Synthesis of Cationic Quaternized Nanolevan Derivative for Small Molecule and Nucleic Acid Delivery.

Levan is a biopolymer composed of fructose chains covalently linked by ß-2,6 glycosidic linkages. This polymer self-assembles into a nanoparticle of uniform size, making it useful for a wide range of applications. Also, levan exhibits various biological activities such as antioxidants, anti-inflammatory, and anti-tumor, that make this polymer very attractive for biomedical application. In this study, levan synthesized from Erwinia tasmaniensis was chemically modified by glycidyl trimethylammonium chloride (GTMAC) to produce cationized nanolevan (QA-levan). The structure of the obtained GTMAC-modified levan was determined by FT-IR, 1H-NMR and elemental (CHN) analyzer. The size of the nanoparticle was calculated using the dynamic light scattering method (DLS). The formation of DNA/QA-levan polyplex was then investigated by gel electrophoresis. The modified levan was able to increase the solubility of quercetin and curcumin by 11-folds and 205-folds, respectively, compared to free compounds. Cytotoxicity of levan and QA-levan was also investigated in HEK293 cells. This finding suggests that GTMAC-modified levan should have a potential application for drug and nucleic acid delivery.

Cassava pullulanase and its synergistic debranching action with isoamylase 3 in starch catabolism.

Pullulanase (EC 3.2.1.41, PUL), a debranching enzyme belonging to glycoside hydrolase family 13 subfamily 13, catalyses the cleavage of α-1,6 linkages of pullulan and ß-limit dextrin. The present work studied PUL from cassava Manihot esculenta Crantz (MePUL) tubers, an important economic crop. The Mepul gene was successfully cloned and expressed in E. coli and rMePUL was biochemically characterised. MePUL was present as monomer and homodimer, as judged by apparent mass of ~ 84 - 197 kDa by gel permeation chromatography analysis. Optimal pH and temperature were at pH 6.0 and 50 °C, and enzyme activity was enhanced by the addition of Ca2+ ions. Pullulan is the most favourable substrate for rMePUL, followed by ß-limit dextrin. Additionally, maltooligosaccharides were potential allosteric modulators of rMePUL. Interestingly, short-chain maltooligosaccharides (DP 2 - 4) were significantly revealed at a higher level when rMePUL was mixed with cassava isoamylase 3 (rMeISA3), compared to that of each single enzyme reaction. This suggests that MePUL and MeISA3 debranch ß-limit dextrin in a synergistic manner, which represents a major starch catabolising process in dicots. Additionally, subcellular localisation suggested the involvement of MePUL in starch catabolism, which normally takes place in plastids.

Synergistic enzyme cocktail between levansucrase and inulosucrase for superb levan-type fructooligosaccharide synthesis.

Inulosucrase (ISC) and levansucrase (LSC) utilise sucrose and produce inulin- and levan-type fructans, respectively. This study aims to propose a new strategy to improve levan-type fructooligosaccharide (L-FOS) production. The effect of ISC/ LSC -mixed reaction was elucidated on L-FOS production. The presence of ISC in the LSC reaction significantly leads to the higher production of L-FOSs as the main products. Furthermore, the different ratios between ISC and LSC affected the distribution of L-FOSs. A greater amount of ISC compared to LSC promoted the synthesis of short-chain L-FOSs. Conversely, when LSC was increased, the synthesis of longer-chain L-FOSs was enhanced. The addition of trisaccharide mixtures obtained from either a single ISC or LSC reaction could enhance L-FOSs synthesis in the LSC reaction. Analysis of these trisaccharides revealed that most species of the oligosaccharides were similar, with 1-kestose being the major one. The supplement of only 1-kestose in the LSC reaction showed similar results to those of the reaction in the presence of trisaccharide mixtures. Moreover, the results were supported by molecular dynamics simulations. This work not only provides an improvement in L-FOS production but also revealed and supported some insights into the mechanism of fructansucrases.

Fruit ripening-associated leucylaminopeptidase with cysteinylglycine dipeptidase activity from durian suggests its involvement in glutathione recycling.

BACKGROUND: Durian (Durio zibethinus L.) is a highly popular fruit in Thailand and several other Southeast Asian countries. It is abundant in essential nutrients and sulphur-containing compounds such as glutathione (GSH) and γ-glutamylcysteine (γ-EC). Cysteinylglycine (Cys-Gly) is produced by GSH catabolism and occurs in durian fruit pulp. Cysteine (Cys) is a precursor of sulphur-containing volatiles generated during fruit ripening. The aforementioned substances contribute to the strong odour and flavour of the ripe fruit. However, the genes encoding plant Cys-Gly dipeptidases are unknown. The aim of this study was to measure leucylaminopeptidase (LAP) activity in durian fruit pulp. RESULTS: We identified DzLAP1 and DzLAP2, which the former was highly expressed in the fruit pulp. DzLAP1 was expressed at various ripening stages and in response to ethephon/1-MCP treatment. Hence, DzLAP1 is active at the early stages of fruit ripening. DzLAP1 is a metalloenzyme ~ 63 kDa in size. It is activated by Mg2+ or Mn2+ and, like other LAPs, its optimal alkaline pH is 9.5. Kinetic studies revealed that DzLAP1 has Km = 1.62 mM for its preferred substrate Cys-Gly. DzLAP1-GFP was localised to the cytosol and targeted the plastids. In planta Cys-Gly hydrolysis was confirmed for Nicotiana benthamiana leaves co-infiltrated with Cys-Gly and expressing DzLAP1. CONCLUSIONS: DzLAP1 has Cys-Gly dipeptidase activity in the γ-glutamyl cycle. The present study revealed that the LAPs account for the high sulphur-containing compound levels identified in fully ripened durian fruit pulp.

Characterisation of insoluble α-1,3-/α-1,6 mixed linkage glucan produced in addition to soluble α-1,6-linked dextran by glucansucrase (DEX-N) from Leuconostoc citreum ABK-1.

Glucansucrases catalyse the formation of glucans from sucrose. The glucansucrase-encoding gene from Leuconostoc citreum ABK-1, dex-N, was successfully cloned and expressed in E. coli BL21 Star (DE3). DEX-N produces 2 types of glucans: soluble (S-dextran) and insoluble (I-glucan) glucans. The S-dextran was determined to be ca. 10 kDa in size and contained >90% α-1,6 linkages; along with its water solubility, this is similar to commercial dextran. On the other hand, I-glucan was water-insoluble, harbouring a block-wise pattern of α-1,3 and α-1,6 linkages in its structure. Notably, the FTIR and powder X-ray diffraction pattern of I-glucan exhibited a combination of features found in α-1,6-linked dextran and α-1,3-linked mutan. Although both I-glucan and mutan are insoluble glucans, their physical characteristics are notably dissimilar.

Identification and Functional Characterization of Genes Involved in the Biosynthesis of Caffeoylquinic Acids in Sunflower (Helianthus annuus L.).

Sunflower (Helianthus annuus L.) sprouts accumulate high amounts of caffeoylquinic acids (CQAs) including chlorogenic acid (5-CQA) and 1,5-diCQA. These compounds, which can be found in many plants, including tomato, globe artichoke, and chicory, have many health benefits, including antioxidant, antihepatotoxic, and antiglycative activities. However, CQA profiles and biosynthesis have not previously been studied in sunflower sprouts. In the present study, we found that 5-CQA and 1,5-diCQA were the major CQAs found in sunflower sprouts. We also identified minor accumulation of other CQAs, namely 3-CQA, 4-CQA, 3,4-diCQA, and 4,5-diCQA. According to genome-wide identification and phylogenetic analysis of genes involved in CQA biosynthesis in sunflower, three genes (HaHQT1, HaHQT2, and HaHQT3) encoding hydroxycinnamoyl CoA:quinate hydroxycinnamoyl transferase (HQT) and two genes (HaHCT1 and HaHCT2) encoding hydroxycinnamoyl CoA:shikimate/quinate hydroxycinnamoyl transferase (HCT) were identified. Expression analysis of these five genes in hypocotyls and cotyledons strongly suggested that HaHQT2 could be the main enzyme responsible for CQA biosynthesis, as HaHQT2 had the highest expression levels. In addition, when transiently expressed in the leaves of Nicotiana benthamiana, all three HaHQTs, which were soluble and not membrane-bound enzymes, could increase the content of 5-CQA by up to 94% compared to that in a control. Overall, our results increase understanding of CQA biosynthesis in sunflower sprouts and could be exploited by plant breeders to enhance accumulation of health-promoting CQAs in these plants.

Temperature-dependent inulin nanoparticles synthesized by Lactobacillus reuteri 121 inulosucrase and complex formation with flavonoids.

Inulin nanoparticles (INNPs) are a biocompatible material which has a potential application for enhancing solubility and preventing degradation of compounds. In this work, we demonstrated that INNPs could be synthesized from sucrose using inulosucrase from Lactobacillus reuteri 121. Noticeably, dynamic light scattering (DLS) analysis showed that the derived INNPs exhibited uniformity in size, which was easily controlled by the reaction temperature. The effect of enzyme and sucrose concentration, as well as reaction time, was explored. Moreover, the solubility of INNPs in various organic solvents was also investigated, and we found that the INNPs were freely regenerated in water even though they had precipitated by organic solvents. Essentially, we demonstrated that the derived INNPs could be applied for flavonoid encapsulation. The solubility and stability of quercetin and fisetin in the INNPs complexes was higher than those of free compounds. These results make the INNPs very promising for many applications.

Modified properties of alternan polymers arising from deletion of SH3-like motifs in Leuconostoc citreum ABK-1 alternansucrase.

Alternansucrase (ALT, EC 2.4.1.140) catalyses the formation of an alternating 〈-1, 3/1, 6-linked glucan, with periodic branch points, from sucrose substrate. Beyond the catalytic domain, this enzyme harbours seven additional C-terminal SH3-like repeats. We herein generated two truncated alternansucrases, possessing deletions of three and seven adjacent SH3 motifs, giving Δ3SHALT and Δ7SHALT. Δ3SHALT and Δ7SHALT exhibited kcat/Km for transglycosylation activity 2.3- and 1.5-fold lower than wild-type ALT (WTALT), while hydrolysis was detected only in the truncated ALTs, oligosaccharide patterns and polymer glycosidic linkage were similar to that of WTALT. The viscosities of ALT polymers increase by ˜100-fold at 15% (w/v), with gel-like states formed at 12.5, 15.0, and 20.0% (w/v) produced by polymer from WTALT, Δ3SHALT, and Δ7SHALT, respectively. The average nanoparticle sizes of Δ3SHALT and Δ7SHALT polymers were 80 nm, compared to 90 nm from WTALT. In conclusion, even relatively subtle differences in the structure of ALT-produced alternan give rise to profound impact on the glucan polymer physicochemical properties.

In Silico Analyses of Rice Thionin Genes and the Antimicrobial Activity of OsTHION15 Against Phytopathogens.

Thionins are a family of antimicrobial peptides. We performed in silico expression analyses of the 44 rice (Oryza sativa) thionins (OsTHIONs). Modulated expression levels of OsTHIONs under different treatments suggest their involvement in many processes, including biotic, abiotic, and nutritional stress responses, and in hormone signaling. OsTHION15 (LOC_Os06g32600) was selected for further characterization based on several in silico analyses. OsTHION15 in O. sativa subsp. indica 'KDML 105' was expressed in all of the tissues and organs examined, including germinating seed, leaves, and roots of seedlings and mature plants, and inflorescences. To investigate the antimicrobial activity of OsTHION15, we produced a recombinant peptide in Escherichia coli Rosetta-gami (DE3). The recombinant OsTHION15 exhibited inhibitory activities toward rice-pathogenic bacteria such as Xanthomonas oryzae pv. oryzae and Pectobacterium carotovorum pv. atroseptica, with minimum inhibitory concentrations of 112.6 and 14.1 µg ml-1, respectively. A significant hyphal growth inhibition was also observed toward Fusarium oxysporum f. sp. cubense and Helminthosporium oryzae. In addition, we demonstrated the in planta antibacterial activity of this peptide in Nicotiana benthamiana against X. campestris pv. glycines. These activities suggest the possible application of OsTHION15 in plant disease control.

Cloning of the full-length isoamylase3 gene from cassava Manihot esculenta Crantz 'KU50' and its heterologous expression in E. coli.

Isoamylase (EC.3.2.1.68), an essential enzyme in starch metabolism, catalyses the cleavage of α-1,6 glucosidic linkages of branched α-polyglucans such as beta-limit dextrin and amylopectin, but not pullulan. Three different isoamylase isoforms have been reported in plants and algae. We herein report on the first success in preparation of full-length isoamylase3 gene (MeISA3) of cassava Manihot esculenta Crantz 'KU50' from 5' Rapid Amplification of cDNA Ends (5' RACE). The MeISA3 was cloned to pET21b and expressed in E. coli. The HistrapTM-purified rMeISA3 appeared as a single band protein with approximate molecular size of 75 kDa on SDS-PAGE and Western blot, while 80 kDa was shown by gel filtration chromatography. This indicated the existence of a monomeric enzyme. Biochemical characterisation of rMeISA3 showed that the enzyme was specific towards beta-limit dextrin, with optimal activity at 37 °C pH 6.0. Activity of rMeISA3 could be significantly promoted by Mg2+ and Co2+. rMeISA3 debranched glucan chains of amylopectin were confirmed by HPAEC-PAD analysis.

Heterologous expression and antimicrobial activity of OsGASR3 from rice (Oryza sativa L.).

According to an in silico analysis, OsGASR3 (LOC_Os03g55290) from rice (Oryza sativa L.) was predicted to be involved in plant defense mechanisms. A semi-quantitative reverse transcription polymerase chain reaction assay revealed that OsGASR3 is highly expressed in the inflorescences of Thai jasmine rice (O. sativa L. subsp. indica 'KDML 105'). To characterize the biological activity of OsGASR3, we produced an OsGASR3-glutathione S-transferase fusion protein in Escherichia coli Rosetta-gami (DE3) cells for a final purified recombinant OsGASR3 yield of 0.65 mg/L. The purified OsGASR3 inhibited the hyphal growth of Fusarium oxysporum f.sp. cubense and Helminthosporium oryzae at a relatively low concentration (7.5 µg/mL). Furthermore, OsGASR3 exhibited in planta inhibitory activity against Xanthomonas campestris, suggesting its involvement in defense mechanisms, in addition to its previously reported functions affecting growth and development. These observations indicate that recombinant OsGASR3 may be useful for protecting agriculturally important crops against pathogenic microbes.

Heterologous co-expression in E. coli of isoamylase genes from cassava Manihot esculenta Crantz 'KU50' achieves enzyme-active heteromeric complex formation.

KEY MESSAGE: Cloning of two isoamylase genes, MeISA1 and MeISA2, from cassava (Manihot esculenta Crantz) tubers, accompanied by their co-expression in E. coli demonstrates a requirement for heteromeric complex formation to achieve debranching activity. Starch debranching enzyme (DBE) or isoamylase (ISA) (EC.3.2.1.68), an important enzyme in starch metabolism, catalyses the hydrolysis of α-1,6 glycosidic linkages of amylopectin. Isoforms of ISAs have been reported in higher plants and algae (Fujita et al. in Planta 208:283-293, 1999; Hussain et al. in Plant Cell 15:133-149, 2003; Ishizaki et al. in Agric Biol Chem 47:771-779, 1983; Mouille et al. in Plant Cell 8:1353-1366, 1996). In the current work, cassava ISA genes were isolated from cDNA generated from total RNA from tubers of Manihot esculanta Crantz cultivar KU50. MeISA1 and MeISA2 were successfully amplified and cloned into a pETDuet1 vector. The putative MeISA1 and MeISA2 proteins comprised 763 and 882 amino acids, with substantial similarity to StISA1 and StISA2 from potato (84.4% and 68.9%, respectively). Recombinant MeISA1 and MeISA2 were co-expressed in Escherichia coli SoluBL21 (DE3). HistrapTM-Purified rMeISA1 and rMeISA2 showed approximate molecular weights of 87 and 99 kDa, respectively, by SDS-PAGE. Debranching activity was only detectable in the column fractions where both recombinant ISA isoforms were present. The heteromeric DBE from crude extracts of 4-5 h induced cultures analysed by gel filtration chromatography and western blot showed combinations of rMeISA1 and rMeISA2 at ratios of 1:1 to 4:1. Pooled fractions with DBE activity were used for enzyme characterisation, which showed that the enzyme was specific for amylopectin, with optimum activity at 37 °C and pH 7.0. Enzyme activity was enhanced by Co2+, Mg2+ and Ca2+, but was strongly inhibited by Cu2+. Debranched amylopectin products showed chain length distributions typical of plant DBE.