N-Acetylchitooligosaccharides,biotic elicitor for phytoalexin production,induce transient membrane depolarization in suspension-cultured rice cells

Summary N-acetylchitooligosaccharides (fragments of chitin) elicit the production of phytoalexin in suspension-cultured rice cells. This oligosaccharide elicitor induced rapid and transient membrane depolarization at sub-nanomolar concentrations. Only the oligomers with a certain degree of polymerization were active, while deacetylated chitooligosaccharides caused no effect. Such specificity coincided well with that for the elicitor activity, suggesting possible involvement of this transient change in membrane potential as one of the initial signals in the signal transduction sequence for the activation of defense responses.     

Pathway Engineering of Bacillus subtilis for Enhanced N‐Acetylneuraminic Acid Production via Whole‐Cell Biocatalysis

N‐acetylneuraminic acid (NeuAc) is a common sialic acid that has a wide range of applications in nutraceuticals and pharmaceuticals. However, low production efficiency and high environmental pollution associated with traditional extraction and chemical synthesis methods constrain the supply of NeuAc. Here, a biological approach is developed for food‐grade NeuAc production via whole‐cell biocatalysis by the generally regarded as safe (GRAS) bacterium Bacillus subtilis (B. subtilis). Promoters for controlling N‐acetylglucosamine 2‐epimerase (AGE) and NeuAc adolase (NanA) are optimized, yielding 32.84 g L^?1 NeuAc production with a molar conversion rate of 26.55% from N‐acetylglucosamine (GlcNAc). Next, NeuAc production is further enhanced to 46.04 g L^?1, which is 40.2% higher than that of the strain with promoter optimization, by expressing NanA from Staphylococcus hominis instead of NanA from Escherichia coli. To enhance the expression level of ShNanA, the N‐terminal coding sequences of genes with high expression levels are fused to the 5′‐end of the ShNanA gene, resulting in 56.82 g L^?1 NeuAc production. Finally, formation of the by‐product acetoin from pyruvate is blocked by deleting the alsS and alsD genes, resulting in 68.75 g L^?1 NeuAc production with a molar conversion rate of 55.57% from GlcNAc. Overall, a GRAS B. subtilis strain is demonstrated as a whole‐cell biocatalyst for efficient NeuAc production.     

Changes of Hexosamine and Acetyl Contents during Cultivation of Extracellular Galactosamine-containing Polymer from Aspergillus parasiticus

The reactions of N-acetylchitooligosaccharides with chitinolytic enzyme were analyzed by HPLC using a Tosoh TSK-Gel amide-80 column with 70% acetonitrile as an eluent. We separated α and β anomeric forms of N-acetylchitooligosaccharides, and obtain the following advantages of this HPLC method.

1. We can easily identify the reaction mechanism of chitinolytic enzymes by this method, distinguishing the inverting mechanism showing α anomer formation from the retaining mechanism showing β anomer formation.

2. We can also estimate the cleavage patterns of N-acetylchitooligosaccharides by chitinolytic enzymes by using natural substrates.     

A reducing-end-acting chitinase from <Emphasis Type="Italic">Vibrio proteolyticus</Emphasis> belonging to glycoside hydrolase family 19

A chitinase gene belonging to the glycoside hydrolase family 19 from Vibrio proteolyticus (chi19) was cloned. The recombinant enzyme (Chi19) showed weak activities against polymeric substrates and considerable activities against fully N-acetylated chitooligosaccharides, (GlcNAc) _n , whose degree of polymerization was greater than or equal to five. It hydrolyzed (GlcNAc) _n at the second linkage position from the reducing ends of the chitooligosaccharides. The hydrolytic products of colloidal chitin were mainly (GlcNAc)₂ from the initial stage of the reaction. The hydrolytic pattern of reduced colloidal chitin clearly suggested that the enzyme hydrolyzed the polymeric substrate from the reducing end.     

Recognition of N-acetylchitooligosaccharide elicitor by rice protoplasts

The response by rice protoplasts to N-acetylchitooligosaccharide elicitor was examined by monitoring the production of reactive oxygen species (ROS), and the expression of the two early-responsive genes, EL2 and EL3. Freshly prepared rice protoplasts produced a high level of ROS in the absence of the elicitor, and did not show further increase of the ROS generation in response to N-acetylchitooligosaccharide elicitor. By incubating protoplasts for 1 d, the background level decreased and the induction of ROS production and the induction of mRNAs for the two genes were observed. The structural requirements of N-acetylchitooligosaccharides for elicitor-activity, as well as the effects of inhibitors of protein kinase (K-252a), protein phosphatase (calyculin A) and protein synthesis (cycloheximide) on the ROS production and gene expression were very similar to those observed in suspension-cultured rice cells, indicating that rice protoplasts retain the machinery for the recognition of, and initial signaling from, N-acetylchitooligosaccharide elicitor.     

Solid-state CP/MASS 13C-NMR spectroscopy: a sensitive method to monitor enzymatic hydrolysis of chitin

The time-course hydrolysis of colloidal chitin by the chitinase complex isolated from Myrothecium verrucaria was monitored using solution and solid-state ¹³C-NMR spectroscopy. The solution NMR studies showed the presence of N-acetylglucosamine (GlcNAc) as the sole product of hydrolysis. Solid-state ¹³C CP/MASS studies, on the other hand, indicated the presence of high molecular weight oligomers as well as GlcNAc. The linewidth of the C₁ carbon of the oligomers obtained after hydrolysis is found to be less than that of the unhydrolysed sample. The linewidths calculated from the spin-spin relaxation times (T₂) of colloidal chitin and its products of hydrolysis were in the restricted range of 40–50 Hz, compared with the observed linewidths of 143–123 Hz. Peak area measurement on monomer to polymer/oligomer indicated an initial slow formation of the monomer, GlcNAc. From the NMR data, the involvement of endo-enzymes in the initial phase of hydrolysis is suggested.     

Chitinous Components of the Cell Wall of Fusarium oxysporum

The cell wall of Fusarium oxysporum f. sp. lycopersici was digested with chitinase to analyze the structure of its chitinous components. In spite of a similar acetylation degree of the cell wall components to that of 25–35% acetylated chitosan, only N-acetylglucosamine disaccharide [(GlcNAc)₂] was obtained from chitinase hydrolyzate of the fungal cell wall by CM-Sephadex C-25 column chromatography, while (GlcNAc)₂ and several types of deacetylated chitooligosaccharides were separated from that of 25–35% acetylated chitosan. The results indicate that N-acetylglucosamine residues in the polysaccharide chains of the fungal cell wall are most likely condensed into some region, while acetylated residues are more scattered in 25–35% acetylated chitosan.     

Preparation of a novel (1→4)-β-D-glycan by Acetobacter xylinum — a proposed mechanism for incorporation of a N-acetylglucosamine residue into bacterial cellulose

A novel polysaccharide having a N-acetylglucosamine (GlcNAc) residue as one of the constituents was synthesized by incubation of Acetobacter xylinum in a modified Schramm-Hestrin medium containing lysozyme-susceptible phosphoryl chitin (P-chitin) andd-glucose. HPLC of the culture medium snowed that the P-chitin added was depolymerized to monomeric and oligomeric P-chitins during the incubation, and the P-chitins with permeable sizes were utilized as a carbon source by the bacteria.¹³C NMR analysis revealed that the P-chitin consists mainly of GlcNAc 6-P residues. Furthermore, monomeric GlcNAc 6-phosphate was also found to enhance the incorporation of GlcNAc residues into the polysaccharide. However, no incorporation of the GlcNAc residues was observed when A. xylinum was incubated in a medium containing either highly phosphorylated chitin (DS = 1.90) or its oligomers produced by acid hydrolysis.     

Glycoconjugate histochemistry of the digestive tract of <Emphasis Type="Italic">Triturus carnifex</Emphasis> (Amphibia,Caudata)

Summary In this study, the variety of sugar residues in the gut glycoconjugates of Triturus carnifex (Amphibia, Caudata) are investigated by carbohydrate conventional histochemistry and lectin histochemistry. The oesophageal surface mucous cells contained acidic glycoconjugates, with residues of GalNAc, Gal β1,3 GalNAc and (GlcNAc β1,4) _n oligomers. The gastric surface cells mainly produced neutral glycoproteins with residues of fucose, Gal β1-3 GalNAc, Gal-αGal, and (GlcNAc β1,4) _n oligomers in N- and O-linked glycans, as the glandular mucous neck cells, with residues of mannose/glucose, GalNAc, Gal β1,3 GalNAc, (GlcNAc β1,4) _n oligomers and fucose linked α1,6 or terminal α1,3 or α1,4 in O-linked glycans. The oxynticopeptic tubulo-vesicular system contained neutral glycoproteins with N- and O-linked glycans with residues of Gal-αGal, Gal β1-3 GalNAc and (GlcNAc β1,4) _n oligomers; Fuc linked α1,2 to Gal, α1,3 to GlcNAc in (poly)lactosamine chains and α1,6 to GlcNAc in N-linked glycans. Most of these glycoproteins probably corresponds to the H⁺K⁺-ATPase β-subunit. The intestinal goblet cells contained acidic glycoconjugates, with residues of GalNAc, mannose/ glucose, (GlcNAc β1,4) _n oligomers and fucose linked α1,2 to Gal in O-linked oligosaccharides. The different composition of the mucus in the digestive tracts may be correlated with its different functions. In fact the presence of abundant sulphation of glycoconjugates, mainly in the oesophagus and intestine, probably confers resistance to bacterial enzymatic degradation of the mucus barrier.     

Rice gene expression in response to N-acetylchitooligosaccharide elicitor: comprehensive analysis by DNA microarray with randomly selected ESTs

N-acetylchitooligosaccharides are potent elicitors to suspension-cultured rice cells, inducing a set of defense reactions. Expression of defense-related genes is considered to play an important role in defense reactions, and we employed microarray analysis of 8987 randomly selected expressed sequence tags to analyze the changes in gene expression caused by N-acetylchitooctaose. In this experiment, 166 genes were significantly induced and 93 genes were repressed. RNA gel blot analysis of 16 of these genes confirmed the microarray results. Of the 259 ESTs identified as responsive to N-acetylchytooctaose, 18 genes are related to signal transduction, including five calcium-dependent protein kinases (CDPKs). Among these, three novel CDPKs responsive to N-acetylchitooctaose were isolated.     

Binding of simple carbohydrates and some N-acetyllactosamine-containing oligosaccharides to Erythrina cristagalli agglutinin as followed with a fluorescent indicator ligand

Erythrina cristagalli agglutinin, a dimeric lectin [J. L. Iglesias, et al. (1982) Eur. J. Biochem.123, 247–252] was shown by equilibrium dialysis to be bivalent for 4-methylumbelliferyl-β-d-galactoside. Upon binding to the lectin, this ligand showed a difference absorption spectrum with two maxima (at 322 and 336 nm) of equal intensity (Δ? = 1.2 × 10³m^?1 cm^?1). A similar spectrum with a comparable value of Δ? was obtained with 4-methylumbelliferyl-N-acetyl-β-d-galactosaminide. Binding of methyl-α-d-galactoside, lactose, and N-acetyllactosamine all produced small but equally intense protein difference spectra with a maximum (Δ? = 2.8 × 10² M^?1 cm^?1) at 291.6 nm. Upon binding of N-dansyl-d-galactosamine to the lectin, there was a fivefold increase in fluorescence intensity of this ligand. The association constant for N-dansyl-d-galactosamine was caused by a very favorable ΔS° of the dansyl group without affecting the strictly carbohydrate-specific character of binding. N-Dansyl-d-galactosamine was employed as a fluorescent indicator ligand in substitution titrations. This involved the use of simple carbohydrates, N-acetyllactosamine, and oligosaccharides which occur in the carbohydrate units of N-glycoproteins; the latter were Gal(β → 4)GlcNAc(β1 → 2)Man, Gal(β1 → 4)GlcNAc(β1 → 6)Man, and Gal(β1 → 4)GlcNAc(β1 → 6)[Gal(β1 → 4)GlcNAc(β1 → 2)]Man. The titrations were performed at two temperatures to determine the thermodynamic parameters. In the series N-acetyl-d-galactosamine, methyl-α-d-galactoside, and lactose, ?ΔH° increased from 24 to 41 kJ mol^?1; it increased further for N-acetyllactosamine and then remained unchanged for the N-acetyllactosamine-containing oligosaccharides (55 ± 1 kJ mol^?1). This indicated that the site specifically accommodated the disaccharide structure with an important contribution of the 2-acetamido group in the penultimate sugar. Beyond this, no additional contacts seemed to be formed. This conclusion also followed from considerations of ΔS° values which became more unfavorable in the above series (?23 to ?101 ± 4 J mol^?1 K^?1); the most negative value of ΔS° was observed with N-acetyllactosamine and the three N-acetyllactosamine-containing oligosaccharides.     

Rhizobial lipochitooligosaccharide nodulation factors activate expression of the legume early nodulin gene ENOD12 in rice

Lipochitooligosaccharide nodulation factors (Nod factors) produced by rhizobia are a major host range determinant. These factors play a pivotal role in the molecular signal exchange, infection and induction of symbiotic developmental responses in legumes leading to the formation of a nodule in which rhizobia carry out N₂ fixation. Determining whether rice ( Oryza sativa ) can respond to Nod factors could lead to strategies that would make rice amenable to develop a nitrogen-fixing endosymbiotic association with rhizobia. We introduced into rice the promoter of the infection-related gene MtENOD12 (from Medicago truncatula ) fused to the β-glucuronidase (GUS) reporter gene to serve as a molecular marker to aid in the detection of Nod factor signal perception by rice cells. Treatment of the transgenic rice roots with Nod factors (10^–6–10^–9 m ) under nitrogen-limiting conditions induced MtENOD12 -GUS expression in cortical parenchyma, endodermis and pericycle. In contrast, chitooligosaccharide backbone alone failed to elicit such a response in the root tissues. These findings demonstrate that rice roots perceive Nod factors and that these lipochitooligosaccharides, but not simple chitin oligomers, act as signal molecules in activating MtENOD12 in cortical parenchyma as in legumes. Exogenous application of N -naphthaleneacetic acid mimicked the Nod factor-elicited tissue-specific expression of MtENOD12 in roots while cytokinins inhibited it, thus evidencing that Nod factors, auxin and cytokinins probably act on similar signaling elements responsible for the regulation of MtENOD12 activation in rice. Taken together, these results suggest that at least a portion of the signal transduction machinery important for legume nodulation is likely to exist in rice.      

Purification and Characterization of β-N-Acetylhexosaminidase from the Liver Prawn,Penaeus japonicus

β-N-Acetvlhexosaminidase (EC 3.2.1.52) was purified from the liver of a prawn, Penaeus japonicus, by ammonium sulfate fractionation and chromatography with Sephadex G-100, hydroxylapatite, DEAE-Cellulofine, and Cellulofine GCL-2000-m. The purified enzyme showed a single band keeping the potential activity on both native PAGE and SDS–PAGE. The apparent molecular weight was 64,000 and 110,000 by SDS–PAGE and gel filtration, respectively. The pI was less than 3.2 by chromatofocusing. The aminoterminal amino acid sequence was NH₂-Thr-Leu-Pro-Pro-Pro-Trp-Gly-Trp-Ala-?-Asp-Gln-Gly-VaI-?-Val-Lys-Gly-Glu-Pro-. The optimum pH and temperature were 5.0 to 5.5 and 50°C, respectively. The enzyme was stable from pH 4 to 11, and below 55°C. It was 39% inhibited by 10mM HgCl₂.

Steady-state kinetic analysis was done with the purified enzyme using N-acetylchitooligosaccharides (GlcNAc_n, n = 2 to 6) and p-nitrophenyl N-acetylchitooligosaccharides (pNp-β-GlcNAc_n, n= 1 to 3) as the substrates. The enzyme hydrolyzed all of these substrates to release monomeric GlcNAc from the non-reducing end of the substrate. The parameters of K_m and k_cat at 25°C and pH 5.5 were 0.137 mM and 598s^–1 for pNp-β-GlcNAc, 0.117 mM and 298s^–1 for GlcNAc₂, 0.055 mM and 96.4s^–1 for GlcNAc₃, 0.044 mM and 30.1 s^–1 for GlcNAc_4, 0.045 mM and 14.7 s^–1 for GlcNAc₅, and 0.047 mM and 8.3 s^–1 for GlcNAc₆, respectively. These results suggest that this β-N-acetylhexosaminidase is an exo-type hydrolytic enzyme involved in chitin degradation, and prefers the shorter substrates.     

The effect of different fertilizer treatments on water quality parameters in rice-cum-fish culture systems

A rice–fish culture experiment was conducted to determine the effects of different fertilizer treatments on water quality parameters, i.e. dissolved oxygen, temperature, pH and conductivity, as well as the nutrients nitrogen, potassium and phosphorus. Data gathered were subjected to analysis of variance. Dissolved oxygen, nitrogen and potassium concentrations showed significant differences (P<0.5) among the various rice‐cum‐fish management systems, with the highest mean concentration in the fish‐only management system. The insignificant difference (P>0.05) in the oxygen concentration between rice‐only and rice‐cum‐fish management systems was probably due to the narrow spacing of rice cultivars (20 × 20 cm). Under different fertilizer treatments, dissolved oxygen, nitrogen and potassium concentrations were not significantly different (P>0.05), indicating that different fertilizer treatments had no effect on their concentrations. Mean temperature, pH, conductivity and phosphorus concentrations in all rice‐cum‐fish management systems and fertilizer treatments did not differ significantly (P>0.05) from each other. This might possibly be due to the hydrogeology and climatic conditions of the area. However, all levels of studied water quality parameters were tolerable to the fish (Oreochromis niloticus) in the rice–fish ecosystem. There was no significant difference (P>0.05) in rice yield between the rice–fish management system and the rice‐only management system. However, there was a significant difference (P<0.05) under different fertilizer treatments, with nitrogen/phosphorus/potassium (NPK) (20 : 10 : 10) giving the highest production of an estimated 14.36 kg ha^?1 year^?1, indicating that fertilizer treatments had an effect on the rice yield. Fish growth was not significantly different (P>0.05) under different rice–fish management systems, but was significant (P<0.05) under different fertilizer treatments. Chicken manure gave the best growth with 17.7±5.97 g; estimated fish yields were 343 and 602 kg ha^?1 year^?1 in fish monoculture and rice–fish culture respectively. It is recommended that farmers who are more inclined to fish or rice production in integrated rice‐cum‐fish management systems could use chicken manure and NPK (20 : 10 : 10) respectively.     

A Highly N-Glycosylated Chitin Deacetylase Derived from a Novel Strain of Mortierella sp. DY-52

Chitin deacetylase (CDA), the enzyme that catalyzes the hydrolysis of acetamido groups of GlcNAc in chitin, was purified from culture filtrate of the fungus Mortierella sp. DY-52 and characterized. The extracellular enzyme is likely to be a highly N-glycosylated protein with a pI of 4.2–4.8. Its apparent molecular weight was determined to be about 52 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and 67 kDa by size-exclusion chromatography. The enzyme had an optimum pH of 6.0 and an optimum temperature of 60 °C. Enzyme activity was slightly inhibited by 1–10 mM Co²⁺ and strongly inhibited by 10 mM Cu²⁺. It required at least two GlcNAc residues for catalysis. When (GlcNAc)₆ was used as substrate, K _m and V _max were determined to be 1.1 mM and 54.6 μmol min^?1 respectively.     

Difucosylation of chitooligosaccharides by eukaryote and prokaryote α1,6-fucosyltransferases

Background

The synthesis of eukaryotic N-glycans and the rhizobia Nod factor both involve α1,6-fucosylation. These fucosylations are catalyzed by eukaryotic α1,6-fucosyltransferase, FUT8, and rhizobial enzyme, NodZ. The two enzymes have similar enzymatic properties and structures but display different acceptor specificities: FUT8 and NodZ prefer N-glycan and chitooligosaccharide, respectively. This study was conducted to examine the fucosylation of chitooligosaccharides by FUT8 and NodZ and to characterize the resulting difucosylated chitooligosaccharides in terms of their resistance to hydrolysis by glycosidases.

Methods

The issue of whether FUT8 or NodZ catalyzes the further fucosylation of chitooligosaccharides that had first been monofucosylated by the other. The oligosaccharide products from the successive reactions were analyzed by normal-phase high performance liquid chromatography, mass spectrometry and nuclear magnetic resonance. The effect of difucosylation on sensitivity to glycosidase digestion was also investigated.

Results

Both FUT8 and NodZ are able to further fucosylate the monofucosylated chitooligosaccharides. Structural analyses of the resulting oligosaccharides showed that the reducing terminal GlcNAc residue and the third GlcNAc residue from the non-reducing end are fucosylated via α1,6-linkages. The difucosylation protected the oligosaccharides from extensive degradation to GlcNAc by hexosamidase and lysozyme, and also even from defucosylation by fucosidase.

Conclusions

The sequential actions of FUT8 and NodZ on common substrates effectively produce site-specific-difucosylated chitooligosaccharides. This modification confers protection to the oligosaccharides against various glycosidases.

General significance

The action of a combination of eukaryotic and bacterial α1,6-fucosyltransferases on chitooligosaccharides results in the formation of difucosylated products, which serves to stabilize chitooligosaccharides against the action of glycosidases.     

Enzymatic properties of wild-type and active site mutants of chitinase A from Vibrio carchariae, as revealed by HPLC-MS

The enzymatic properties of chitinase A from Vibrio carchariae have been studied in detail by using combined HPLC and electrospray MS. This approach allowed the separation of alpha and beta anomers and the simultaneous monitoring of chitooligosaccharide products down to picomole levels. Chitinase A primarily generated beta-anomeric products, indicating that it catalyzed hydrolysis through a retaining mechanism. The enzyme exhibited endo characteristics, requiring a minimum of two glycosidic bonds for hydrolysis. The kinetics of hydrolysis revealed that chitinase A had greater affinity towards higher Mr chitooligomers, in the order of (GlcNAc)6 > (GlcNAc)4 > (GlcNAc)3, and showed no activity towards (GlcNAc)2 and pNP-GlcNAc. This suggested that the binding site of chitinase A was probably composed of an array of six binding subsites. Point mutations were introduced into two active site residues - Glu315 and Asp392 - by site-directed mutagenesis. The D392N mutant retained significant chitinase activity in the gel activity assay and showed approximately 20% residual activity towards chitooligosaccharides and colloidal chitin in HPLC-MS measurements. The complete loss of substrate utilization with the E315M and E315Q mutants suggested that Glu315 is an essential residue in enzyme catalysis. The recombinant wild-type enzyme acted on chitooligosaccharides, releasing higher quantities of small oligomers, while the D392N mutant favored the formation of transient intermediates. Under standard hydrolytic conditions, all chitinases also exhibited transglycosylation activity towards chitooligosaccharides and pNP-glycosides, yielding picomole quantities of synthesized chitooligomers. The D392N mutant displayed strikingly greater efficiency in oligosaccharide synthesis than the wild-type enzyme.     

Induction of nodule primordia on Phaseolus and Acacia by lipo-chitin oligosaccharide nodulation signals from broad-host-range Rhizobium strain GRH2

Rhizobium wild-type strain GRH2 was originally isolated from the tree, Acacia cyanophylla, and has a broad host-range which includes herbaceous legumes, such as Phaseolus and Trifolium species. Here we show that strains of Rhizobium sp. GRH2, into which heterologous nodD alleles have been introduced, produce a large diversity of both sulphated and non-sulphated lipo-chitin oligosaccharides (LCOs). Most of the molecular species contain an N-methyl group on the reducing-terminal N-acetyl-glucosamine. The LCOs vary in the nature of the fatty acyl chain and in the length of the chitin backbone. The majority of the LCOs have an olgosaccharide chain length of five GlcNAc residues, but a few are oligomers having six GlcNAc units. LCOs purified from GRH2 are able to induce root hair formation and deformation on Acacia cyanophylla and A. melanoxylon plants. We show that an N-vaccenoyl-chitopentaose bearing an N-methyl group is able to induce nodule primordia on Phaseolus vulgaris, A. cyanophylla, and A. melanoxylon, indicating that for these plants an N-methyl modification is sufficient for nodule primordia induction.