed as NaCl level increased. These results suggest that expression of SeMIPS1 is down-regulated by salt stress during sesame seed germination.

Chromium halides mediated production of hydroxymethylfurfural from starch-rich acorn biomass in an acidic ionic liquid.

Chromium halides were introduced for the sustainable production of hydroxymethylfurfural (HMF) from raw acorn biomass using an acidic ionic liquid. The free sugars (glucose and maltose) released by the acidic hydrolysis of the biomass were confirmed by the FT-IR absorption bands around 995-1014cm(-1) and HPLC. FESEM analysis showed that the acorn biomass contains various sizes of starch granules and their structures were severely changed by the acidic hydrolysis. An optimal concentration of HCl for the HMF yields was 0.3M. The highest HMF yield (58.7+1.3dwt%) was achieved in the reaction mixture of 40% [OMIM]Cl+10% ethyl acetate+50% 0.3M HCl extract containing a mix of CrBr(3)/CrF(3). The combined addition of two halide catalysts was more effective in the synthesis of HMF (1.2-fold higher on average) than their single addition. The best productivity of HMF was found at 15% concentration of the biomass and at 50%, its relative productivity declined down to ca. 0.4-fold.

Expressional characterization of dehydroascorbate reductase cDNA in transgenic potato plants

In plants ascorbic acid (AsA) is a strong antioxidant or reductant that can be converted to dehydroascorbate (DHA) by oxidation. DHA, a very short-lived chemical, can either be hydrolyzed irreversibly to 2,3-diketogulonic acid or recycled to AsA by dehydroascorbate reductase (DHAR).DHAR cDNA, isolated from sesame hairy roots, was inserted into two plant expression vector syrtems with theCaMV35S promoter (CaMV35S : :DHAR) and a potato tuber-specific promoter,Patatin (Patatin : :DHAR). Southern and northern blot hybridization analyses indicated thatDHAR cDNA was successfully integrated into the potato genome and actively transcribed. High levels of sesameDHAR transcript and DHAR enzyme activity were determined, by thePatatin promoter, in regenerated potato tubers, but their levels in leaves were very low. In contrast, much higher amounts of transcript were accumulated in the leaves of CaMV35S : :DHAR regenerants than in the tubers while the activity of DHAR enzyme was higher in the latter. AsA content in the tubers of Patatin : :DHAR transgenic lines was also increased (1.1- to 1.3-fold) compared with that of non-transgenic plants. However, this was not true for the transgenic leaves. In contrast, theCaMV35S promoter was associated with AsA accumulations in both the tubers (up to 1.6-fold) and the leaves (up to 1.5-fold). However, more detailed analyses indicated that this increased enzyme activity was not always accompanied by an elevation in AsA content from transgenic plants. This suggests that other factors may limit the accumulation of vitamin C via ascorbate-recycling in transgenic potato plants.

Statistical Optimization for Production of Carboxymethylcellulase of Bacillus amyloliquefaciens DL-3 by a Recombinant Escherichia coli JM109/DL-3 from Rice Bran Using Response Surface Method

The optimal conditions for production of carboxymethylcellulase (CMCase) of Bacillus amyloliquefaciens DL-3 by a recombinant Escherichia coli JM109/DL-3 were established at a flask scale using the response surface method (RSM). The optimal conditions of rice bran, tryptone, and initial pH of the medium for cell growth extracted by Design Expert Software were 66.1 g/L, 6.2 g/L, and 7.2, respectively, whereas those for production of CMCase were 58.0 g/L, 5.0 g/L, and 7.1. The analysis of variance (ANOVA) of results from central composite design (CCD) indicated that significant factor (“probe > F” less than 0.0500) for cell growth was rice bran, whereas those for production of CMCase were rice bran and initial pH of the medium. The optimal temperatures for cell growth and the production of CMCase by E. coli JM109/DL-3 were found to be 37°C. The optimal agitation speed and aeration rate of 7 L bioreactors for cell growth were 498 rpm and 1.4 vvm, whereas those for production of CMCase were 395 rpm and 1.1 vvm. The ANOVA of results indicated that the aeration rate was more significant factor (“probe > F” less than 0.0001) than the agitation speed for cell growth and production of CMCase. The optimal inner pressure for cell growth was 0.08 MPa, whereas that for the production of CMCase was 0.06 MPa. The maximal production of CMCase by E. coli JM109/DL-3 under optimized conditions was 871.0 U/mL, which was 3.0 times higher than the initial production of CMCase before optimization.

Kinetic Study on the Pretreatment and Enzymatic Saccharification of Rice Hull for the Production of Fermentable Sugars

The production of fermentable sugars from rice hull was studied by dilute acid pretreatment and enzymatic saccharification. Rice hull (15%, w/v) was pretreated by 1% (v/v) sulfuric acid at high temperature (120∼160u2009°C) for 15, 30, 45, and 60xa0min, respectively. The maximum sugar concentration from rice hull in the prehydrolysate was obtained at 140u2009°C for 30xa0min, but the enzymatic saccharification yield from the corresponding pretreated rice hull is not high. To another aspect, the maximum enzymatic saccharification yield was achieved at 160u2009°C for 60xa0min, while the recovery of fermentable sugars was the poorest. To take account of fermentable sugars from pretreatment and enzymatic saccharification, the maximum yield of sugars was obtained only when rice hull was treated at 140u2009°C for 30xa0min. Under this condition, 72.5% (w/w) of all sugars generated from the raw material can be recovered. The kinetic study on the enzymatic saccharification of dilute acid pretreated rice hull was also performed in this work by a modified Michaelis–Menten model and a diffusion-limited model. After calculation by a linear and a non-linear regression analysis, both models showed good relation with the experimental results.

Optimization of salts in medium for production of carboxymethylcellulase by a psychrophilic marine bacterium, Psychrobacter aquimaris LBH-10 using two statistical methods

The concentration of four mineral salts in the medium for the production of carboxymethylcellulase (CMCase) by Psychrobacter aquimaris LBH-10 were optimized using orthogonal array method (OAM) and response surface method (RSM) and their results from two statistical methods were compared. The analysis of variance (ANOVA) of data from central composite design (CCD) based on OAM indicated that potassium phosphate gave the highest percentage participation for cell growth as well as production of CMCase. However, their relative participations of four salts for cell growth were different from those for production of CMCase. The ANOVA of results from RSM indicated that highly significant factors (“probe>F” less than 0.0001) for cell growth were K2HPO4 and (NH4)2SO4, whereas those for production of CMCase were K2HPO4, NaCl, and MgSO4·7H2O. The optimal concentration of K2HPO4, NaCl, MgSO4·7H2O, and (NH4)2SO4 for cell growth extracted by Design Expert Software based on RSM were 7.10, 0.84, 0.24, and 0.33 g/L, respectively, whereas those for production of CMCase were 3.00, 0.52, 0.34, and 0.45 g/L. The optimal concentrations of salts for cell growth and production of CMCase using RSM basically coincided with those using OAM as well as those from ‘one-factor-at-a-time’ method. The production of CMCase by P. aquimaris LBH-10 with optimized concentrations of salts was 273.0 U/mL, which was enhanced by 1.27 times higher than the previous report.

Protein variation and efficient in vitro culture of scale segments from Hyacinthus orientalis L. cv. Carnegie

Abstract SDS–PAGE analysis of scale extracts of Hyacinthus orientalis L. cv. Carnegie exhibited various polypeptide bands in electrophoresis gels. As the culture period of the hyacinth bulb scales increased, some polypeptide bands disappeared and some new bands appeared. The most outstanding feature was that a very strong 29xa0kD polypeptide band was observed during the early stages of scale culture, but after 8 weeks of culture, the polypeptide band was nearly undetectable in the gel, indicating that this polypeptide may be one of a family of major storage proteins in the hyacinth scales. Treatment with IBA at concentrations of 1.5 and 3.0xa0mg/l showed higher regeneration and growth of the bulblets formed from the scale segments of bulbs than those treated with IAA at the same concentrations. No recognizable difference was observed between cultures on either perlite or agar media. The normal (base-down) orientation of the explants had greater influence on the root growth than their inverted (base-up) orientation. The above data suggested that a two-step culture system (first cultured in the agar medium, then in the perlite medium) may be more useful for efficient in vitro culture of this hyacinth cultivar.

Characterisation of a methionine-rich storage protein cDNA from perilla (Perilla frutescens) seeds

We have cloned and characterised a cDNA (PrLeg) coding for a methionine-rich storage protein, which is reported for the first time in perilla (Perilla frutescens (L.) Britt. var. Japonica) seeds, homologous to the 11S legumin-like storage proteins. The most significant feature of the PrLeg precursor protein is that it has the highest content of methionine residues among the 11S legumin-like storage proteins examined so far. Another feature is that the deduced amino acid sequences of the PrLeg protein are phylogenetically close to the sequence groups derived from evolutionally ancient states of the 11S legumin-like storage proteins, or from gymnospermous seed storage proteins, such as Magnolia, Asarum, Dioscorea, Cryptomeria, Metasequoia and Ginkgo. In contrast, with the exception of sesame, relatively low phylogenetic relationships are determined between the PrLeg sequence group and those derived from crop plants, such as soybean, pea, broad bean, rape, pumpkin, rice, coffee and citrus. Southern blot analysis suggests that there may be several copy numbers of thePrLeg genes and their seed-specific expression patterns at the transcriptional level were confirmed by northern hybridisation analysis.

Conversion of plant materials into hydroxymethylfurfural using ionic liquids.

AbstractnThe use of fossil fuels now induces two major issues. First, fossil fuel burning is increasing atmospheric carbon dioxide (CO2) concentrations and, in turn, global warming. Second, fossil fuel resources are limited and will thus decrease in the long run. As a potential solution, there is a need for ecological manufacturing processes that convert raw plant materials into chemical products. For instance, raw plants can be directly converted into hydroxymethylfurfural, which is a versatile intermediate for the synthesis of valuable biofuels such as dimethylfuran and 5-ethoxymethyl-2-furfural. This technology has two benefits for chemical sustainability. First, the pretreatment step is eliminated, thus contributing to reduction of CO2 emissions. Second, plants are sustainable resources versus fossil fuels, which are limited. Here, we review current sustainable technologies for the production of biobased products and hydroxymethylfurfural from plants, using in particular ionic liquids. Plant sources include poplar, switchgrass, miscanthus, weed plants, and agave species.