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Nam Soo Han - One of the best experts on this subject based on the ideXlab platform.
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screening of endogenous strong promoters of Leuconostoc citreum efel2700 based on transcriptome analysis and its application for food grade production of β galactosidase
Journal of Biotechnology, 2021Co-Authors: Seo Yeon Kim, Ye Ji Jang, Seul-ah Kim, Seung Oh Seo, Nam Soo HanAbstract:Leuconostoc citreum is a heterofermentative lactic acid bacterium frequently found in the various fermented foods. L. citreum EFEL2700 isolated from Korean kimchi has been used as a host strain for biotechnological applications. For the use as a food-grade host to over-produce food ingredients or enzymes, strong endogenous promoters guarantying high expression levels of target genes are necessary. In this study, transcriptomic analysis of L. citreum EFEL2700 was performed using RNA-Seq and three promoters of the most highly expressed genes were selected: glyceraldehyde 3-phosphate dehydrogenase (G3PD), 6-phosphogluconate dehydrogenase (6PGD), and phosphoketolase (PPK). Thereafter, they were used as promoters to express β-galactosidase gene from Lactobacillus plantarum WCFS1 in L. citreum EFEL2700 and the levels were compared with the control promoter P710 from L. mesenteroides ATCC 8293. As results, the β-galactosidase activities of the transformants were 2.73, 0.27, 37.43, and 9.25 units/mg under the P710, G3PD, 6PGD, and PPK promoters, respectively. The expression level of endogenous promoter 6PGD was superior to the heterologous P710 promoter previously used in a Leuconostoc-Escherichia coli shuttle vector. The 6PGD developed in this study can be used as the most suitable promoter for β-galactosidase expression in L. citreum EFEL2700.
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Plasmid curing resulted in improved heterologous gene expression in Leuconostoc citreum EFEL2700.
Letters in applied microbiology, 2019Co-Authors: Ye Ji Jang, Seul-ah Kim, Seung Oh Seo, Nam Soo HanAbstract:Leuconostoc citreum EFEL2700 isolated from kimchi was used as a host strain for genetic and metabolic engineering in our previous studies, but the cells of EFEL2700 contained a cryptic plasmid (P-cells). Thus, we created plasmid-free cells (F-cells) using the CRISPR/Cas9 system. In this study, we compared the microbial characteristics of P- and F-cells in terms of growth rate, biochemical properties, transformation efficiency, plasmid copy number and protein expression level. When the growth rate was measured in MRS medium at 30°C, no significant difference (P > 0·01) was observed. Biochemical properties, tested using an API 50CHL kit, showed no differences. Transformation efficiency of F-cells, measured using pCB4270, was higher (1·3 × 104 CFU per μg DNA) than that of P-cells (5·0 × 103 CFU per μg DNA). Copy number after transformation of pCBBgl was 4-fold higher for F-cells than for P-cells. When β-glucosidase activity was assayed in the above experiment, F-cells showed 3·4-fold higher values than P-cells. In conclusion, this study demonstrates that plasmid curing in L. citreum EFEL2700 improves its characteristics as a gene expression host. SIGNIFICANCE AND IMPACT OF THE STUDY: Leuconostoc citreum EFEL2700 (P-cell) isolated from kimchi is a useful food-grade host for expressing heterologous genes. The presence of a cryptic plasmid is thought to limit efficient gene expression. In this study, we compared the microbial and genetic changes after plasmid curing in this strain. The plasmid-free strain showed improved levels of transformation efficiency, copy number and heterologous gene expression without alterations in phenotypes such as the growth rates and biochemical properties. The resulting strain of L. citreum EFEL2701 (F-cell) can be used as an efficient host for genetic engineering.
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Complete genome sequence of Leuconostoc citreum EFEL2700, a host strain for transformation of pCB vectors
Journal of biotechnology, 2018Co-Authors: Seul-ah Kim, Jin Seok Moon, Ye Ji Jang, Ji Eun Heo, Nam Soo HanAbstract:Abstract Leuconostoc citreum is an important lactic acid bacterium used as a starter culture for producing kimchi, the traditional Korean fermented vegetables. An efficient host strain for plasmid transformation, L. citreum EFEL2700, was isolated from kimchi, and it has been frequently used for genetic engineering of L. citreum. In this study, we report the whole genome sequence of the strain and its genetic characteristics. Genome assembly yielded 5 contigs (1 chromosome and 4 plasmids), and the complete genome contained 1,923,830 base pairs (bp) with a G + C content of 39.0%. Average nucleotide identity analysis showed high homology (≥ 99%) to the reference strain L. citreum KM 20. The smallest plasmid (4.3 kbp) was used as an Escherichia coli shuttle vector (pCB) for heterologous gene expression, and L. citreum EFEL2700 showed the highest transformation efficiency, 6.7 × 10⁴ CFU μg−1 DNA. Genetic analysis of the genome enabled the construction of primary metabolic pathway showing a typical hetero-type lactic acid fermentation. Notably, no core genes for primary metabolism were observed in plasmid 4 and it could be eliminated to create an efficient host for gene transformation. This report will facilitate the understanding and application of L. citreum EFEL2700 as a food-grade microbial cell factory.
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development of bicistronic expression system for the enhanced and reliable production of recombinant proteins in Leuconostoc citreum
Scientific Reports, 2018Co-Authors: Seung Hoon Jang, Nam Soo Han, Ji Won Cha, Ki Jun JeongAbstract:The lactic acid bacteria (LAB) Leuconostoc citreum are non-sporulating hetero-fermentative bacteria that play an important role in the fermented food industry. In this study, for the enhanced and reliable production of recombinant proteins in L. citreum, we developed a bicistronic design (BCD) expression system which includes a short leader peptide (1st cistron) followed by target genes (2nd cistron) under the control of a single promoter. Using superfolder green fluorescent protein (sfGFP) as a reporter, the functionality of BCD in L. citreum was verified. Further, to improve the expression in BCD, we tried to engineer a Shine-Dalgarno sequence (SD2) for the 2nd cistron and a promoter by FACS screening of random libraries, and both strong SD2 (eSD2) and promoter (P710V4) were successfully isolated. The usefulness of the engineered BCD with P710V4 and eSD2 was further validated using three model proteins—glutathione-s-transferase, human growth hormone, and α-amylase. All examined proteins were successfully produced with levels highly increased compared with those in the original BCD as well as the monocistronic design (MCD) expression system.
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elimination of the cryptic plasmid in Leuconostoc citreum by crispr cas9 system
Journal of Biotechnology, 2017Co-Authors: Ye Ji Jang, Seul-ah Kim, Seung Oh Seo, Taejip Kim, Sun Chang Kim, Yong Su Jin, Nam Soo HanAbstract:Leuconostoc spp. are important lactic acid bacteria for the fermentation of foods. In particular, L. citreum strains isolated from various foods have been used as host strains for genetic and metabolic engineering studies. In order to develop a food-grade genetic engineering system, L. citreum CB2567 was isolated from Kimchi. However, the isolated bacterium contained a cryptic plasmid which was difficult to eliminate. As the existence of the plasmid might hinder strain engineering, we eliminated the plasmid using an RNA-guided DNA endonuclease CRISPR/Cas9 system. We demonstrated that a plasmid-free L. citreum CB2567 host strain could be efficiently constructed through a two-step procedure: 1) transformation of the “killer” plasmid expressing Cas9 endonuclease and a guide RNA (gRNA) targeting for a specific sequence in the cryptic plasmid, and 2) serial subculture without antibiotics for curing the killer plasmid. When the crude extract of L. citreum expressing Cas9 and the guide RNA was incubated with a PCR fragment containing the specific sequence recognized by the guide RNA, the PCR fragment was cleaved. Also, the cryptic plasmid pCB42 was successfully eliminated from the host strain after transforming the plasmid harboring Cas9 and the guide RNA. The Cas9 and gRNA expression plasmid used in this study can be applied for genome engineering purposes by additionally introducing an editing DNA template to repair the double strand DNA breakage caused by Cas9 in the genome of L. citreum. This study demonstrates the feasibility of developing CRISPR/Cas9-based genetic engineering tools to develop a safe host strain and construct food-grade lactic acid bacteria without residual antibiotic markers.
Karan Wangpaiboon - One of the best experts on this subject based on the ideXlab platform.
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unravelling regioselectivity of Leuconostoc citreum abk 1 alternansucrase by acceptor site engineering
International Journal of Molecular Sciences, 2021Co-Authors: Karan Wangpaiboon, Thassanai Sitthiyotha, Surasak Chunsrivirot, Thanapon Charoenwongpaiboon, Rath PichyangkuraAbstract:Alternansucrase (ALT, EC 2.4.1.140) is a glucansucrase that can generate α-(1,3/1,6)-linked glucan from sucrose. Previously, the crystal structure of the first alternansucrase from Leuconostoc citreum NRRL B-1355 was successfully elucidated; it showed that alternansucrase might have two acceptor subsites (W675 and W543) responsible for the formation of alternating linked glucan. This work aimed to investigate the primary acceptor subsite (W675) by saturated mutagenesis using Leuconostoc citreum ABK-1 alternansucrase (LcALT). The substitution of other residues led to loss of overall activity, and formation of an alternan polymer with a nanoglucan was maintained when W675 was replaced with other aromatic residues. Conversely, substitution by nonaromatic residues led to the synthesis of oligosaccharides. Mutations at W675 could potentially cause LcALT to lose control of the acceptor molecule binding via maltose–acceptor reaction—as demonstrated by results from molecular dynamics simulations of the W675A variant. The formation of α-(1,2), α-(1,3), α-(1,4), and α-(1,6) linkages were detected from products of the W675A mutant. In contrast, the wild-type enzyme strictly synthesized α-(1,6) linkage on the maltose acceptor. This study examined the importance of W675 for transglycosylation, processivity, and regioselectivity of glucansucrases. Engineering glucansucrase active sites is one of the essential approaches to green tools for carbohydrate modification.
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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
International Journal of Biological Macromolecules, 2020Co-Authors: Karan Wangpaiboon, Thanapon Charoenwongpaiboon, Robert A Field, Nisachon Waiyaseesang, Pawinee Panpetch, Sergey A Nepogodiev, Sanong Ekgasit, Rath PichayangkuraAbstract: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.
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an α 1 6 and α 1 3 linked glucan produced by Leuconostoc citreum abk 1 alternansucrase with nanoparticle and film forming properties
Scientific Reports, 2018Co-Authors: Karan Wangpaiboon, Thanapon Charoenwongpaiboon, Panuwat Padungros, Santhana Nakapong, Martin Rejzek, Robert A Field, Rath PichyangkuraAbstract:Alternansucrase catalyses the sequential transfer of glucose residues from sucrose onto another sucrose molecule to form a long chain polymer, known as “alternan”. The alternansucrase-encoding gene from Leuconostoc citreum ABK-1 (Lcalt) was successfully cloned and expressed in Escherichia coli. Lcalt encoded LcALT of 2,057 amino acid residues; the enzyme possessed an optimum temperature and pH of 40 °C and 5.0, respectively, and its’ activity was stimulated up to 2.4-fold by the presence of Mn2+. Kinetic studies of LcALT showed a high transglycosylation activity, with Km 32.2 ± 3.2 mM and kcat 290 ± 12 s−1. Alternan generated by LcALT (Lc-alternan) harbours partially alternating α-1,6 and α- 1,3 glycosidic linkages confirmed by NMR spectroscopy, methylation analysis, and partial hydrolysis of Lc-alternan products. In contrast to previously reported alternans, Lc-alternan can undergo self-assembly, forming nanoparticles with an average size of 90 nm in solution. At concentrations above 15% (w/v), Lc-alternan nanoparticles disassemble and form a high viscosity solution, while this polymer forms a transparent film once dried.
Steve W Cui - One of the best experts on this subject based on the ideXlab platform.
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Leuconostoc citreum sk24 002 glucansucrase biochemical characterisation and de novo synthesis of α glucan
International Journal of Biological Macromolecules, 2016Co-Authors: Liping Song, Ming Miao, Bo Jiang, Steve W Cui, Tao ZhangAbstract:The cell-associated glucansucrase from Leuconostoc citreum SK24.002 was isolated, purified, characterized and used for de novo synthesis of α-glucan and acceptor-products. The final specific glucansucrase activity was 1.4U/mg protein with 13.2-fold purification. The obtained glucansucrase had a molecular weight of 186kDa,Tm of 61.7 °C and △H of 176.7kJ/mol. The enzyme showed maximum activity at pH 5.0-6.0 and 45°C. The enzyme activity was enhanced by Ca(2+), Mn(2+) or Co(2+) ions, whereas the activity decreased as the methanol, ethanol, n-butanol, DMSO or isopropanol concentration increased. The chemical inhibitors including BD, DTNB, EDC or NBS also significantly inhibited enzyme activity. Km, Vmax and kcat of glucansucrase were 10.9mM, 3.6U/mg and 306.6 1/s, respectively. For de novo synthesis from sucrose, α-glucan polymer with molecular weight of 1.5×10(7)g/mol and maltose acceptor products (trisaccharide, tetrasaccharide and pentasaccharide) were obtained by glucansucrase via glucosyltransfer reactions, respectively.
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structure prebiotic properties relationship for α d glucan from Leuconostoc citreum sk24 002
Food Hydrocolloids, 2016Co-Authors: Ming Miao, Bo Jiang, Steve W Cui, Xue Jia, Bruce R Hamaker, Chao HuangAbstract:Effects of non-chemical modification on structure and fermentability of α-D-glucan from Leuconostoc citreum SK24.002 were investigated. It was found that the apparent viscosity, molecular weight and percentage of α-1,6 linkage of α-D-glucan decreased after endodextranase or ultrasonic treatment. During in vitro fermentation, a strong increase in bacterial population was observed and the largest changes took place during the first 12 h of incubation. A decreased pH was also observed and gas production after 48 h fermentation increased at the following sequence: enzymatic treated α-D-glucan < ultrasonic treated α-D-glucan < α-D-glucan. The fermentation of modified sample resulted in a consistent generation of SCFA, particularly in the 12–48 h part of incubation. The concentrations of propionic acid and butyric acid at 48 h of fermentation were 60.4 and 19.1 mM for enzymatic treated α-D-glucan, respectively, higher than ultrasonic treated α-D-glucan or α-D-glucan. This showed that enzymatic hydrolysis could be a promising degradation method for the production of potential prebiotics.
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elucidating molecular structure and prebiotics properties of bioengineered α d glucan from Leuconostoc citreum sk24 002
Food Hydrocolloids, 2016Co-Authors: Ming Miao, Bo Jiang, Xue Jia, Steve W CuiAbstract:Abstract The objective of this work was to produce the bioengineered α-D-glucan from Leuconostoc citreum SK24.002 and study its molecular structure and prebiotics properties. The connectivities and substitutions of α-D-glucan were measured using 2D NMR and the chain structure of the repeating unit was deduced. During in vitro fecal fermentation, a strong increase in bacterial population was observed and the largest changes took place during the first 24 h of incubation. A decrease pH (approximately 2.0) was also observed for the fermentation from 0 h to 48 h compared to the blank (pH 6.8–6.6). Total short-chain fatty acid (SCFA) increased during batch fecal fermentation in the presence of α-D-glucan and it reached the maximum level of 30.01 mM after 48 h of fermentation. The lowest gas production and highest butyric acid production at 48 h of incubation was recorded with α-D-glucan as compared with dextran or pullulan. Moreover, human fecal microbiota on α-D-glucan produced more butyric acid between 24 and 48 h than other test carbohydrates. These results suggest that α-D-glucan would be a good candidate slowly fermentable fiber and may have a positive effect for the gut microbiota homeostasis.
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physicochemical characteristics of a high molecular weight bioengineered α d glucan from Leuconostoc citreum sk24 002
Food Hydrocolloids, 2015Co-Authors: Ming Miao, Bo Jiang, Steve W Cui, Chao Huang, Xue Jia, Tao ZhangAbstract:Abstract A high molecular weight bioengineered α-D-glucan was obtained from Leuconostoc citreum SK24.002 and certain physicochemical properties were investigated. Scanning electron microscopy of α-D-glucan demonstrated a cementitious material with agglutinated granules. Atomic force microscopy showed the irregularly dendritic shaped structure, revealing some side chains. The glucan solution (3%, v/w) has opalescent, bluish-white colour and the concentration affected the depth of colour. The degradation process of 265–345 °C and melting point of 133.9 °C were determined from the thermal analysis. The glucan viscosity increased exponentially with concentration in the range of 5%–10% (w/v). The glucan solution (9%, w/v) showed a non-Newtonian pseudoplastic behaviour at shear rates of 0.01–100 1/s. The changes of elastic modulus and viscous modulus indicated the glucan could form a weak gel. The glucan solution also had good resistance to acidic or thermal hydrolysis. Three-dimensional model revealed α-D-glucan molecule has a helical conformation with some inter-molecular hydrogen bonds, suggesting a high viscosity in solution and this could make it suitable for usage as a biothickener or stabilizer agent in food systems.
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characterisation of a novel water soluble polysaccharide from Leuconostoc citreum sk24 002
Food Hydrocolloids, 2014Co-Authors: Ming Miao, Bo Jiang, Aijuan Bai, Yang Song, Steve W Cui, Tao ZhangAbstract:Abstract Water-soluble polysaccharides that are produced from sucrose by lactobacillus strains isolated from Chinese traditional pickled vegetables were investigated. Strain SK24.002, which secreted an exopolysaccharide with a high dextranase resistance, was identified as Leuconostoc citreum via 16S rDNA sequence analysis. A polysaccharide yield of 35 g/L was achieved at 20 h in Man-Rogosa-Sharpe medium containing 100 g/L sucrose. The water-soluble polysaccharide consisted exclusively of glucose, and the weight-average molecular weight was 4.62 × 10 7 Da. Spectroscopy results demonstrated that the water-soluble exopolysaccharide was mainly composed of α-1,3 and α-1,6 linked d -glucopyranose units. Methylation analysis revealed that the ratio of α-1,6 linkages to α-1,3 linkages was 5:4. These results suggested a possible molecular structure for the water-soluble exopolysaccharide that consists of a backbone chain of alternating α-1,3 and α-1,6 linkages with a branched point at the C6 of the 1,3,6-linked d -glucopyranose unit. This novel polysaccharide may have applications as a functional ingredient in processed foods.
Rath Pichyangkura - One of the best experts on this subject based on the ideXlab platform.
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unravelling regioselectivity of Leuconostoc citreum abk 1 alternansucrase by acceptor site engineering
International Journal of Molecular Sciences, 2021Co-Authors: Karan Wangpaiboon, Thassanai Sitthiyotha, Surasak Chunsrivirot, Thanapon Charoenwongpaiboon, Rath PichyangkuraAbstract:Alternansucrase (ALT, EC 2.4.1.140) is a glucansucrase that can generate α-(1,3/1,6)-linked glucan from sucrose. Previously, the crystal structure of the first alternansucrase from Leuconostoc citreum NRRL B-1355 was successfully elucidated; it showed that alternansucrase might have two acceptor subsites (W675 and W543) responsible for the formation of alternating linked glucan. This work aimed to investigate the primary acceptor subsite (W675) by saturated mutagenesis using Leuconostoc citreum ABK-1 alternansucrase (LcALT). The substitution of other residues led to loss of overall activity, and formation of an alternan polymer with a nanoglucan was maintained when W675 was replaced with other aromatic residues. Conversely, substitution by nonaromatic residues led to the synthesis of oligosaccharides. Mutations at W675 could potentially cause LcALT to lose control of the acceptor molecule binding via maltose–acceptor reaction—as demonstrated by results from molecular dynamics simulations of the W675A variant. The formation of α-(1,2), α-(1,3), α-(1,4), and α-(1,6) linkages were detected from products of the W675A mutant. In contrast, the wild-type enzyme strictly synthesized α-(1,6) linkage on the maltose acceptor. This study examined the importance of W675 for transglycosylation, processivity, and regioselectivity of glucansucrases. Engineering glucansucrase active sites is one of the essential approaches to green tools for carbohydrate modification.
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an α 1 6 and α 1 3 linked glucan produced by Leuconostoc citreum abk 1 alternansucrase with nanoparticle and film forming properties
Scientific Reports, 2018Co-Authors: Karan Wangpaiboon, Thanapon Charoenwongpaiboon, Panuwat Padungros, Santhana Nakapong, Martin Rejzek, Robert A Field, Rath PichyangkuraAbstract:Alternansucrase catalyses the sequential transfer of glucose residues from sucrose onto another sucrose molecule to form a long chain polymer, known as “alternan”. The alternansucrase-encoding gene from Leuconostoc citreum ABK-1 (Lcalt) was successfully cloned and expressed in Escherichia coli. Lcalt encoded LcALT of 2,057 amino acid residues; the enzyme possessed an optimum temperature and pH of 40 °C and 5.0, respectively, and its’ activity was stimulated up to 2.4-fold by the presence of Mn2+. Kinetic studies of LcALT showed a high transglycosylation activity, with Km 32.2 ± 3.2 mM and kcat 290 ± 12 s−1. Alternan generated by LcALT (Lc-alternan) harbours partially alternating α-1,6 and α- 1,3 glycosidic linkages confirmed by NMR spectroscopy, methylation analysis, and partial hydrolysis of Lc-alternan products. In contrast to previously reported alternans, Lc-alternan can undergo self-assembly, forming nanoparticles with an average size of 90 nm in solution. At concentrations above 15% (w/v), Lc-alternan nanoparticles disassemble and form a high viscosity solution, while this polymer forms a transparent film once dried.
Bo Jiang - One of the best experts on this subject based on the ideXlab platform.
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di glycosyl stevioside production via Leuconostoc citreum sk24 002 alternansucrase enzymatic reaction and structural characterization
Journal of Food Measurement and Characterization, 2019Co-Authors: Abubakr Musa, Bo Jiang, Mohammed Abdalbasit A Gasmalla, Mandour H Abdalhai, Ammar AlalfargaAbstract:In this work, alternansucrase enzyme from Leuconostoc citreum SK24.002 was used to produce di-glycosyl-stevioside through acceptor reaction. This was detected by high-performance liquid chromatography (HPLC) and was isolated using AB-8 macroporous resin and semi-preparative HPLC. With the help of mass spectroscopy, 1D NMR (H1 and C13) and 2D NMR (COSY, HMQC, HMBC) the di-glycosyl-stevioside structure was identified to be 13-{[α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-β-D glucopyranosyl-(1→2)-β-D-glucopyranosyl]oxy} kaur-16-en-19-oic acid β-D glucopyranosyl eater. In order to increase the modified stevioside (di-glycosyl-stevioside) product and reduce production time, the impact of velocity of shaking on the di-glycosyl-stevioside production was also evaluated, using 75–150 rpm for 24 h at 25 °C. The results showed that the di-glycosyl-stevioside production increased significantly (p < 0.05) from 0.35 ± 0.0.01 mg/mL for 75 rpm to 1.0667 ± 0.0152 mg/mL for 150 rpm at 6 h during the glycosylation reaction.
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Leuconostoc citreum sk24 002 glucansucrase biochemical characterisation and de novo synthesis of α glucan
International Journal of Biological Macromolecules, 2016Co-Authors: Liping Song, Ming Miao, Bo Jiang, Steve W Cui, Tao ZhangAbstract:The cell-associated glucansucrase from Leuconostoc citreum SK24.002 was isolated, purified, characterized and used for de novo synthesis of α-glucan and acceptor-products. The final specific glucansucrase activity was 1.4U/mg protein with 13.2-fold purification. The obtained glucansucrase had a molecular weight of 186kDa,Tm of 61.7 °C and △H of 176.7kJ/mol. The enzyme showed maximum activity at pH 5.0-6.0 and 45°C. The enzyme activity was enhanced by Ca(2+), Mn(2+) or Co(2+) ions, whereas the activity decreased as the methanol, ethanol, n-butanol, DMSO or isopropanol concentration increased. The chemical inhibitors including BD, DTNB, EDC or NBS also significantly inhibited enzyme activity. Km, Vmax and kcat of glucansucrase were 10.9mM, 3.6U/mg and 306.6 1/s, respectively. For de novo synthesis from sucrose, α-glucan polymer with molecular weight of 1.5×10(7)g/mol and maltose acceptor products (trisaccharide, tetrasaccharide and pentasaccharide) were obtained by glucansucrase via glucosyltransfer reactions, respectively.
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structure prebiotic properties relationship for α d glucan from Leuconostoc citreum sk24 002
Food Hydrocolloids, 2016Co-Authors: Ming Miao, Bo Jiang, Steve W Cui, Xue Jia, Bruce R Hamaker, Chao HuangAbstract:Effects of non-chemical modification on structure and fermentability of α-D-glucan from Leuconostoc citreum SK24.002 were investigated. It was found that the apparent viscosity, molecular weight and percentage of α-1,6 linkage of α-D-glucan decreased after endodextranase or ultrasonic treatment. During in vitro fermentation, a strong increase in bacterial population was observed and the largest changes took place during the first 12 h of incubation. A decreased pH was also observed and gas production after 48 h fermentation increased at the following sequence: enzymatic treated α-D-glucan < ultrasonic treated α-D-glucan < α-D-glucan. The fermentation of modified sample resulted in a consistent generation of SCFA, particularly in the 12–48 h part of incubation. The concentrations of propionic acid and butyric acid at 48 h of fermentation were 60.4 and 19.1 mM for enzymatic treated α-D-glucan, respectively, higher than ultrasonic treated α-D-glucan or α-D-glucan. This showed that enzymatic hydrolysis could be a promising degradation method for the production of potential prebiotics.
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elucidating molecular structure and prebiotics properties of bioengineered α d glucan from Leuconostoc citreum sk24 002
Food Hydrocolloids, 2016Co-Authors: Ming Miao, Bo Jiang, Xue Jia, Steve W CuiAbstract:Abstract The objective of this work was to produce the bioengineered α-D-glucan from Leuconostoc citreum SK24.002 and study its molecular structure and prebiotics properties. The connectivities and substitutions of α-D-glucan were measured using 2D NMR and the chain structure of the repeating unit was deduced. During in vitro fecal fermentation, a strong increase in bacterial population was observed and the largest changes took place during the first 24 h of incubation. A decrease pH (approximately 2.0) was also observed for the fermentation from 0 h to 48 h compared to the blank (pH 6.8–6.6). Total short-chain fatty acid (SCFA) increased during batch fecal fermentation in the presence of α-D-glucan and it reached the maximum level of 30.01 mM after 48 h of fermentation. The lowest gas production and highest butyric acid production at 48 h of incubation was recorded with α-D-glucan as compared with dextran or pullulan. Moreover, human fecal microbiota on α-D-glucan produced more butyric acid between 24 and 48 h than other test carbohydrates. These results suggest that α-D-glucan would be a good candidate slowly fermentable fiber and may have a positive effect for the gut microbiota homeostasis.
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physicochemical characteristics of a high molecular weight bioengineered α d glucan from Leuconostoc citreum sk24 002
Food Hydrocolloids, 2015Co-Authors: Ming Miao, Bo Jiang, Steve W Cui, Chao Huang, Xue Jia, Tao ZhangAbstract:Abstract A high molecular weight bioengineered α-D-glucan was obtained from Leuconostoc citreum SK24.002 and certain physicochemical properties were investigated. Scanning electron microscopy of α-D-glucan demonstrated a cementitious material with agglutinated granules. Atomic force microscopy showed the irregularly dendritic shaped structure, revealing some side chains. The glucan solution (3%, v/w) has opalescent, bluish-white colour and the concentration affected the depth of colour. The degradation process of 265–345 °C and melting point of 133.9 °C were determined from the thermal analysis. The glucan viscosity increased exponentially with concentration in the range of 5%–10% (w/v). The glucan solution (9%, w/v) showed a non-Newtonian pseudoplastic behaviour at shear rates of 0.01–100 1/s. The changes of elastic modulus and viscous modulus indicated the glucan could form a weak gel. The glucan solution also had good resistance to acidic or thermal hydrolysis. Three-dimensional model revealed α-D-glucan molecule has a helical conformation with some inter-molecular hydrogen bonds, suggesting a high viscosity in solution and this could make it suitable for usage as a biothickener or stabilizer agent in food systems.