Conformation of (2→1)-β-d-fructan in aqueous solution

The conformation and dilute solution properties of (2→1)-β-d-fructan in aqueous solution were studied by gel permeation chromatography, low-angle laser light-scattering photometry, viscometry, small-angle X-ray scattering and electron microscopy. Fractions covering a broad range of weight-average molecular weights (M_w) from 1.49 × 10⁴ to 5.29 × 10⁶ were obtained from a native sample by ultrasonic degradation and fractional precipitation. For M_w < 4 × 10⁴, the intrinsic viscosity [η] varies with M_w^0.71, indicating that the fructan chain behaves as a random coil expanded by an excluded-volume effect in this molecular weight region. For M_w > 10⁵, [η] exhibits an unusually weak dependence on M_w and finally becomes almost independent of molecular weight. This behaviour is interpreted in terms of a globular conformation of the high-molecular-weight fructan molecules. Small-angle X-ray-scattering measurements and electron microscopic observations support this interpretation of the values of [η] observed.     

Molecular mass and antitumor activities of sulfated derivatives of α-glucan from Poria cocos mycelia

Two kinds of water-insoluble (1 → 3)-α-d-glucan samples, ab-PCM3-I and ac-PCM3-I, isolated from different Poria cocos mycelia were sulfated, to produce two series of water-soluble derivatives ab-PCM3-I-S1–S5 and ac-PCM3-I-S1–S5, respectively. The derivatives having different weight-average molecular mass (M_w) were produced by changing reaction temperature and time as well as molar ratios between chlorosulfonic acid and number of hydroxyl groups in the glucan. The degrees of substitution (DS) of the sulfated derivatives were analyzed by elemental analysis (EA) to be 0.39–0.67 for ab-PCM3-I-S and 0.73–0.96 for ac-PCM3-I-S, respectively. The M_w and the intrinsic viscosity ([η]) of the samples ab-PCM3-I-S and the ac-PCM3-I-S were measured by size exclusion chromatography combined with laser light scattering (SEC–LLS) and viscometry in phosphate buffer solution (PBS) at 37 °C. The results indicated that their M_w ranged from 2.0 to 11.3 × 10⁴ for the samples ab-PCM3-I-S, and 4.7 to 40.0 × 10⁴ for the samples ac-PCM3-I-S. Moreover, the antitumor activities of the sulfated derivatives ab-PCM3-I-S and ac-PCM3-I-S against Sarcoma 180 tumor cell tested both in vitro and in vivo are significantly higher than those of the native α-d-glucans. Therefore, a moderate range of molecular mass from 2.0 × 10⁴ to 40.0 × 10⁴, relatively high chain stiffness and good water solubility of the sulfated derivatives are beneficial to the enhancement of their antitumor activities.     

Molecular mass and chain conformations of Rhizoma Panacis Japonici polysaccharides

The molecular mass and size of five water-soluble polysaccharides isolated from Rhizoma Panacis Japonici (RPJ) were determined with laser light scattering (LLS), size-exclusion chromatography (SEC) combined with LLS (SEC–LLS), dynamic light scattering (DLS), as well as transmission electron microscope (TEM). Their weight-average molecular masses (M_w) were 3.5 × 10⁴, 1.47 × 10⁵, 1.24 × 10⁶, 9.26 × 10⁵ and 1.36 × 10⁶, radii of gyration (<s²>_z^1/2) were 14.7, 31.7, 50.8, 41.8 and 40.4 nm, and hydrodynamic radii (R_h) were 19.9, 37.5, 66.2, 52.1, and 55.2 nm, respectively. The results showed that molecular masses and sizes of the polysaccharides were influenced by the pH and temperature of the extraction mediums. The conformation parameters were calculated from the above data according to polymer solution theory. The values of ρ (= <s²>_z^1/2/R_h) were from 0.7 to 0.8, exponents (ν) of <s²>_z^1/2 = k M_w^ν were from 0.31 to 0.43, and fractal dimension (d_f) were from 2.3 to 3.2, respectively. The results revealed that all of the polysaccharides existed as spheres in 0.15 M NaCl aqueous solution. TEM and atomic force microscope (AFM) further confirmed the spherical morphologies of these molecules. The spherical conformations of the polysaccharides were a result of their highly branched structures.     

Properties of a novel β-glucosidase from Fusarium proliferatum ECU2042 that converts ginsenoside Rg3 into Rh2

A novel β-glucosidase from Fusarium proliferatum ECU2042 (FPG) was successfully purified to homogeneity with a 506-fold increase in specific activity. The molecular mass of the native purified enzyme (FPG) was estimated to be approximately 78.7 kDa, with two homogeneous subunits of 39.1 kDa, and the pI of this enzyme was 4.4, as measured by two-dimensional electrophoresis. The optimal activities of FPG occurred at pH 5.0 and 50 °C, respectively. The enzyme was stable at pH 4.0–6.5 and temperatures below 60 °C, and the deactivation energy (E_d) for FPG was 88.6 kJ mo1⁻¹. Moreover, it was interesting to find that although the purified enzyme exhibited a very low activity towards p-nitrophenyl β-d-glucoside (pNPG), and almost no activity towards cellobiose, a relatively high activity was observed on ginsenoside Rg₃. The enzyme hydrolyzed the 3-C, β-(1 → 2)-glucoside of ginsenoside Rg₃ to produce ginsenoside Rh₂, but did not sequentially hydrolyze the β-d-glucosidic bond of Rh₂. The K_m and V_max values of FPG for ginsenoside Rg₃ were 2.37 mM and 0.568 μmol (h mg protein)⁻¹, respectively. In addition, this enzyme also exhibited significant activities towards various alkyl glucosides, aryl glucosides and several natural glycosides.     

Characterization and anti-tumor activities of sulfated polysaccharide SRBPS2a obtained from defatted rice bran

A novel chemically sulfated polysaccharide SRBPS2a with potent anti-tumor activity was derived from defatted rice bran by chlorosulfonic acid–pyridine (CSA–Pyr) method. The average molecular weight of SRBPS2a was 3.5 × 10⁵ Da and the degree of sulfation (DS) was 1.29. The Fourier-transform infrared spectra (FT-IR) and ¹³C NMR spectroscopy analysis revealed that SRBPS2a was mainly consist of β-(1 → 3)-d-galactopyranosyl residues, the sulfate substitution site was on C-2 and C-4 while the side chains were cut off during the sulfated reaction. Furthermore, SRBPS2a exhibited evident growth inhibition on mouse mammary tumor EMT-6 cells both in vitro and in vivo.     

Rapid method to determine the molecular weight of dextrins and dextrans

A rapid method was developed to determine the molecular weight (M_n) of β-limit dextrin and dextrans (Leuconostoc mesenteroides) using a reducing power approach. The M_n of the β-limit dextrin was also estimated from high performance liquid chromatography (HPLC). Chromatograms were pre-calibrated with the dextrans. The three dextrins had a M_n of 2.09, 2.40 and 2.63 × 10⁵ using the reducing method and 4.80, 5.90 and 2.80 × 10⁵ by HPLC. The method could be employed to estimate M_n of dextrins where chromatographic systems were not available.     

Global conformation analysis of irradiated xyloglucans

Xyloglucan isolated and purified from tamarind seed was subjected to various degrees of γ-irradiation treatments, from 10 to 70 kGy, monitored for radiation damage and then studied using a new combined hydrodynamic approach with regards to conformation and flexibility. Radiation products were analysed with regard to molecular weight (weight average) M_w from size exclusion chromatography coupled to multi-angle laser light scattering (SEC–MALLs), intrinsic viscosity [η] and sedimentation coefficient s^o_20,w. Sedimentation coefficient distributions and elution profiles from SEC–MALLs confirmed the unimodal nature of the molecular weight distribution for each sample in solution. The chain flexibility was then investigated in terms of the persistence length, L_p of the equivalent worm-like chain model. The traditional Bushin–Bohdanecky (intrinsic viscosity) and Yamakawa–Fujii (sedimentation coefficient) relations were used separately then combined together by minimisation of a target function according to a recently published procedure [Ortega, A., & García de la Torre, J. (2007). Equivalent radii and ratios of radii from solution properties as indicators of macromolecular conformation, shape, and flexibility. Biomacromolecules, 8, 2464–2475 [see also Ortega, A. Metodologías computacionales para propiedades en disolución de macromoléculas rígidas y flexibles. Ph.D. Dissertation, Universidad de Murcia, 2005]] and yielded an estimate for L_p in the range 4–9 nm using floated and fixed mass per unit length analysis protocols and “point” global analysis: irradiated xyloglucans behave as flexible structures in common with pressure/heat treated materials.     

Quantification of hydrogen peroxide and glucose using 3-methyl-2-benzothiazolinonehydrazone hydrochloride with 10,11-dihydro-5H-benz(b,f)azepine as chromogenic probe

A sensitive, selective, and rapid enzymatic method is proposed for the quantification of hydrogen peroxide (H₂O₂) using 3-methyl-2-benzothiazolinonehydrazone hydrochloride (MBTH) and 10,11-dihydro-5H-benz(b,f)azepine (DBZ) as chromogenic cosubstrates catalyzed by horseradish peroxidase (HRP) enzyme. MBTH traps free radical released during oxidation of H₂O₂ by HRP and gets oxidized to electrophilic cation, which couples with DBZ to give an intense blue-colored product with maximum absorbance at 620 nm. The linear response for H₂O₂ is found between 5 × 10⁻⁶ and 45 × 10⁻⁶ mol L⁻¹ at pH 4.0 and a temperature of 25 °C. Catalytic efficiency and catalytic power of the commercial peroxidase were found to be 0.415 × 10⁶ M⁻¹ min⁻¹ and 9.81 × 10⁻⁴ min⁻¹, respectively. The catalytic constant (k_cat) and specificity constant (k_cat/K_m) at saturated concentration of the cosubstrates were 163.2 min⁻¹ and 4.156 × 10⁶ L mol⁻¹ min⁻¹, respectively. This method can be incorporated into biochemical analysis where H₂O₂ undergoes catalytic oxidation by oxidase. Its applicability in the biological samples was tested for glucose quantification in human serum.     

Methyllycaconitine: a selective probe for neuronal α-bungarotoxin binding sites

The ability of methyllycaconitine (MLA) to inhibit the binding of [¹²⁵I]α-bungarotoxin to rat brain membranes, frog and human muscle extracts and the human muscle cell line TE671 has been measured. MLA showed a markedly higher affinity for the rat brain site (K_i 1.4 × 10⁻⁹ M) than for the muscle receptors (K_i; 10⁻⁵-10⁻⁶ M). Structure modelling techniques were used to fit the structure of MLA to a nicotinic pharmacophore model. MLA is the first low molecular weight ligand to be shown to discriminate between muscle nicotinic receptors and their α-bungarotoxinbinding counterpart in the brain, and as such may be a useful structural probe for pursuing the structural and functional properties of the neuronal protein.     

Chain conformation of carboxymethylated derivatives of (1 → 3)-β-d-glucan from Poria cocos sclerotium

A water-insoluble (1 → 3)-β-d-glucan (PCSG) isolated from the fresh sclerotium of Poria cocos was carboxymethylated to afford a water-soluble derivative coded as C-PCSG. The carboxymethylated (1 → 3)-β-d-glucan was fractionated to obtain eight fractions according to the nonsolvent addition method. The weight-average molecular mass (M_w), radius of gyration and intrinsic viscosity ([η]) of the fractions were determined by size-exclusion chromatography combined with laser light scattering (SEC-LLS) and viscometry in 0.2 M NaCl aqueous solution at 25 °C. The dependences of [η] and on M_w for C-PCSG were found to be , and (nm), respectively. Analysis of M_w and [η] in terms of the known theories for wormlike chain model yielded 633 nm⁻¹ for molar mass per unit contour length (M_L), 5.5 nm for persistence length (q), and 20.2 for characteristic ratio (C_∞). These results indicated that C-PCSG exists as a relatively extended flexible chain in 0.2 M NaCl aqueous solution. Therefore, the introduction of the carboxymethyl groups into the β-glucan improved significantly the water solubility and enhanced the stiffness of the chains.     

Physicochemical properties and antitumor activities for sulfated derivatives of lentinan

Five fractions of lentinan, a β-(1→3)-d-glucan bearing β-(1→6)-d-glucopyranosyl branches, were treated with chlorosulfonic acid for 90 min at 60 °C in pyridine medium to synthesize water-soluble sulfated derivatives having the substitution degree of 1.44–1.76. The ¹³C NMR spectra of the sulfated β-glucans indicated that the C-6 position was preferentially substituted by the sulfate groups. The values of the weight-average molecular weight (M_w), radius of gyration (), and intrinsic viscosity ([η]) of the sulfated lentinan fractions were determined by size-exclusion chromatography with multi-angle laser light scattering (SEC–MALLS) and viscometry in 0.15 M aq NaCl at 25 °C, respectively. The dependence of [η] on M_w for the sulfated lentinan was found to be [η] = 8.93 × 10⁻³ (mL/g) in 0.15 M aq NaCl (for M_w ranging from 14.6 × 10⁴ to 50.4 × 10⁴). On the basis of the Yamakawa–Fujii–Yoshizaki (YFY) theory, the conformational parameters of the sulfated lentinan were calculated as 950 nm⁻¹ for the molar mass per unit contour length (M_L), 4.8 nm for the persistence length (q), and 13.9 for the characteristic ratio (C_∞), indicating relatively extended single flexible chains in solution. The sulfated glucan fractions exhibited in vitro antiproliferative activities against sarcoma 180 (S-180) cells, and their inhibition ratios were lower than that of the triple-helix lentinan, but higher than that for the one with single random-coil lentinan chains.     

Mutagenicity of bisphenol A and its suppression by interferon-α in human RSa cells

Bisphenol A is used as a monomer in the production of polycarbonate plastic products. The widespread use of bisphenol A has raised concerns about its effects in humans. Since there is little information on the mutagenic potential of the chemical, the mutagenicity of bisphenol A was tested using human RSa cells, which has been utilized for identification of novel mutagens. In genomic DNA from cells treated with bisphenol A at concentrations ranging from 1×10⁻⁷ to 1×10⁻⁵ M, base substitution mutations at K-ras codon 12 were detected using PCR and differential dot-blot hybridization with mutant probes. Mutations were also detected using the method of peptide nucleic acid (PNA)-mediated PCR clamping. The latter method enabled us to detect the mutation in bisphenol A-treated cells at a dose (1×10⁻⁸ M) equivalent to that typically found in the environment. Induction of ouabain-resistant (Oua^R) phenotypic mutation was also found in cells treated with 1×10⁻⁷ and 1×10⁻⁵ M of bisphenol A. The induction of K-ras codon 12 mutations and Oua^R mutations was suppressed by pretreating RSa cells with human interferon (HuIFN)-α prior to bisphenol A treatment. The cells treated with bisphenol A at the concentration of 1×10⁻⁶ M elicited unscheduled DNA synthesis (UDS). These findings suggested that bisphenol A has mutagenicity in RSa cells as well as mutagens that have been tested in these cells, and furthermore, that a combination of the PNA-mediated PCR clamping method with the human RSa cell line may be used as an assay system for screening the mutagenic chemicals at very low doses.     

Gene cloning, purification, and characterization of α-methylserine aldolase from Bosea sp. AJ110407 and its applicability for the enzymatic synthesis of α-methyl-l-serine and α-ethyl-l-serine

The gene encoding α-methylserine aldolase was isolated from Bosea sp. AJ110407. Sequence analysis revealed that the predicted amino acid sequence encoded by the 1320-bp open reading frame was 65.0% similar to the corresponding sequence of the enzyme isolated from Ralstonia sp. AJ110405. The gene was expressed in Escherichia coli, and the recombinant enzyme was purified. Gel filtration revealed the molecular mass of the purified enzyme to be approximately 78 kDa, suggesting that the enzyme is a homodimer. The enzyme exhibited a specific peak at 429 nm in the spectrum and contained 1 mol pyridoxal 5′-phosphate per mole of the subunit. The V_max value was 1.40 μmol min⁻¹ mg⁻¹, and the K_m value was 1.5 mM for the reaction wherein formaldehyde was released from α-methyl-l-serine. This enzyme could also catalyze the reverse reaction, i.e., the synthesis of α-methyl-l-serine from l-alanine and formaldehyde. This activity was inhibited in the excess of formaldehyde; however, α-methyl-l-serine was efficiently produced from l-alanine in the presence of formaldehyde. This method was also applicable for producing α-ethyl-l-serine from l-2-aminobutyric acid.     

Expression of recombinant human tumor necrosis factor-α in a baculovirus expression system

A cDNA fragment coding human tumor necrosis factor-alpha (TNF-α) was inserted into the vector pSXIVVI⁺X3 with the control of Syn XIV promoter. The Sf9 cells (Spodoptera frugiperda) were co-transfected with the recombinant plasmid and TnNPV DNA (Trichoplusia ni nuclear polyhedrosis virus DNA). Cells infected with recombinant virus synthesized TNF-α protein at a level of about 38% of total cellular protein. TNF-α activity in infected cells was measured by L929 cytotoxic assay, the highest expression level, 1.5 × 10⁴ U/10⁶ cells, was obtained at 76 h after infection. Western blot analysis of protein extracts from infected larvae showed that the virus-mediated TNF-α had immunoreactivity.     

Effect of dietary chitosans on trace iron, copper and zinc in mice

Dietary chitosans with different molecular weight M_w and the degree of deacetylation DDA (high molecular weight chitosan HCS with M_w 7.60 × 10⁵ and DDA 85.5%, middle molecular weight chitosan MCS with M_w 3.27 × 10⁴ and DDA 85.2%, chito-oligomer COS with M_w 0.99 × 10³ and DDA 85.7% and water-soluble chitosan WSC with M_w 3.91 × 10⁴ and DDA 52.6%) were used at the 1.05% level to feed mice for 90 days. Afterwards no pathological symptoms, clinical signs or deaths were observed. The body weight of mice in chitosan group and control group showed no significant difference. Although HCS, COS and WSC had no significant effect on the level of Fe, Zn and Cu in the tested mice’s liver, spleen, heart and kidney, MCS significantly increased the level of Fe, Zn and Cu in liver. Therefore dietary ingestion of chitosan did not depress the level of Fe, Zn and Cu in mice.     

NMR spectroscopy of α-crystallin. Insights into the structure, interactions and chaperone action of small heat-shock proteins

The subunit molecular mass of α-crystallin, like many small heat-shock proteins (sHsps), is around 20 kDa although the protein exists as a large aggregate of average mass around 800 kDa. Despite this large size, a well-resolved ¹H NMR spectrum is observed for α-crystallin which arises from short, polar, highly-flexible and solvent-exposed C-terminal extensions in each of the subunits, αA- and αB-crystallin. These extensions are not involved in interactions with other proteins (e.g. β- and γ-crystallins) under non-chaperone conditions. As determined by NMR studies on mutants of αA-crystallin with alterations in its C-terminal extension, the extensions have an important role in acting as solubilising agents for the relatively-hydrophobic α-crystallin molecule and the high-molecular-weight (HMW) complex that forms during the chaperone action. The related sHsp, Hsp25, also exhibits a flexible C-terminal extension. Under chaperone conditions, and in the HMW complex isolated from old lenses, the C-terminal extension of the αA-crystallin subunit maintains its flexibility whereas the αB-crystallin subunit loses, at least partially, its flexibility, implying that it is involved in interaction with the ‘substrate’ protein. The conformation of ‘substrate’ proteins when they interact with α-crystallin has been probed by ¹H NMR spectroscopy and it is concluded that α-crystallin interacts with ‘substrate’ proteins that are in a disordered molten globule state, but only when this state is on its way to large-scale aggregation and precipitation. By monitoring the ¹H and ³¹P NMR spectra of α-crystallin in the presence of increasing concentations of urea, it is proposed that α-crystallin adopts a two-domain structure with the larger C-terminal domain unfolding first in the presence of denaturant. All these data have been combined into a model for the quaternary structure of α-crystallin. The model has two layers each of approximately 40 subunits arranged in an annulus or toroid. A large central cavity is present whose entrance is ringed by the flexible C-terminal extensions. A large hydrophobic region in the aggregate is exposed to solution and is available for interaction with ‘substrate’ proteins during the chaperone action.     

Characterization of a root-specific β-thioglucoside glucohydrolase gene in Carica papaya and its recombinant protein expressed in Pichia pastoris

Plant β-thioglucoside glucohydrolases (TGGs or myrosinases) are a young class of enzymes in the glycosyl hydrolase family 1 and have a narrow distribution. TGG genes have mainly been cloned from crucifers, while TGGs in other species have received little attention. The TGG gene CpTGG2 and its recombinant protein from papaya were characterized in this paper. This is the first plant TGG gene without unusual intron splicing borders, as present in all other available TGG genes. Phylogenetic analysis indicated that plant myrosinases are divided into two major lineages. CpTGG2 is located in the lineage constituted by AtTGG4–6 from Arabidopsis thaliana, while the rest of myrosinases (including MA, MB and MC subfamilies) are grouped into another lineage. RT-PCR analysis indicated that CpTGG2 was specifically expressed in the root. The recombinant CpTGG2 expressed in yeast had a subunit mass of 70 kDa, and had low basal TGG activity without addition of ascorbate. Low concentrations of ascorbate stimulated CpTGG2 activity, while high concentrations were inhibitory. CpTGG2 was active in broad pH and temperature ranges, similar to AtTGG4 and AtTGG5. The apparent K_m and V_max were 2.24 mM and 24.3 μmol min⁻¹ mg⁻¹ when sinigrin was the substrate. The calculated k_cat/K_m value was 1.3 × 10⁴ S⁻¹ M⁻¹_. Our results reshaped and expanded the myrosinase family structure and provided clues to the evolution of myrosinase genes.     

Characterization of galactomannans isolated from legume endosperms of Caesalpinioideae and Faboideae subfamilies by multidetection aqueous SEC

Galactomannans isolated from legume seed endosperms, including those of commercial interest, have been characterized by multidetection aqueous SEC. Galactomannans derived from seeds of the Faboideae subfamily had substantially higher M_w than those from Caesalpinioideae seeds (M_w,Fab = 2.4–3.1 × 10⁶ g/mol, M_w,Caes. = 0.86–2.1 × 10⁶ g/mol) and within the latter botanical subfamily, an apparent correlation between M_w and the degree of galactose substitution DG was found. The molar mass distributions were unimodal and differed primarily by a scale factor, with distributional widths narrower than a true Flory ‘most-probable distribution’; good fits to Schulz–Zimm model were obtained. Across subfamilies no differences were found in the exponents of [η]–M and R_v–M relationships (0.61 ± 0.02, 0.54 ± 0.01, respectively), the Flory chain stiffness ratio (C_∞ = 20 ± 1 (BSF analysis)), or the persistence length (L_p = 5.5 ± 0.2 nm) obtained from SEC fraction data. However, it was found that prefactors in the [η]–M and R_v–M relationships as well as the unperturbed parameter K_Θ decrease in proportion to DG and therefore chain density. Generalized relationships incorporating galactose-dependent prefactors were therefore developed to model SEC fraction data of native galactomannans ([η]_GM = (1800 ± 200) × M_o^−1.61 × M^0.61±0.02, R_v,GM = 0.63 ± 0.05 × M_o^−0.54 × M^0.54±0.01) as well as lower-M fractions obtained by ultrasonication ([η]_GM = (730 ± 100) × M_o^−1.71 × M_w^0.71±0.02, R_v,GM = 0.49 ± 0.05 × M_o^−0.57 × M_w^0.57±0.01, M ≈ 1 × 10⁵-native). As a consequence of this dependence and the observed patterns in molar mass variation, [η] varies within a narrow range for galactomannans as a whole despite substantial M_w differences.     

In vitro antioxidant activities of a water-soluble polysaccharide derived from Dendrobium nobile Lindl. extracts

A water-soluble polysaccharide (DNP), isolated from the aqueous extracts of the stem of Dendrobium nobile Lindl., was found to have an average molecular weight (Mw) of about 8.76 × 10⁴ Da. Monosaccharides analysis revealed that DNP was composed of rhamnose, arabinose, xylose, mannose, glucose and galactose in a molar ratio of 1.00:2.80:2.20:30.76:117.96:31.76. The evaluation of antioxidant activity in vitro revealed that DNP is a novel potential antioxidant. The NMR spectra suggested that the main structure of DNP was possible as     

Significance of metal ion supplementation in the fermentation medium on the structure and anti-tumor activity of Tuber polysaccharides produced by submerged culture of Tuber melanosporum

The significance of metal ion supplementation in the fermentation medium on the structure and anti-tumor activity of Tuber polysaccharides was systematically studied in the submerged fermentation of Tuber melanosporum. The lowest weight-average molecular weight (M_w) (i.e., 115.3 × 10⁴ g/mol) of intracellular polysaccharides (IPS) was obtained when Mg²⁺ and K⁺ was added in the fermentation medium. The IPS with the lower M_w exhibited a higher inhibition ratio against S-180 tumor cells. The compact conformation of extracellular polysaccharides (EPS) was formed when only K⁺ was supplied in the fermentation medium. Interestingly, EPS with compact conformation exhibited a higher inhibition ratio (i.e., 59.2%) than EPS with branched polymer chain (i.e., 9.2%) against A549 tumor cells. The highest inhibition ratio for EPS with α-glycosidic linkages against the tumor cell line HepG2 reached 32.2% when Mg²⁺ or K⁺ was supplied in the fermentation medium. The addition of metal ion Mg²⁺, K⁺, and their combination to the fermentation medium is a vital factor affecting the structures of Tuber polysaccharides, which further determine their anti-tumor activities. The information obtained in this work will be useful for the efficient and directed production of polysaccharides with anti-tumor activities by the submerged fermentation of edible fungi mycelium.