• Journal of Life Science
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• v.27 no.7
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• pp.849-856
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• 2017

The ${\beta}$-D-fructofuranosidase (EC 3.2.1.26) is an important enzyme from a historical point of view, discovered by French biologist Berthelot in 1860 and was first used to study enzymology. ${\beta}$-D-fructosfuranosidase catalyzes the hydrolysis of sucrose into D-glucose and D-fructose. Four biochemical subgroups of ${\beta}$-D-fructofuranosidase have been investigated in plants. There are vacuolar (soluble acid), cytoplasmic (soluble alkaline), membrane-bound (insoluble alkaline), and cell wall-bound (insoluble acid) ${\beta}$-D-fructofuranosidase by purification. Their biochemical characteristics are distinct. It suggested that those enzymes might be different gene products. The contribution of each of these enzymes to sucrose management in the plant is likely to be correlated with their localization. Common localization in developing cells in tissues from a range of developmental stages and plant parts suggests that all of the isoforms may be closely involved in nutrient transport. The ${\beta}$-D-fructofuranosidases were most commonly found associated with maturing tissues in developing fruits, leaves, and roots. The ${\beta}$-D-fructofuranosidase activity varies in the relationship between growth and expansion through cell division, development of storage organs and tissues, and the relationship of plant defense responses. It is necessary to summarize more researches in order to know the definite physiological function.

• The Journal of the Convergence on Culture Technology
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• v.9 no.4
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• pp.587-592
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• 2023

Plant growth is regulated by a variety of factors, including organic matter availability. Organic nutrients are carbohydrate molecules from photosynthetic products produced by tissues associated with carbon and energy fixation called "sources". These compounds flow through plant vascular bundles into non-photosynthetic or growing tissues called "sinks". Among these possible compounds, the disaccharide fructosyl glucose, sucrose, is the most representative. During the transport of sucrose, the pathway from the source to the sinks can include hydrolysis of sucrose into glucose and fructose derivatives or direct transfer of sucrose. Among the enzymes involved in this, β-D-fructofuranosidase is the most important. Soluble neutral β-D-fructofuranosidase, one of several isoenzymes, is located in intracellular protoplasts and helps plant cells metabolize sucrose to produce energy. In order to track the activity of this enzyme during the course of plant growth, histological methods were used for the most effective immunolocalization. As a result, the activity was higher in the phloem and epidermis than in the mesophyll tissue in the leaf. In the growing stem, activity was high in the phloem, epidermis, and cortex. The activity of the root, which is a sink tissue, was high in all parts, but especially the highest in the root tip part. It is thought that this is because it helps unloading of sucrose in sink tissues that require sucrose degradation and plays a role in hydrolysising sucrose.

• Journal of Microbiology and Biotechnology
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• v.10 no.2
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• pp.238-243
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• 2000

An exoinulinase (${\beta}-D-fructofuranosidase$) gene was cloned by chromosome walking along the upstream region of the endoinulinase gene of Pseudomonas mucidolens isolated from soil. the exoinulinase gene consisted of an ORF of 0,506 bp encoding a polypeptide of 501 amino acids with a deduced molecular weight of 55,000. The exoinulinase produced by the recombinant Escherichia coli $DH5{\alpha}$ strain was also purified to homogeneity as determined by SDS-PAGE and a zymogram. The molecular weight of the purified exoinulinase according to both SDS-PAGE and gel filtration matched the deduced molecular weight of the protein described above, thereby indicating that the native form of the exoinulinase was a monomer. The purified enzyme hydrolyzed activity value of 2.0. Furthermore, no inulo-oligomers were liberated from the inulin substrate in the enzymatic reaction mixtures incubated for 90 min at $55^{\circ}C$. Taken together, these results indicate that the purified ${\beta}-D-fructofuranosidase$ was an exoinulinase. The pH and temperature optima of the exoinulinase were pH 6.0 and $55^{\circ}C$, respectively. the enzymehad no apparent requirement for a cofactor, and its activity was completely inactivated by $Ag^{+},{\;}Hg^{2+},{\;}and{\;}Zn^{2+}$. Kinetic experiments gave $K_m,{\;}V_{max},{\;}and{\;}K_{cat}$ values for inulin of 11.5 mM, 18 nM/s, and $72{\;}s^{-1}$, respectively. the exoinulinase was fairly stable in broad pH conditions (pH 5-9), and at pH 6.0 it showed a residual activity of about 70% after 4 h incubation at $55^{\circ}C$.

• Journal of Life Science
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• v.17 no.1 s.81
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• pp.6-11
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• 2007

[ $\beta$ ]-Fructofuranosidases, a family 32 of glycoside hydrolases (GH32), share three conserved domains including the W(L/M)(C/N)DP(Q/N), FRDPK, and ECP(D/G) motifs. The functional role of the conserved acidic residues within three domains of levan fructotransferase, one of the $\beta-fructofuranosidases$, from Microbacterium sp. AL-210 was studied by site-directed mutagenesis. Each mutant was overexpressed in E. coli BL21(DE3) and purified by using Hi-Trap chelating affinity chromatography and fast performance liquid chromatography. Substitution of Asp-63 by Ala, Asp-195 by Asn, and Glu-245 by Ala and Asp decreased the enzyme activity by approximately 100-fold compared to the wild-type enzyme. This result indicates that three acidic residues Asp-63, Asp-195, and Glu-245 play a major role in catalysis. Since the three acidic residues are present in a conserved position in inulinase, levanase, levanfructotransferase, and invertase, they are likely to have a common functional role as nucleophile, transition state stabilizer, and general acid in $\beta-fructofuranosidases$.

• Microbiology and Biotechnology Letters
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• v.18 no.5
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• pp.490-495
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• 1990

The extracellular inulinase from Bacillus spp. was purified to a single protein through a sequence of operations including ammonium sulfate fractionation, heat treatment, DEAE Sepharose C1-6B ion exchange chromatography, Sephadex 6-100 and Sephadex 6-150 gel filtration. The purified enzyme was confirmed to be a $\beta$ -D-fructofuranosidase(EC 3.2.1.26) which was much more active on sucrose than on inulin(I/S = 0.2). The maximal inulinase activity was observed at pH 6.0 and at the temperature of $50^{\circ}C$. The mo1ecular weight of the enzyme was about 56, 000. Tryptophan and histidine residues of the enzyme molecule were found to be essential for its catalytic activity.

• Microbiology and Biotechnology Letters
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• v.16 no.4
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• pp.297-302
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• 1988

The protoplasts of Saccharomyces cerevisiae D-71, a thermophilic strain and Zygosaccharomyces rouxii SR-S, an osmotolerant strain were fused, and a fusant (FS-RN 1) was selected, then was characterized for its genetic stability, DNA content, cell capacity, growth rate, tolerance to salts and chemicals, $\beta$-fructofuranosidase level and ethanol fermenting activity. After 6 months of preservation, 5.8% of the fusant clones were segregated to parental types. The DNA content and cell capacity of the fusant were greater than those of the parental strains. Lag period of growth for the fusant was longer than those for the parents. The fusant colonies showed pink-color reaction to triphenyltetrazolium chloride(TTC) test. The fusant appeared to have resistances to NaCl at moderate levels between both parental strains, and resistances to KCI, sodium propionate and cycloheximide similar to either one of the parents. $\beta$-Fructofuranosidase activity of the fusant was 24.2$\times$10$^{-3}$/IU/g(dry wt) for 3 days culture. Ethanol yields ofter 4 days of fermentation by the fusant at 3$0^{\circ}C$ were : 6.0%(v/v) from 40% of glucose and 8.8%(v/v) from 20% of sucrose.

• Journal of Plant Biology
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• v.38 no.4
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• pp.349-357
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• 1995

The alkaline invertase ($\beta$-D-fructofuranoside fructohydrolase, EC 3.2.1.26) was isolated and characterized from the hypocotyls of mung bean (Phaseolus radiatus L.). The enzyme was purified by consecutive step using diethylaminoethyl (DEAE)-cellulose anion exchange, 1st Sephadex G-200, DEAE-Sephadex A50 and 2nd Sephadex G-200 chromatography. The overall purification was about 77-fold with a yield of about 6%. The finally purified enzyme exhibited a specific activity of about 48 $\mu$mol of glucose produced mg-1 protein min-1 at pH 7.0 and appeared to be a single protein by nondenaturing polyacrylamide gel electrophoresis (PAGE). The enzyme had the native molecular weight of 450 kD and subunits molecular weight of 63 kD and 38 kD as estimated by Sephadex G-200 chromatography and SDS-PAGE, respectively, suggesting that the enzyme is a heteromultimeric protein composed of two types of subunits. On the other hand, the enzyme appeared to be not a glycoprotein according to the results of Con A chromatography and glycoprotein staining. The enzyme had a Km for sucrose of 19.7 mM at pH 7.0 and maximum activity around pH 7.5. The enzyme was most active with sucrose as substrate, compared to raffinose, cellobiose, maltose and lactose. These results indicate the alkaline invertase is a $\beta$-fructofuranosidase.

• Journal of Plant Biology
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• v.38 no.3
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• pp.251-258
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• 1995

The soluble acid invertase ($\beta$-D-fructofuranoside fructohydrolase, EC 3.2.1.26) was isolated and characterized from the hypocotyls of mung bean (Phaseolus radiatus L.). The enzyme was purified to apparent homogeneity by consecutive step using diethylaminoethyl (DEAE)-cellulose anion exchange, Concanavalin (Con) A affinity and Sephacryl S-300 chromatography. The overall purification was about 148-fold with a yield of about 15%. The finally purified enzyme exhibited a specific activity of about 139 $\mu$mol of glucose produced mg-1 protein min-1 at pH 5.0 and appeared to be a single protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and nondenaturing PAGE. The enzyme had the native molecular weight of 70 kD and subunit molecular weight of 70 kD as estimated by Sephadex G-200 chromatography and SDS-PAGE, respectively, suggesting that the enzyme was composed of a monomeric protein. On the other hand, the enzyme appeared to be a glycoprotein containing N-linked high mannose oligosaccharide chain on the basis of its ability to bind to the immobilized C on A. The enzyme had a Km for sucrose of 1.8 mM at pH 5.0 and maximum activity around pH 5.0. The enzyme showed highest enzyme activity with sucrose as substrate, but the activity was slightly measured with raffinose and cellobise. No activity was measured with maltose and lactose. These results indicate the soluble acid invertase is a $\beta$-fructofuranosidase.

• Molecules and Cells
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• v.26 no.3
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• pp.236-242
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• 2008

Invertase (${\beta}$-D-fructofuranosidase; EC 3.2.1.26) catalyzes the conversion of sucrose into glucose and fructose and is involved in an array of important processes, including phloem unloading, carbon partitioning, the response to pathogens, and the control of cell differentiation and development. Its importance may have caused the invertases to evolve into a multigene family whose members are regulated by a variety of different mechanisms, such as pH, sucrose levels, and inhibitor proteins. Although putative invertase inhibitors in the Arabidopsis genome are easy to locate, few studies have been conducted to elucidate their individual functions in vivo in plant growth and development because of their high redundancy. In this study we assessed the functional role of the putative invertase inhibitors in Arabidopsis by generating transgenic plants harboring a putative invertase inhibitor gene under the control of the CaMV35S promoter. A transgenic plant that expressed high levels of the putative invertase inhibitor transcript when grown under normal conditions was chosen for the current study. To our surprise, the stability of the invertase inhibitor transcripts was shown to be down-regulated by the phytohormone ABA (abscisic acid). It is well established that ABA enhances invertase activity in vivo but the underlying mechanisms are still poorly understood. Our results thus suggest that one way ABA regulates invertase activity is by down-regulating its inhibitor.