Characterization of Alternaria and Penicillium species from similar substrata based on growth at different temperature,pH and water activity

Fifty-eight Alternaria isolates representing 10 species or species-groups and 66 Penicillium isolates representing 18 species were examined for their growth response to the combined effects of water activity, temperature and pH in an extended Central Composite Design. Growth responses were recorded as colony diameter after one and two weeks of growth and analysed using different multivariate statistical analyses. The isolates, when analysed by Principal Component Analysis, clustered according to their genus and to some degree to species or species groups and not according to substratum as excepted. Soft Independent Modelling of Class Analogy and Response Surface Analysis showed that growth responses or growth profiles may be used as classification tool. Partial Least Squares Regression showed that a combination of two different media based on Dichloran Rose bengal Yeast Extract Sucrose agar incubated at two different temperatures were enough to get genus segregation and to some extent species segregation. The results also showed that water activity, temperature and pH interact strongly in their effect on growth rates and that the squared products (optima) of water activity, temperature and pH for each isolate were important for modelling the data sufficiently.     

Penicillium aurantiogriseum Dierckx 1901: Producer of Diketopiperazine Alkaloids (Roquefortine and 3,12-Dihydroroquefortine), Isolated from Permafrost

Secondary metabolites of three strains of Penicillium aurantiogriseumisolated from permafrost sediments were identified. It was found that these fungi synthesized the diketopiperazine alkaloids roquefortine and 3,12-dihydroroquefortine. The strain VKM FW-766 synthesized alkaloids in the course of certain growth-related processes. When the strain was grown on a mineral medium, the time courses of the roquefortine and 3,12-dihydroroquefortine concentrations were characterized by biphasic curves.     

Aldo-keto reductase from Helicobacter pylori--role in adaptation to growth at acid pH

Pyridine-linked oxidoreductase enzymes of Helicobacter pylori have been implicated in the pathogenesis of gastric disease. Previous studies in this laboratory examined a cinnamyl alcohol dehydrogenase that was capable of detoxifying a range of aromatic aldehydes. In the present work, we have extended these studies to identify and characterize an aldoketo reductase (AKR) enzyme present in H. pylori. The gene encoding this AKR was identified in the sequenced strain of H. pylori, 26695. The gene, referred to as HpAKR, was cloned and expressed in Escherichia coli as a His-tag fusion protein, and purified using nickel chelate chromatography. The gene product (HpAKR) has been assigned to the AKR13C1 family, although it differs in specificity from the two other known members of this family. The enzyme is a monomer with a molecular mass of approximately 39 kDa on SDS/PAGE. It reduces a range of aromatic aldehyde substrates with high catalytic efficiency, and exhibits dual cofactor specificity for both NADPH and NADH. HpAKR can function over a broad pH range (pH 4-9), and has a pH optimum of 5.5. It is inhibited by sodium valproate. Its substrate specificity complements that of the cinnamyl alcohol dehydrogenase activity in H. pylori, giving the organism the capacity to reduce a wide range of aldehydes. Generation of an HpAKR isogenic mutant of H. pylori demonstrated that HpAKR is required for growth under acidic conditions, suggesting an important role for this enzyme in adaptation to growth in the gastric mucosa. This AKR is a member of a hitherto little-studied class.     

Isolation, purification and characterization of a novel glucose oxidase from Penicillium sp. CBS 120262 optimally active at neutral pH

A novel glucose oxidase (GOX), a flavoenzyme, from Penicillium sp. was isolated, purified and partially characterised. Maximum activities of 1.08U mg(-1)dry weight intracellular and 6.9U ml(-1) extracellular GOX were obtained. Isoelectric focussing revealed two isoenzymes present in both intra- and extracellular fractions, having pI's of 4.30 and 4.67. GOX from Penicillium sp. was shown to be dimeric with a molecular weight of 148kDa, consisting of two equal subunits with molecular weight of 70k Da. The enzyme displayed a temperature optimum between 25 and 30 degrees C, and an optimum pH range of 6-8 for the oxidation of beta-d-glucose. The enzyme was stable at 25 degrees C for a minimum of 10h, with a half-life of approximately 30 min at 37 degrees C without any prior stabilisation. The lyophilized enzyme was stable at -20 degrees C for a minimum of 6 months. GOX from Penicillium sp. Tt42 displayed the following kinetic characteristics: Vmax, 240.5U mg(-1); Km, 18.4mM; kcat, 741 s(-1) and kcat/Km, 40 s(-1)mM(-1). Stability at room temperature, good shelf-life without stabilisation and the neutral range for the pH optimum of this GOX contribute to its usefulness in current GOX-based biosensor applications.     

Dunaliella acidophila: an algae with a positive zeta potential at its optimal pH for growth*

Abstract. The zeta potential (which approximates the surface potential) of the acid resistant green alga Dunaliella acidophila (optimal growth at pH 1.0) and the salt resistant D. parva (grown at pH 7.6) were calculated from the electrophoretic mobility of cells as determined by means of free-flow electrophoresis. Dunaliella acidophila cells exhibit a positive zeta potential (+5 to +20mV) at acidic external pH values, whereas negative zeta potentials (-30 mV) were measured at neutral pH values. Negative zeta potentials of the same order of magnitude were also measured for D. parva cells (pH 7.6). Low concentrations of La³⁺ and A1³⁺ did not affect the positive zeta potential of D. acidophila at acidic pH values, whereas higher concentrations caused a shift to more positive potentials. However, at neutral pH these cations caused a significant decrease of the negative zeta potential. The impermeant polycation poly-L-lysine acted in a similar manner to A1³⁺ or La³⁺. The effect of Impermeant cations and anions on various physiological reactions also supports the existence of a positive zeta potential for D. acidophila and of a negative zeta potential for D. parva: polycations such as DEAE-dextran and poly-L-lysine strongly inhibitied photosynthesis and mobility of D. parva, but did not affect these reactions in D. acidophila. Comparable differential inhibitions were also observed for A1³⁺ and La³⁺. Impermeant anions such as Dextran-sulfate exhibited effects in the opposite direction: inhibition was stronger with D. acidophila and weaker with D. parva. However, the differential inhibition by impermeant anions was much less pronounced in comparison with impermeant cations due to the relatively high pK_a values of anionic solutes and consequently relatively high protonation at pH 1.0. The physiological consequences of an asymmetrically charged plasma membrane (excess of positive charges outside, excess of negative charges on the cytoplasmic side) of D. acidophila are discussed in regard to the extreme acid resistance of this alga and its resistance to cationic toxic solutes in industrial wastes.     

Isolation of viridicatin from Penicillium palitans

Summary Viridicatin, C₁₅H₁₁O₂N, has been shown to be a metabolic product of several fungi including P. palitans, P. olivino-viride, P. puberulum, P. martensii, P. crustosum, P. granulatum, and P. cyclopium. Although these strains also produce the tremorgenic mycotoxin, tremortin, no toxicity could be shown for viridicatin. Viridicatin does not appear to be a component of the tremortin molecule. A quantitative colorimetric assay has been developed for viridicatin.This is a laboratory of the Northern Utilization Research and Development Division, Agricultural Research Service, U.S. Department of Agriculture. Mention of firm names or trade products does not imply that they are endorsed or recommended by the Department of Agriculture over other firms or similar products not mentioned.     

Isolation and Properties of Cellobiase from Penicillium verruculosum

Cellobiase (-D-glucosidase) with a molecular weight of 100 kDa and pI 5.2 was isolated from the cellulolytic system of Penicillium verruculosum. Kinetic parameters of enzymatic hydrolysis of cellobiose, gentiobiose, sophorose, and synthetic substrates, i. e. methylumbelliferyl and p-nitrophenyl sugar derivatives were determined. Glucose and D-glucose--lactone competitively inhibited cellobiase (K _i0.19 mM and 17 M, respectively). Glucosyl transfer reactions were studied with cellobiose as a single substrate and in the mixture of cellobiose and methylumbelliferyl cellobioside. The product composition was determined in these systems. The ratio of hydrolysis and transfer reaction rates for cellobiose conversion was calculated.     

Isolation of Yersinia enterocolitica from well water and growth in distilled water.

Yersinia enterocolitica was recovered from well water during a large water-borne outbreak of gastrointestinal illness. Isolates were predominantly Nilehn biotype 1, of which 57% were serologically nontypable. Isolation and enumeration of these Y. enterocolitica strains were made on M-Endo broth. Laboratory studies were conducted on selected isolates to establish the growth of Y. enterocolitica in distilled water and the competitive growth of this organism in various enteric media. Growth was obtained in sterile distilled water without added nutrients at 4, 25, and 37 degrees C. M-Endo medium gave equal or better recovery of Y. enterocolitica in competitive growth studies than did other commonly used enteric media using the membrane filter technique and incubating at 35 degrees C. All well water isolates were confirmed biochemically at 25 and 35 degrees C and serotyped, and antibiotic susceptibility tests were performed.     

Isolation of Yersinia enterocolitica from well water and growth in distilled water.

Yersinia enterocolitica was recovered from well water during a large water-borne outbreak of gastrointestinal illness. Isolates were predominantly Nilehn biotype 1, of which 57% were serologically nontypable. Isolation and enumeration of these Y. enterocolitica strains were made on M-Endo broth. Laboratory studies were conducted on selected isolates to establish the growth of Y. enterocolitica in distilled water and the competitive growth of this organism in various enteric media. Growth was obtained in sterile distilled water without added nutrients at 4, 25, and 37 degrees C. M-Endo medium gave equal or better recovery of Y. enterocolitica in competitive growth studies than did other commonly used enteric media using the membrane filter technique and incubating at 35 degrees C. All well water isolates were confirmed biochemically at 25 and 35 degrees C and serotyped, and antibiotic susceptibility tests were performed.     

Isolation and properties of cellobiase from Penicillium verruculosum]

Cellobiase (beta-D-glucosidase) with a molecular weight of 100 kDa and pI 5.2 was isolated from the cellulolytic system of Penicillium verruculosum. Kinetic parameters of enzymatic hydrolysis of cellobiose, gentiobiose, sophorose, and synthetic substrates, i.e. methylumbelliferyl and p-nitrophenyl sugar derivatives were determined. Glucose and D-glucose-delta-lactone competitively inhibited cellobiase (Ki = 0.19 mM and 17 microM, respectively). Glucosyl transfer reactions were studied with cellobiose as a single substrate and in the mixture of cellobiose and methylumbelliferyl cellobioside. The product composition was determined in these systems. The ratio of hydrolysis and transfer reaction rates for cellobiose conversion was calculated.     

Effect of water activity and temperature on growth of Penicillium citreoviride and Penicillium citrinum on MiGao (rice cake)

Contamination by mold is a serious problem in steam-cooked rice cake, a traditional Chinese food. Growth responses to different temperatures and water activity values for Penicillium citreoviride and Penicillium citrinum, two of the most common molds, were investigated. Partial least square regression analysis showed that the growth of the two fungi did not differ in response to changes in water activity and temperature. Optimum water activity for growth was 0.90 and optimum temperatures for growth were 30 degrees C in most cases.     

Isolation of a Pure Dextranase from Penicillium funiculosum

A dextranase, produced by Penicillium funiculosum, was purified 1,000-fold to yield the enzyme which was demonstrated by gel electrophoresis and electrofocusing to be a homogeneous protein. The purification method included acetone partition, ammonium sulfate fractionation, gel filtration, iron defecation and precipitation, and diethylaminoethyl-cellulose chromatography. The pure enzyme was also obtained by preparative gel electrophoresis. Gel-permeation chromatography indicates a molecular weight of 41,000. An isoelectric pH of 4.6 was established by electrofocusing. A 1-mg amount of the enzyme hydrolyzes a dextran substrate to yield 27,000 isomaltose reducing units in 2 hr.     

Isolation and characterization of a beta-primeverosidase-like enzyme from Penicillium multicolor

p-Nitrophenyl and eugenyl beta-primeveroside (6-O-beta-D-xylopyranosyl-beta-D-glucopyranoside) hydrolytic activity was found in culture filtrate from Penicillium multicolor IAM7153, and the enzyme was isolated. The enzyme was purified as a beta-primeverosidase-like enzyme by precipitation with ammonium sulfate followed by successive chromatographies on Phenyl Sepharose, Mono Q, and beta-galactosylamidine affinity columns. The molecular mass was estimated to be 50 kDa by SDS-PAGE and gel filtration. The purified enzyme was highly specific toward the substrate p-nitrophenyl beta-primeveroside, which was cleaved in an endo-manner into primeverose and p-nitrophenol, but a series of beta-primeveroside as aroma precursors were hydrolyzed only slightly as substrates for the enzyme. In analyses of its hydrolytic action and kinetics, the enzyme showed narrow substrate specificity with respect to the aglycon and glycon moieties of the diglycoside. We conclude that the present enzyme is a kind of beta-diglycosidase rather than beta-primeverosidase.