Functional analysis of fructosyl-amino acid oxidases of Aspergillus oryzae

Three active fractions of fructosyl-amino acid oxidase (FAOD-Ao1, -Ao2a, and -Ao2b) were isolated from Aspergillus oryzae strain RIB40. N-terminal and internal amino acid sequences of FAOD-Ao2a corresponded to those of FAOD-Ao2b, suggesting that these two isozymes were derived from the same protein. FAOD-Ao1 and -Ao2 were different in substrate specificity and subunit assembly; FAOD-Ao2 was active toward N(epsilon)-fructosyl N(alpha)-Z-lysine and fructosyl valine (Fru-Val), whereas FAOD-Ao1 was not active toward Fru-Val. The genes encoding the FAOD isozymes (i.e., FAOAo1 and FAOAo2) were cloned by PCR with an FAOD-specific primer set. The deduced amino acid sequences revealed that FAOD-Ao1 was 50% identical to FAOD-Ao2, and each isozyme had a peroxisome-targeting signal-1, indicating their localization in peroxisomes. The genes was expressed in Escherichia coli and rFaoAo2 showed the same characteristics as FAOD-Ao2, whereas rFaoAo1 was not active. FAOAo2 disruptant was obtained by using ptrA as a selective marker. Wild-type strain grew on the medium containing Fru-Val as the sole carbon and nitrogen sources, but strain Delta faoAo2 did not grow. Addition of glucose or (NH(4))(2)SO(4) to the Fru-Val medium did not affect the assimilation of Fru-Val by wild-type, indicating glucose and ammonium repressions did not occur in the expression of the FAOAo2 gene. Furthermore, conidia of the wild-type strain did not germinate on the medium containing Fru-Val and NaNO(2) as the sole carbon and nitrogen sources, respectively, suggesting that Fru-Val may also repress gene expression of nitrite reductase. These results indicated that FAOD is needed for utilization of fructosyl-amino acids as nitrogen sources in A. oryzae.     

Thermostabilization of bacterial fructosyl-amino acid oxidase by directed evolution

We succeeded in isolating several thermostable mutant fructosyl-amino acid oxidase (FAOX; EC 1.5.3) without reduction of productivity by directed evolution that combined an in vivo mutagenesis and membrane assay screening system. Five amino acid substitutions (T60A, A188G, M244L, N257S, and L261M) occurred in the most thermostable mutant obtained by a fourth round of directed evolution. This altered enzyme, FAOX-TE, was stable at 45 degrees C, whereas the wild-type enzyme was not stable above 37 degrees C. The K(m) values of FAOX-TE for D-fructosyl-L-valine and D-fructosyl-glycine were 1.50 and 0.58 mM, respectively, in contrast with corresponding values of 1.61 and 0.74 mM for the wild-type enzyme. This altered FAOX-TE will be useful in the diagnosis of diabetes.     

Amino acid immobilization of fungal motile cells

Summary A 0.2 M mixture of L-leucine and L-lysine, a pair of amino acids which Machlis (1969) had shown could attract the zoospores of Allomyces in much lower concentrations, was found to immobilize zoospores by stopping flagellar motion. While the age of the spores does not affect the response to the amino acid mixture, the time for 100% immobilization does increase with increasing numbers of spores. Viability of the spores is not altered by treatment with the mixture of L-leucine and L-lysine and subsequent germling development is highly synchronized.Several other amino acid mixtures had a similar effect upon the Allomyces' flagellum. Indeed, L-lysine by itself seems to be the most effective compound tested. Immobilization of flagella in other fungi, algae, and one protozoan was also caused by treatment with L-leucine and L-lysine. Nothing is known of the mechanism of action of this amino acid treatment.     

Characterisation of three fungal glucuronoyl esterases on glucuronic acid ester model compounds

Applied Microbiology and Biotechnology - The glucuronoyl esterases (GEs) that have been identified so far belong to family 15 of the carbohydrate esterases in the CAZy classification system and are...     

Effects of boron-containing compounds in the fungal kingdom

BackgroundThe number of known boron-containing compounds (BCCs) is increasing due to their identification in nature and innovative synthesis procedures. Their effects on the fungal kingdom are interesting, and some of their mechanisms of action have recently been elucidated.MethodsIn this review, scientific reports from relevant chemistry and biomedical databases were collected and analyzed.ResultsIt is notable that several BCC actions in fungi induce social and economic benefits for humans. In fact, boric acid was traditionally used for multiple purposes, but some novel synthetic BCCs are effective antifungal agents, particularly in their action against pathogen species, and some were recently approved for use in humans. Moreover, most reports testing BCCs in fungal species suggest a limiting effect of these compounds on some vital reactions.ConclusionsNew BCCs have been synthesized and tested for innovative technological and biomedical emerging applications, and new interest is developing for discovering new strategic compounds that can act as environmental or wood protectors, as well as antimycotic agents that let us improve food acquisition and control some human infections.     

Occurrence of fungal endophytes in cultivars ofTriticum aestivum in South Africa

Fungal endophytes were isolated from leaves, roots and stems of four wheat cultivars and a breeding line at three different sampling dates during the 1993 growing season. Of the 55 different fungal taxa encountered, 19 were present at relative importance values of more than 5%. No cultivar-related differences in the assembleges of endophytes were observed.Phoma glomerata was not restricted to only one tissue type, whereasAlternaria alternata, basidiomycete sp. 1,Pleospora herbarum andEpicoccum nigrum occurred primarily in the leaves, andFusarium avenaceum was extremely frequent in roots. In general, colonization by endophytes increased with the age of the plants. Most endophytes were isolated from wheat leaves. Successional colonization of a given tissue type was quantitative rather than qualitative, with a given fungal taxon increasing or decreasing over the period sampled, rather than replacing the fungi initially encountered.Present address: Téra d'Sott 5, CH-6949, Comano, Switzerland.     

Occurrence of brominated compounds in soft-bottom benthic organisms

The abundant species of infaunal soft-bodied organisms from two geographically separated sites with very different predatory regimes were examined for the occurrence of volatile brominated compounds. Of the 10 species, three contained substantial amounts of brominated compounds while two others contained trace amounts. A greater proportion of species from the Western Atlantic site with large, sediment-disturbing predators and much lower infaunal densities contained these compounds. The occurrence of the compounds did not follow phylogenetic lineages or food sources. The toxicity of such compounds and potential for predator deterrence may be major determinants of their occurrence.     

Controlled mycelial growth for kojic acid production using Ca-alginate-immobilized fungal cells

Summary Conidia of Aspergillus oryzae were immobilized in Ca-alginate beads and then incubated in a nutrient medium to yield an immobilized biocatalyst producing kojic acid. The immobilized cell cultures produced kojic acid linearly during cultivation. Regardless of the size of the immobilized particles, there existed an optimal nitrogen concentration for the maximum production rate of kojic acid, at which smaller bead sizes resulted in a higher production rate. When the growth of mycelia were confined within the bead surface and segregated from each other by gel material, they produced kojic acid with maximal catalytic activity and exhibited the highest conversion yield of glucose. The extent of mycelial segregation was especially higher in cultures of smaller bead particles, and the depth of mycelial growth was 150 to 250 m from the gel bead surface in all cultures of different nitrogen concentrations and bead sizes. Therefore, for the maximum expression of catalytic activities of immobilized mycelial cultures, it was found very critical to optimally control the mycelial distribution in gel beads by the culture conditions affecting mycelial growth.     

紫杉醇产生菌的细胞总核酸提取

树状多节孢（Nodulisporium sylviforme)为中国新记录属种,首次发现它可产生特效抗癌药物紫杉醇,国内外尚未见其DNA提取等方面的研究报道。为了获得高质量的DNA,利用单因素试验对其细胞总核酸的提取方法进行了探索。找到了一套适合紫杉醇产生菌DNA的提取较为简便易行可靠的方法。用此法直接从紫杉醇产生菌中提取DNA,并将所提取DNA进行随机引物扩增（RAPD）,得到了较清晰的扩增图谱。     

Biodegradation of octyltin compounds by Cochliobolus lunatus and influence of xenobiotics on fungal fatty acid composition

The perturbation in the growth and fatty acid profile of the microscopic fungus Cochliobolus lunatus IM 4417 in the presence of octyltin compounds (trioctyltin – TOT, dioctyltin – DOT and monooctyltin – MOT) was studied. Fungal resistance to the tested organotins decreased with a reduction in the number of octyl groups bonded with a tin atom. Also, the fatty acid unsaturation index decreased according to the mentioned scheme. Among all tested octyltin compounds, TOT was removed with the highest efficiency. The efficiency of MOT removal was correlated with the initial concentration of the compounds and for concentrations 20 and 100 mg l⁻¹ reached the value of 75% and 40%, respectively. Elimination of octyltins depended on the metabolic activity of the fungus and was not the result of passive sorption. During bioconversion of TOT the hydroxylated derivative of substrate was detected. Moreover, the addition of cytochrome P-450 inhibitors significantly reduced the metabolism of octyltin compounds. Thus, it is postulated that the process of degradation of octyltin compounds is similar to that described for tributyltin (TBT) and it is mediated by cytochrome P-450.     

The fungal metabolite culmorin and related compounds

This paper reviews the toxicology of culmorins, a family of compounds found in grains contaminated by Fusarium graminearum and related fungi. We include the results of an Ames test and studies based on Quantitative Structure-Activity Relationships. Culmorin has low toxicity in several in vitro assays and in one study in swine and is Ames test negative. Culmorin is moderately antifungal. QSAR analysis suggested that the plant compound longifolene was similar. Longifolene is a GRAS compound used in cosmetics and is also moderately antifungal.     

Complementary action of jasmonic acid on salicylic acid in mediating fungal elicitor-induced flavonol glycoside accumulation of Ginkgo biloba cells

The antagonistic action between jasmonic acid (JA) and salicylic acid (SA) in plant defence responses has been well documented. However, their relationship in secondary metabolite production is largely unknown. Here, we report that PB90, a protein elicitor from Phytophthora boehmeriae , triggers JA generation, SA accumulation and flavonol glycoside production of Ginkgo biloba cells. JA inhibitors suppress not only PB90-triggered JA generation, but also the elicitor-induced flavonol glycoside production. However, the elicitor can still enhance flavonol glycoside production even though the JA generation is totally inhibited. Over-expression of SA hydrolase gene NahG not only abolishes SA accumulation, but also suppresses the elicitor-induced flavonol glycoside production when JA signalling is inhibited. Interestingly, expression of NahG does not inhibit the elicitor-induced flavonol glycoside accumulation in the absence of JA inhibitors. Moreover, JA levels are significantly enhanced when SA accumulation is impaired in the transgenic cells. Together, the data suggest that both JA and SA are involved in PB90-induced flavonol glycoside production. Furthermore, we demonstrate that JA signalling might be enhanced to substitute for SA to mediate the elicitor-induced flavonol glycoside accumulation when SA signalling is impaired, which reveals an unusual complementary relationship between JA and SA in mediating plant secondary metabolite production.     

Cloning and expression of fructosyl-amino acid oxidase gene from Corynebacterium sp. 2-4-1 in Escherichia coli

The gene encoding the fructosyl-amino acid oxidase (fructosyl-alpha-L-amino acid: oxygen oxidoreductase (defructosylating); EC 1.5.3) of Corynebacterium sp. 2-4-1 was cloned and expressed in Escherichia coli. The gene consists of 1,116 nucleotides and encodes a protein of 372 amino acids with a predicted molecular mass of 39,042. The open reading frame was confirmed as the gene of the fructosyl-amino acid oxidase by comparison with the N-terminal amino acid sequence of the purified fructosyl-amino acid oxidase from Corynebacterium sp. 2-4-1. The sequence of the AMP-binding motif, GXGXXG, was found in the deduced N-terminal region. The amino acid sequence of the fructosyl-amino acid oxidase showed no similarity to that of fungal fructosyl-amino acid oxidases. In addition, substrate specificities of this fructosyl-amino acid oxidase were different from those of other fructosyl-amino acid oxidases. The fructosyl-amino acid oxidase of Corynebacterium sp. 2-4-1 is an enzyme that has unique substrate specificity and primary structure in comparison with fungal fructosyl-amino acid oxidases.     

Enhancing fungal production of galactaric acid

Galactaric (mucic) acid is a symmetrical six carbon diacid which can be produced by oxidation of galactose with nitric acid, electrolytic oxidation of d-galacturonate or microbial conversion of d-galacturonate. Both salts and the free acid of galactarate have relatively low solubility, which may create challenges for a microbial host. Galactaric acid was most soluble at pH values around 4.7 in the presence of ammonium or sodium ions and less soluble in the presence of potassium ions. Solubility increased with increasing temperature. Production of galactaric acid by Trichoderma reesei D-161646 was dependent on temperature, pH and medium composition, being best at pH 4 and 35 °C. Up to 20 g L^?1 galactaric acid were produced from d-galacturonate using a fed-batch strategy with lactose as co-substrate and both ammonium and yeast extract as nitrogen sources. Crystals of galactaric acid were observed to form in the broth of some fermentations.