Optimization of a chemically defined medium for mycelial growth and polysaccharide production by medicinal mushroom <Emphasis Type="Italic">Phellinus igniarius</Emphasis>

A chemically defined medium for mycelial growth and exopolysaccharide (EPS) production by submerged culture of Phellinus igniarius was investigated. The mainly defined medium compositions were optimized by using orthogonal matrix method. The optimal defined medium (per liter) was 40.0 g glucose, 4.0 g. glutamic acid, 4.0 g (NH₄)₂SO₄, and initial pH 6.0. Under the optimal medium, the maximal mycelial biomass and EPS production were 12.33 ± 0.89 and 1.21 ± 0.08 g l⁻¹ at 192 h in shake flask, while the maximal mycelial biomass and EPS production reached 13.86 ± 0.52 and 1.92 ± 0.07 g l⁻¹ at 168 h in 3 l fermenter, respectively. The molecular weights (g mol⁻¹) of four fractions isolated from EPS by gel permeation were about 6.4 × 10⁶, 3.3 × 10⁵, 2.7 × 10⁵ and 2.9 × 10³. This study should be widely applied to other secondary metabolites production from higher fungus in a chemically defined medium and quantitative regulation of the metabolic flux in polysaccharide biosynthesis.     

Structural investigation of a polysaccharide released by the cyanobacterium <Emphasis Type="Italic">Nostoc insulare</Emphasis>

The structural investigation of an extracellular polysaccharide released during photoautotrophic growth by the cyanobacterium Nostoc insulare is reported. After 60 days of cultivation, an average yield of purified, desalted, and freeze-dried released polysaccharide (RPS) of 0.9 g L⁻¹ medium was obtained. The apparent hydrodynamic volume, determined for RPS, was 1.1 × 10⁶ Da, and the average molecular weight was 2.8 × 10⁶ Da. No sulfate and only traces of pyruvate and acetate groups were detectable. A protein content of only 0.7% indicates a high degree of purity of RPS. The following constituent uronic acids and sugars were identified: glucuronic acid (GlcA), glucose (Glc), arabinose (Ara), and for the first time, cyanobacterial RPSs 3-O-methyl-arabinose (3-O-Methyl-Ara). Adapted from linkage analyses of untreated RPS and of RPS treated by means of reduction of uronic acids, mild acid hydrolysis with oxalic acid, or lithium degradation, respectively, the following partial structure of RPS is proposed, which possesses an arborisation built by 1,3,4-Glcp and a side chain built by 3-O-Methyl-Araf: →1)-Glcp-(3→1)-Glcp-[(3→1)-3-O-Methyl-Araf](4→1)-GlcAp-(4→).     

Exopolysaccharide production of <Emphasis Type="Italic">Lactobacillus salivarius</Emphasis> BCRC 14759 and <Emphasis Type="Italic">Bifidobacterium bifidum</Emphasis> BCRC 14615

In the present study, the production of exopolysaccharides (EPS) by 13 strains of Lactobacillus and 6 strains of Bifidobacterium in a chemical defined medium (CDM) supplemented with 30 g lactose/l was first compared. The highest EPS production of the Lactobacillus strains was found in L. salivarius BCRC 14759 while among the Bifidobacterium strains examined, B. bifidum BCRC 14615 showed the highest EPS production. Analyzes of the effect of lactose concentration and cultivation temperature on EPS production revealed that L. salivarius produced the highest amount of EPS (45.3 mg/l) in CDM supplemented with 5 g lactose/l at 40°C while B. bifidum produced the highest EPS (17.0 mg/l) in CDM supplemented with 40 g lactose/l at 35°C. α-Phosphoglucomutase, UDP-glucose pyrophosphorylase and UDP-galactose-4-epimerase exhibited a markedly notable activity compared with other enzymes examined in the cell extract of both test organisms. This indicates their possible involvement in the biosynthesis of EPS.     

Di-<Emphasis Type="Italic">tert</Emphasis>-butylsilylene-directed α-selective synthesis of <Emphasis Type="Italic">p</Emphasis>-nitrophenyl T-antigen analogues

Seven analogues of p-nitrophenyl T-antigen [Galβ(1→3)GalNAcα(1→O)PNP] have been synthesized as potential substrates for elucidation of the substrate specificity of endo-α-N-acetylgalactosaminidase. These compounds, which are commercially unavailable, include: GlcNAcβ(1→3){GlcNAcβ(1→6)}GalNAcα(1→O)PNP [core 4 type], GalNAcα(1→3)GalNAcα(1→O)PNP [core 5 type], GlcNAcβ(1→6)GalNAcα(1→O)PNP [core 6 type], GalNAcα(1→6)GalNAcα(1→O)PNP [core 7 type], Galα(1→3)GalNAcα(1→O)PNP [core 8 type], Glcβ(1→3)GalNAcα(1→O)PNP and GalNAcβ(1→3)GalNAcα(1→O)PNP. The assembly of these synthetic probes was accomplished efficiently, based on di-tert-butylsilylene(DTBS)-directed α-galactosylation as a key reaction.     

Prospects of using marine actinobacteria as probiotics in aquaculture

In the present study, optimum culture conditions for the production of extracellular polysaccharides (EPS) in submerged culture of an edible mushroom, Laetiporus sulphureus var. miniatus and their stimulatory effects on insulinoma cell (RINm5F) proliferation and insulin secretion were investigated. The maximum mycelial growth (4.1 g l⁻¹) and EPS production (0.6 g l⁻¹) in submerged flask culture were achieved in a medium containing 30 g l⁻¹ maltose, 2 g l⁻¹ soy peptone, and 2 mM MnSO₄·5H₂O at an initial pH 2.0 and temperature 25°C. In the stirred-tank fermenter under optimized medium, the concentrations of mycelial biomass and EPS reached a maximum level of 8.1 and 3.9 g l⁻¹, respectively. Interestingly, supplementation of deep sea water (DSW) into the culture medium significantly increased both mycelial biomass and EPS production by 4- and 6.7-fold at 70% (v/v) DSW medium, respectively. The EPS were proved to be glucose-rich polysaccharides and were able to increase proliferation and insulin secretary function of rat insulinoma RINm5F cells, in a dose-dependent manner. In addition, EPS also strikingly reduced the streptozotocin-induced apoptosis in RINm5F cells indicating the mode of the cytoprotective role of EPS on RINm5F cells.     

Comparative analysis of four <Emphasis Type="Italic">β</Emphasis>-galactosidases from <Emphasis Type="Italic">Bifidobacterium bifidum</Emphasis> NCIMB41171: purification and biochemical characterisation

Four different β-galactosidases (previously named BbgI, BbgII, BbgIII and BbgIV) from Bifidobacterium bifidum NCIMB41171 were overexpressed in Escherichia coli, purified to homogeneity and their biochemical properties and substrate preferences comparatively analysed. BbgI was forming a hexameric protein complex of 875 kDa, whereas BbgII, BbgIII and BbgIV were dimers with native molecular masses of 178, 351 and 248 kDa, respectively. BbgII was the only enzyme that preferred acidic conditions for optimal activity (pH 5.4–5.8), whereas the other three exhibited optima in more neutral pH ranges (pH 6.4–6.8). Na⁺ and/or K⁺ ions were prerequisite for BbgI and BbgIV activity in Bis–Tris-buffered solutions, whereas Mg⁺⁺ was strongly activating them in phosphate-buffered solutions. BbgII and BbgIII were slightly influenced from the presence or absence of cations, with Mg⁺⁺, Mn⁺⁺ and Ca⁺⁺ ions exerting the most positive effect. Determination of the specificity constants (k _cat/K _m) clearly indicated that BbgI (6.11 × 10⁴ s⁻¹ M⁻¹), BbgIII (2.36 × 10⁴ s⁻¹ M⁻¹) and especially BbgIV (4.01 × 10⁵ s⁻¹ M⁻¹) are highly specialised in the hydrolysis of lactose, whereas BbgII is more specific for β-d-(1→6) galactobiose (5.59 × 10⁴ s⁻¹ M⁻¹) than lactose (1.48 × 10³ s⁻¹ M⁻¹). Activity measurements towards other substrates (e.g. β-d-(1→6) galactobiose, β-d-(1→4) galactobiose, β-d-(1→4) galactosyllactose, N-acetyllactosamine, etc.) indicated that the β-galactosidases were complementary to each other by hydrolysing different substrates and thus contributing in a different way to the bacterial physiology.     

Gene analysis,optimized production and property of marine lipase from <Emphasis Type="Italic">Bacillus pumilus</Emphasis> B106 associated with South China Sea sponge <Emphasis Type="Italic">Halichondria rugosa</Emphasis>

Gene cloning, optimized production and property of marine lipase from Bacillus pumilus B106 associated with South China Sea sponge Halichondria rugosa were investigated in this paper. A lipase gene with whole ORF encoding 215 amino acids was obtained by PCR, protein domain prediction suggested that the deduced lipase belongs to α/β hydrolases family. Based on single factor Seriatim-Factorial test and Plackett–Burman experimental design, the optimal medium consisted of (per l) 12.5 ml maize oil, 5.0 g beef extract, 2.0 g PO₄ ³⁻ (0.6 g KH₂PO₄, 1.4 g K₂HPO₄), 17.15 g Mg²⁺, 5.0 g yeast extract, 2.282 g CaCl₂ and 5.0 ml Tween80 with artificial sea water. Using this optimum medium, lipase activity and cell concentration were increased by 3.54- and 1.31-fold over that of the basal medium, respectively. This lipase showed tolerance to high salinity, pH and temperature. About 10–20% methanol exhibited a stimulatory effect on the lipase activity, while activity was inhibited by 30–40% methanol, 2-propanol, DMSO, and ethanol. This study provides a valuable resource for marine lipase production and extends our understanding of the possible role of sponge-associated bacteria in the biotransformation of chemical compounds for the sponge host.     

Structural analysis of the exopolysaccharide produced by <Emphasis Type="Italic">Streptococcus thermophilus</Emphasis> ST1 solely by NMR spectroscopy

The use of lactic acid bacteria in fermentation of milk results in favorable physical and rheological properties due to in situ exopolysaccharide (EPS) production. The EPS from S. thermophilus ST1 produces highly viscous aqueous solutions and its structure has been investigated by NMR spectroscopy. Notably, all aspects of the elucidation of its primary structure including component analysis and absolute configuration of the constituent monosaccharides were carried out by NMR spectroscopy. An array of techniques was utilized including, inter alia, PANSY and NOESY-HSQC TILT experiments. The EPS is composed of hexasaccharide repeating units with the following structure: → 3)[α-d-Glcp-(1 → 4)]-β-d-Galp-(1 → 4)-β-d-Glcp-(1 → 4)[β-d-Galf-(1 → 6)]-β-d-Glcp-(1 → 6)-β-d-Glcp-(1 →, in which the residues in square brackets are terminal groups substituting backbone sugar residues that consequently are branch-points in the repeating unit of the polymer. Thus, the EPS consists of a backbone of four sugar residues with two terminal sugar residues making up two side-chains of the repeating unit. The molecular mass of the polymer was determined using translational diffusion experiments which resulted in M_w = 62 kDa, corresponding to 64 repeating units in the EPS.     

Mathematical modeling of the indole-3-butyric acid applications on rooting of northern highbush blueberry (<Emphasis Type="Italic">Vaccinium corymbosum</Emphasis> L.) softwood-cuttings

The objective of the present study is to develop a mathematical model to predict the effect of indole-3-butyric acid (IBA) on mean rooting (%) and mean root growth of northern highbush blueberry cultivars (Vaccinium corymbosum L.). The best estimating equations for the rooting (%) and root growth are formulized as: RG = (5.672183) + [0.002851 × (IBA)] − [2.0E⁻⁶ × (IBA)2] + (−0.27211 × Cv.) and R = (82.00649) + [0.030801 × (IBA)] − [2,4E⁻⁵ × (IBA)2] − [2.36218 × (Cv.)] where RG is root growth, R is rooting, IBA is indole-3-butyric acid (ppm) and Cv. is cultivar. Cultivars are Ivanhoe [1], Jersey [2], Rekord [3], Northland [4], Berkeley [5] and Bluejay [6]. The numbers given in square brackets represent the blueberry cultivars for the equations. Multiple regression analysis was carried out until the least sum of squares (R2) was obtained. R ² value 0.90 for rooting and 0.95 for root growth. Standard errors were found to be significant at the p < 0.001 level. The actual rooting differed to the blueberry cultivars and it was between 57.76 and 83.23% while estimated rooting percentage calculated by the produced mathematical model was between 59.04 and 83.80%.     

Effect of fungal infection on reproductive potential and survival time of <Emphasis Type="Italic">Tetranychus urticae</Emphasis> (Acari: Tetranychidae)

The effect of fungal infection on the reproductive potential of two-spotted spider mite, Tetranychus urticae, was evaluated as part of the full biocontrol potential of three entomopathogenic fungi by modeling of fecundity probability. Female mites (≤2-day-old) on leaves were exposed to the sprays of Beauveria bassiana, Paecilomyces fumosoroseus and Metarhizium anisopliae at the concentrations of 1.13 × 10³, 1.55 × 10³ and 0.95 × 10³ deposited conidia mm⁻² and then individually reared at 25°C and 12:12 L:D for oviposition. Mite mortalities 10 days after spraying were 73.1, 75.4 and 67.9% in the fungal treatments versus 15.5% in control. On average, females infected by the three fungal species survived 5.8, 6.2 and 6.3 days, and laid 3.1, 4.0 and 4.0 eggs per capita, respectively. These were 3–4 fold lower than the control fecundity at 12.3. The cumulative probabilities [P(m ≤ N)] for the counts of infected and non-infected (control) females laying m eggs per capita (m ≤ N) during 10 days fit very well the equation P(m ≤ N) = 1/[1 + exp(a + bm)] (r ² ≥ 0.98), yielding a solution to the probability for the female mites to achieve a specific fecundity {P(m ≤ N)−P[m ≤ (N − 1)]}. Consequently, the infected mites had 71–78% chance to lay ≤5 eggs per capita but only 5–8% to deposit >10 eggs despite some variation among the tested fungi. In contrast, the chances for the non-infected mites to achieve the low and high fecundities were 23 and 55%. The fitted probabilities provide a full coverage of the fecundity potential of infected versus non-infected mites and are more informative than the mean fecundities.     

Optimization of medium composition for the production of exopolysaccharides from Phellinus baumii Pilát in submerged culture and the immuno-stimulating activity of exopolysaccharides

Optimization of medium composition for the production of exopolysaccharides (EPS) from Phellinus baumii Pilát in submerged culture and the immuno-stimulating activity of EPS were carried out. Firstly, the medium components having significant effect on EPS production were screened out to be glucose, yeast extract and diammonium oxalate monohydrate by using a 2⁽⁷⁻³⁾ fractional factorial design. Secondly, the concentrations of the three factors were optimized using central composite design in response surface methodology. As results, a quadratic model was found to fit for EPS production, and the optimal medium composition was determined as following (g/l): 34.12 glucose, 4 peptone, 5.01 yeast extract, 0.88 diammonium oxalate monohydrate, 0.75 MgSO₄ and 1 KH₂PO₄ and 0.0075 thiamine (VB₁). A yield of 2.363 ± 0.04 g/l for EPS was observed in verification experiment. Finally, EPS from P. baumii Pilát was found to have direct immuno-stimulating activity in vitro on splenocyte proliferative response and acid phosphatase activity in peritoneal macrophages in a dose-dependent manner.     

Production of a novel glycolipid biosurfactant,mannosylmannitol lipid,by <Emphasis Type="Italic">Pseudozyma parantarctica</Emphasis> and its interfacial properties

The development of a novel glycolipid biosurfactant was undertaken using the high-level producers of mannosylerythritol lipids (MELs) such as Pseudozyma parantarctica, Pseudozyma antarctica, and Pseudozyma rugulosa. Besides the conventional MELs (MEL-A, MEL-B, and MEL-C), these yeasts produced an unknown glycolipid when they were cultivated in a medium containing 4% (w/v) olive oil and 4% (w/w) mannitol as the carbon source. The unknown glycolipid extracted from the culture medium of P. parantarctica JCM 11752^T displayed the spot with lower mobility than that of known MELs on TLC and provided mainly two peaks identical to mannose and mannitol on high-performance liquid chromatography after acid hydrolysis. Based on structural analysis by ¹H and ¹³C nuclear magnetic resonance, the novel glycolipid was composed of mannose and mannitol as the hydrophilic sugar moiety and was identified as mannosylmannitol lipid (MML). Of the strains tested, P. parantarctica JCM 11752^T gave the best yield of MML (18.2 g/L), which comprised approximately 35% of all glycolipids produced. We further investigated the interfacial properties of the MML, considering the unique hydrophilic structure. The observed critical micelle concentration (CMC) and the surface tension at CMC of the MML were 2.6 × 10⁻⁶ M and 24.2 mN/m, respectively. In addition, on a water-penetration scan, the MML efficiently formed not only the lamella phase (Lα) but also the myelins at a wide range of concentrations, indicating its excellent self-assembling properties and high hydrophilicity. The present glycolipid should thus facilitate the application of biosurfactants as new functional materials.     

Characterisation and bioactivity of protein-bound polysaccharides from submerged-culture fermentation of Coriolus versicolor Wr-74 and ATCC-20545 strains

The protein-bound polysaccharides of Coriolus versicolor (CPS) have been reported to stimulate overall immune functions against cancers and various infectious diseases by activating specific cell functions. A New Zealand isolate (Wr-74) and a patented strain (ATCC-20545) of C. versicolor were compared in this study. The fruit bodies of both strains were grown for visual verification. Both strains were grown in submerged-culture using an airlift fermentor with milk permeate as the base medium supplemented with glucose, yeast extract and salt. Metabolic profiles of both strains obtained over 7-day fermentation showed very similar trends in terms of biomass production (8.9–10.6 mg/ml), amounts of extracellular polysaccharide (EPS) from the culture medium (1150–1132 μg/ml), and intracellular polysaccharide (IPS) from the mycelium (80–100 μg/ml). Glucose was the dominant sugar in both EPS and IPS, and the polymers each consisted of three molecular weight fractions ranging from 2 × 10⁶ to 3 × 10³ Da. Both the EPS and IPS were able to significantly induce cytokine production (interleukin 12 and γ interferon) in murine splenocytes in vitro. Highest levels of interleukin 12 (291 pg/ml) and γ interferon (6,159 pg/ml) were obtained from samples containing Wr-74 IPS (0.06 μg/ml) and ATCC 20545 IPS (0.1 μg/ml), respectively. The results indicated that lower levels of EPS and IPS generally resulted in higher immune responses than did higher polymer concentrations.     

Inhibition of degradation and aggregation of recombinant human consensus interferon-α mutant expressed in <Emphasis Type="Italic">Pichia pastoris</Emphasis> with complex medium in bioreactor

The methylotrophic yeast Pichia pastoris has been used for the expression of many proteins. However, limitations such as protein degradation and aggregation became obvious when secreting heterologous protein-recombinant human consensus interferon-α mutant. Here, we investigate the effect of induction temperature on the yield and stability of interferon mutant expressed by P. patoris with buffered complex medium. The best results in terms of interferon mutant bioactivity and specific bioactivity were obtained when the microorganism was induced at 15°C, which were 2.91 × 10⁸ ± 0.3 × 10⁸ and 2.26 × 10⁸± 0.23 × 10⁸ IU mg⁻¹, respectively. At the same time, the cells grew fast owing to high AOX1-specific activity, and interferon mutant expression level reached 1.23 g l⁻¹, which was almost 30 times higher than that in the flask. Also, the proteolytic degradation of interferon mutant was inhibited completely because of lower protease bioactivity probably due to a reduced cell death rate at lower temperatures as well as protection of yeast extract and peptone in complex medium. In addition, interferon mutant aggregation was repressed significantly by the addition of Tween-80, and a specific bioactivity of 7.35 × 10⁸ ± 0.56 × 10⁸ IU mg⁻¹ was obtained. These results should be applicable to other low-stability recombinant proteins expressed in P. pastoris.     

Optimization and mechanism of diacetyl accumulation by <Emphasis Type="Italic">Enterobacter</Emphasis><Emphasis Type="Italic">aerogenes</Emphasis> mutant UV-3

A mutant designated as UV-3 was obtained from wild-type Enterobacter aerogenes 10293 through u.v. radiation. The activities of α-acetolactate decarboxylase (Ald), lactate dehydrogenase (Ldh) and diacetyl reductase (Dr) in UV-3 were strongly attenuated, with the lowest activities at pH 7.0–7.5, and temperature between 36 and 39°C. Compared to the wild-type, the yield of diacetyl by UV-3 was increased 18.7-fold, up to 1.05 ± 0.01 g l⁻¹. Acetoin and ethanol productions were decreased by 48.4 and 71.4%, respectively, but acetate yield was increased by 34.6%. Optimum medium for diacetyl production by UV-3 contained 10% glucose, 0.5% peptone, 0.5% yeast extract powder, 0.01% (NH₄)₂SO₄, 0.1% citric acid, 0.2% MnSO₄ and 0.2% MgSO_4, and this was determined by one-factor-at-a-time approach. Data from the five level central composite designs demonstrated that initial pH of 7.0, temperature of 37°C and rotational speed of 180 rev/min were optimum processing parameters for diacetyl production. The maximum yield of diacetyl could reach 1.35 g l⁻¹ in a 5-l bioreactor. These results showed an enhancement of the non-enzymatic oxidative decarboxylation of α-acetolactate and a decrease in the activities of Ald, Ldh and Dr as a consequence of diacetyl accumulation in UV-3.     

Improved simultaneous production of mycelial biomass and polysaccharides by submerged culture of <Emphasis Type="Italic">Hericium erinaceum</Emphasis>: optimization using a central composite rotatable design (CCRD)

The aim of this study was to optimize the culture medium used for the mycelial growth and production of intracellular polysaccharides (IPS) and exopolysaccharides (EPS) in a submerged culture of Hericium erinaceum. Of the various factors examined, including carbon and nitrogen sources, vitamins, mineral elements, and initial pH, those that proved to have a significant effect were then tested using a 2⁴ central composite rotatable design (CCRD). Under the optimal culture conditions, the maximal yield of biomass reached 14.24 ± 0.45 g l⁻¹ and was 1.85-fold higher than in the basal medium. The kinetics of EPS biosynthesis in a bioreactor showed that although the highest yield of EPS (2.75 ± 0.27 g l⁻¹) could be obtained on day 8, the process of biosynthesizing high molecular weight polysaccharides proceeded until the depletion of the carbon source in the medium (after 14 days of cultivation). Our results could be very helpful in the large-scale production of bioactive polysaccharides from H. erinaceum.     

Tumor-inhibitory and liver-protective effects of <Emphasis Type="Italic">Phellinus igniarius</Emphasis> extracellular polysaccharides

The tumor-inhibitory and liver-protective effects of crude extracellular polysaccharides (EPS) extracted from the liquid mycelial culture of the mushroom Phellinus igniarius were studied in mice. The mice were injected with murine sarcoma S180 and murine hepatoma H22. Crude EPS at 100, 200, 400 mg kg⁻¹ body weight was administered to EPS groups each day in the twelve consecutive days. The result showed that EPS 200 mg kg⁻¹ body weight significantly inhibited S180 and H22 at 65.0 and 46.3%, respectively. Moreover, EPS could not only keep the numbers of WBC, RBC, PLT and the concentration of HGB in a normal range, but also normalize the activities of AST, ALT and ALP. For example, in EPS-treated mice, AST significantly reduced with the percentage of A/G reverse in S180 (P < 0.05) and H22 (P < 0.01) when the mice took EPS 200 mg kg⁻¹ body weight. In conclusion, it was remarkable that P. igniarius EPS exhibited antitumor activity related to dosage and protected liver function by sustaining the blood routine as well as keeping the blood biochemical indexes normal.     

Enhancement of polysaccharides production in <Emphasis Type="Italic">Ganoderma lucidum</Emphasis> by the addition of ethyl acetate extracts from <Emphasis Type="Italic">Eupolyphaga sinensis</Emphasis> and <Emphasis Type="Italic">Catharsius molossus</Emphasis>

To screen stimulators from Chinese medicinal insects for mycelial growth and polysaccharides production of Ganoderma lucidum, G. lucidum was inoculated into the media with and without supplementation of medicinal insect extracts. The ethyl acetate extract of Eupolyphaga sinensis at 55 mg l⁻¹ lead to significant increase in both biomass and intracellular polysaccharides (IPS) concentration from 8.53 ± 0.41 to 14.16 ± 0.43 and 1.28 ± 0.09 to 2.13 ± 0.11 g l⁻¹, respectively. In addition, the ethyl acetate extract of Catharsius molossus at 55 mg l⁻¹ significantly enhanced extracellular polysaccharides (EPS) production; the EPS yield increased from 350.9 ± 14.1 to 475.1 ± 15.3 mg l⁻¹. There were no new components in the two types of polysaccharides obtained by the addition of the insect extracts.     

Cloning,expression, and characterization of an insoluble glucan-producing glucansucrase from <Emphasis Type="BoldItalic">Leuconostoc mesenteroides</Emphasis> NRRL B-1118

We have cloned a glucansucrase from the type strain of Leuconostoc mesenteroides (NRRL B-1118; ATCC 8293) and successfully expressed the enzyme in Escherichia coli. The recombinant processed enzyme has a putative sequence identical to the predicted secreted native enzyme (1,473 amino acids; 161,468 Da). This enzyme catalyzed the synthesis of a water-insoluble α-D-glucan from sucrose (K _M 12 mM) with a broad pH optimum between 5.0 and 5.7 in the presence of calcium. Removal of calcium with dialysis resulted in lower activity in the acidic pH range, effectively shifting the pH optimum to 6.0–6.2. The enzyme was quickly inactivated at temperatures above approximately 45°C. The presence of dextran offered some protection from thermal inactivation between room temperature and 40°C but had little effect above 45°C. NMR and methylation analysis of the water-insoluble α-d-glucan revealed that it had approximately equal amounts of α(1 → 3)-linked and α(1 → 6)-linked d-glucopyranosyl units and a low degree of branching.     

Biosynthesis of yeast mannan. Characterization of mannan-synthesizing enzyme systems from mutants defective in mannan structure

The yeastSaccharomyces cerevisiae X2180-1A (wild) and its mutants X2180-1A-4 (mnn 1) and X2180-1A-5 (mnn 2) defective in mannan biosynthesis were used as enzyme sources to catalyzein vitro mannosyl transfer from GDP-[¹⁴C-U]-mannose to endogenous glycoproteins as well as to exogenous, low-molecular weight acceptors. While the enzyme preparation from the wild strain exhibited all mannosyl transferase activities involved in mannan biosynthesis by catalyzing the synthesis of characteristic mannoprotein, the enzyme frommnn 1 mutant failed to catalyze the synthesis of α(1→3) mannoside linkages both with endogenous as well as with exogenous acceptors. The enzyme preparation from themnn 2 mutant catalyzed the formation of mannoprotein very similar to that obtained with the enzyme from the wild strain. The most important difference was the formation of a higher number of unsubstituted mannosyl units in the α(1→ 6) linked mannan backbone. The observed results support the hypothesis that in themnn 1 the mutation has altered the structural gene involved in biosynthesis of an α(1→3) mannosyl transferase catalyzing the addition of α(1→3) linked mannosyl units to α(1→2) linked mannotrioses in the polysaccharide side chains and in the oligosaccharides attached to serine and/or threonine in the protein part of mannan molecule. Themnn 2 mutant represents most probably a kind of regulatory mutation where the activity of an α(1→2) mannosyl transferase adding the mannosyl units directly to α(1→6) linked backbone in the outer region of polysacoharide part of yeast mannan is repressedin vivo but becomes significantin vitro.