The appearance of an enzyme activity catalysing the conversion of norsolorinic acid to averantin in Aspergillus parasiticus cultures

The activity of the enzyme responsible for the conversion of norsolorinic acid to averantin was studied in two strains of Aspergillus parasiticus. Cell-free extracts of the enzyme were purified from different aged mycelia and little activity was found prior to 24 hours after inoculation but this quickly reached a maximum at 48 hours and declined thereafter. Both strains of A. parasiticus, one in aflatoxin producing strain, the other a versicolorin A accumulating mutant, showed this trend. It was concluded that the enzyme responsible for this conversion was a secondary metabolic enzyme and was distinct from alcohol and mannitol dehydrogenases.     

湘江河岸土壤中高产甲壳素脱乙酰酶菌株的筛选及鉴定

【目的】甲壳素脱乙酰酶(CDA)是将天然甲壳素生物转化为可商品化利用的壳聚糖的关键酶。本文旨在从湘江河岸的土壤中筛选可高产CDA的新菌株。【方法】以甲壳素为唯一碳源,利用4'-硝基乙酰苯胺为显色剂,通过变色圈法进行产CDA菌株初筛,产酶活性分析复筛;通过形态学和ITS区序列特征对菌株进行鉴定。【结果】从湘江(长沙段)河岸边的土壤中分离出的117株菌株中筛选到可产CDA的菌株30株,其中4株具有较强产CDA的能力。进一步经发酵产酶分析验证,菌株A1具有较强的产CDA能力,其胞外CDA酶活高达13.21 U/m L。结合形态学和ITS区序列特征,菌株A1初步鉴定为层生镰孢菌。【结论】从湘江河岸边的土壤中筛选到可高产CDA的菌株A1,具有较好的开发应用前景。     

Enumeration of gut associated extracellular enzyme‐producing yeasts in some freshwater fishes

Extracellular enzyme‐producing yeasts might be involved in the supplementation of enzymes within the gastrointestinal tract of fish. The present study was intended to detect yeasts in the intestine of three Indian major carps (Labeo rohita, Catla catla, Cirrhinus mrigala), three exotic carps (Hypophthalmichthys molitrix, Ctenopharyngodon idella, Cyprinus carpio), as well as Nile tilapia (Oreochromis niloticus), and to identify the most promising extracellular enzyme‐producing (e.g. amylase, protease, lipase, cellulase, xylanase and phytase) yeast strains by 18S rDNA sequence analysis. Selected for qualitative enzyme assay were 121 yeast strains, from which 28 were further studied for quantitative enzyme assay. The strain CMH6A isolated from C. mrigala exhibited the best extracellular enzyme activities except for amylase and cellulase. The strain ONF19B isolated from O. niloticus was noted as the best extracellular enzyme producer among the strains that produced all of the extracellular enzymes studied. Sequencing of the 18S rDNA fragment followed by nucleotide blast in the National Centre for Biotechnology Information (NCBI) GenBank revealed that strains CMH6A and ONF19B were similar to Pichia kudriavzevii (Accession no. KF479403 ) and Candida rugosa (Accession no. KF479404 ), respectively. The test of antagonism (in vitro) revealed that the isolated yeasts could not affect the growth of the autochthonous gut bacteria. This might indicate likely co‐existence of autochthonous yeasts and bacteria in the fish gut. Further research is necessary to explore the possibilities of utilizing the extracellular enzyme‐producing yeasts detected in the present study for commercial aquaculture.     

Serologic Characteristics of Glucokinases from Entamoeba histolytica and Related Species1

SYNOPSIS. An enzyme inhibition technic was employed for quantitative comparison of the serologic properties of glucokinases from 4 groups of amebas which are structurally indistinguishable species: Entamoeba histolytica, E. moshkovskii, E. invadens and E. terrapinae. Antiglucokinase was prepared by immunizing rabbits with crude extracts of DKB and Laredo strains of E. histolytica. The combination of amebal glucokinase and homologous antibody was a pseudoirreversible reaction. The inhibition was proportional to the amount of antibody until at least 60% of the enzyme was inhibited, and the inhibition was 96–92% in the region of antibody excess. The nature of the inhibition was uncompetitive with respect to substrate. The presence of substrate had no effect upon the inhibition. Anti-DKB glucokinase inhibited equally glucokinases from DKB, JH, K9, 200, NRS, BH, JI, F22, and N strains. Anti-Laredo glucokinase equally inhibited glucokinases from Laredo, Huff, JA, AG, and 403 strains; 2.5–2.9 times as much antiserum was required to produce the same degree of inhibition between antisera and strains of heterologous group as with homologous antigen. Anti-DKB and anti-Laredo glucokinases cross-reacted with the enzyme from E. moshkovskii, but not with enzymes from reptilian amebas. A new glucokinase-anti-glucokinase dissociation test was developed which provides a method for qualitative differentiation of antiglucokinase against DKB strain from anti-glucokinase against Laredo strain.     

广西三种真红树植物可培养细菌多样性及其生物活性初筛

为了挖掘真红树植物潜在细菌新物种和生物活性物质,丰富红树林微生物多样性,为新型活性产物开发提供菌株资源。该文从秋茄、木榄和红海榄三种广西来源的真红树植物及其生境中,按根、茎、叶、花、果实和泥土分成22份样品,选用8种不同培养基分离可培养细菌,通过16S rRNA基因序列鉴定,分析其多样性,采用纸片法筛选细菌发酵粗提物的抑菌活性,点植法测试其酶活性。结果表明：(1)共分离获得可培养细菌35株,隶属于23个科28个属,芽孢杆菌属占细菌总数的14.3%,为优势菌属,同时发现11株潜在的新细菌资源。(2)活性筛选获得4株细菌具有抑菌活性,16株细菌具有酶活性,芽孢杆菌属是酶活性优势菌属。综上所述,广西真红树植物可培养细菌多样性丰富,部分细菌具有抑菌活性和酶活性,在新型抗生素和酶应用方面具有一定的开发潜力。     

The product of theade1 gene inSchizosaccharomyces pombe: A bifunctional enzyme catalysing two distinct steps in purine biosynthesis

Summary The assignment of the knownade genes to steps in purine biosynthesis inSchizosaccharomyces pombe has been completed with the demonstration that anade3 mutants lacks FGAR amidotransferase,ade1A mutants lack GAR synthetase andade1B mutants lack AIR synthetase. A comparison of enzyme activity with map position forade1 mutants shows that (1) complementingade1A mutants lack GAR synthetase but possess wild type amounts of AIR synthetase, (2) complementingade1B mutants lack AIR synthetase but posses variable amounts of GAR synthetase, (3) non-complementing mutants lack both activities. In wild type strains the two activities fractionate together throughout a hundred-fold purification. Hence theade1 gene appears to code for a bifunctional enzyme catalysing two distinct steps in purine biosynthesis. The two activities are catalysed by two different regions of the polypeptide chain which can be altered independendently by mutation. Gel filtration studies on partially purified enzymes from wild type and various complementing mutant strains, indicate that the bifunctional enzyme is a multimer consisting of between four and six sub-units of 40,000 daltons each. GAR synthetase activity is associated with both the monomeric and multimeric forms but AIR synthetase is only associated with the multimer. A comparison of enzyme levels between diploids and their original complementing haploid strains suggests that complementation is due to hybrid enzyme formation.     

Lytic Enzyme toward Aniline-assimilating Rhodococcus erythropolis AN-13: Screening and Production

A bacterial strain, SH-548, that produces a lytic enzyme toward intact cells of aniline-assimilating Rhodococcus erythropolis AN-13, was isolated from soil. The isolated bacterium was identified as a Flavobacterium species. The growth conditions for the enzyme production by Flavobacterium sp. SH-548 were examined; organic nitrogen compounds, such as meat extract and Polypepton, were effective for its production. The lytic enzyme of this strain lysed intact cells of Rhodococcus, Bacillus, Nocardia, Corynebacterium, Brevibacterium, Streptococcus, Micrococcus, Cellulomonas and DAB (diaminobutyric acid)-type coryneform bacterial strains. However, it did not act on those of Staphylococcus aureus or gram-negative bacteria, Enterobacter, Escherichia, Klebsiella, Proteus or Pseudomonas strains. Bacterial strains having cell walls of the glycolyl type were readily lysed by this enzyme.     

Studies on Xylanase from Microorganisms

As xylanase-producing microorganisms, 64 strains belonging to the genus Streptomyces were isolated from the barn-yard manures, silages and litters collected in Hokkaido district. Among these isolates the strain 102–1–4, which was found to be a new species under taxonomical studies and named Streptomyces xylophagus nov. sp., had the most outstanding ability for the enzyme production. In addition to the isolates, 38 strains of Streptomyces and 480 strains of filamentous fungi which have been preserved in our culture collection were also examined on their ability to produce the enzyme. 1) Among the strains of Streptomyces tested, only two strains, St. albogriseolus IAM 0031 and St. olivaceus IAM 0025 were found to have the ability, but their abilities were less than that of St. xylophagus nov. sp. 2) Out of 480 strains of fungi tested, Chaetomium, Schyzophyllum, Trametes, Echinodontium, Alternaria, Cepharosporium, Cercospora, Gibberella, Glomerella and Macrosporium produced the enzyme. Especially, Ch. trilateral 2264 was the most excellent.     

Lysinoalanine Formation in Gluten: A Conformational Effect

The production of pectolytic enzyme in the genus Kluyveromyces was investigated. The production of the enzyme was dependent on the strain, and some strains belonging to K. fragilis, K. Marxianus, and K. Wickerhamii produced this enzyme among 11 species (29 strains) of the genus Kluyveromyces. K. Fragilis IFO 0288 produced at least four endo-polygalacturonases which have different molecular weights. The dominant endo-polygalacturonase in the culture filtrate of the strain was purified and isolated as crystals. The purified enzyme was homogeneous based on analysis by polyacrylamide gel electrophoresis and ultracentrifugation. The enzyme was a glycoprotein having an isoelectric point around pH 5.6. The sedimentation coefficient (s_2o,w) was 3.77S, and the molecular weight was around 33,000. The enzyme contained aspartic acid (asparagine), serine,threonine, and glycine at relatively high levels. The enzyme showed the highest activity around pH 5.0 and was stable at pH 5.0 up to 30°C. With the enzyme, and activity which releases highly polymerized pectin from various protopectins (protopectinase activity) was found.     

Virulence and Enzyme Production of Erwinia carotovora ssp. atroseptica on Potato Tuber Tissue

The elicitation of pathogenesis on potato tuber slices by 6 strains of Erwinia carotovora ssp. atroseptica was investigated by neutral red vital staining and has been compared with bacterial growth rate, penetration ability, enzyme production and enzyme spectrum. The induction of enzyme synthesis (particularly, of the extracellular polygalacturonase) elicites the rot attack on tuber tissue and this requires a sufficient bacterial density. Due to wound healing, the inductors of enzyme production are removed, and after 48 h enzymes do not attack tuber tissue any more. Therefore, growth rate and penetration ability (to get the necessary bacterial density and inductor substances) may limit virulence. A similar influence of enzyme production and enzyme spectrum of the strains on the virulence was not detected.     

Localisation and distribution of alcohol-cytochrome 553 reductase in acetic acid bacteria

Alcohol-cytochrome 553 reductase was detected in several strains of the mesoxydans and oxydans group ofAcetobacter. A similar enzyme, but with a higher optimum pH, was detected inAcetobacter aceti (liquefaciens) and in two strains ofGluconobacter.Intermittent ultrasonic disruption ofAcetobacter aceti cells, strainsrancens T-5 andmobilis 6428, showed that the alcohol-cytochrome 553 reductase was mainly localized on the cell hull. The NADP-linked aldehyde dehydrogenase appeared to be present as a cytoplasmic component.The oxidation rate of ethanol and acetaldehyde by intact resting cells which have been grown with either glucose or ethanol as a carbon source under either neutral or acidic conditions was nearly identical. The ethanol oxidizing enzyme system thus behaved as constitutive enzymes, as would be expected if they were bound to the cell hull.The results support the hypothesis that the alcohol-cytochrome 553 reductase is one of the important components of the enzyme system responsible for the physiological production of acetic acid from ethanol by acetic acid bacteria.     

The involvement of alcohol dehydrogenase and aldehyde dehydrogenase in alcohol/aldehyde metabolism in Drosophila melanogaster

In this study we have examined the roles of alcohol dehydrogenase, aldehyde oxidase, and aldehyde dehydrogenase in the adaptation of Drosophila melanogaster to alcohol environments. Fifteen strains were characterized for genetic variation at the above loci by protein electrophoresis. Levels of in vitro enzyme activity were also determined. The strains examined showed considerable variation in enzyme activity for all three gene-enzyme systems. Each enzyme was also characterized for coenzyme requirements, effect of inhibitors, subcellular location, and tissue specific expression. A subset of the strains was chosen to assess the physiological role of each gene-enzyme system in alcohol and aldehyde metabolism. These strains were characterized for both the ability to utilize alcohols and aldehydes as carbon sources as well as the capacity to detoxify such substrates. The results of the above analyses demonstrate the importance of both alcohol dehydrogenase and aldehyde dehydrogenase in the in vivo metabolism of alcohols and aldehydes.     

VARIATION OF ENZYME ACTIVITIES AT A BRANCHED PATHWAY INVOLVED IN THE UTILIZATION OF GLUCONATE IN ESCHERICHIA COLI

Abstract.— Twenty‐four strains of Escherichia coli from the ECOR collection were characterized for growth rate in gluconate minimal salts medium and for Vmax and Km of the three enzymes (gluconokinase, 6‐phosphogluconate dehydrogenase, and 6‐phosphogluconate dehydratase) that form a branch point for the utilization of gluconate. A total of 11 characters–growth rate, three Vmax values, four Km values, and three Vmax/Km values–were determined for these 24 ECOR strains. Most of the characters were normally distributed. Statistical tests showed that growth rate is significantly less variable than enzyme activities. Also, analyses of variance showed significant differences among strains and among the extant five genetic groups of E. coli for the characters measured. A Mantel test showed that, for some characters, closely related strains shared similar character values. Two hypotheses regarding the relationships between growth rate and enzyme activity and between various enzyme activities were tested. None of the expected correlations between growth rate and enzyme activity or between enzyme activities was detected. The results were discussed in terms of metabolic control analysis and neutral theory.     

Degradation of Nucleic Acids and their Related Compounds by Microbial Enzymes

Seven strains of microorganisms selected by the previous screening tests were further compared on their ability to produce extracellular enzyme systems capable of degrading RNA into 5′-ribonucleotides. As a result, two strains of Streptomyces were finally concluded to be most preferable. When these two were applied, the rate of 5′-nucleotide production reached up to 70%.

Bacillus subtilis was outstanding in its activity to degrade RNA, but its PDase activity producing 5′-nucleotides from RNA was found to be lower than those of Streptomyces strains. A pathway involving 3′- and 5′-nucleotides as intermediates was proposed for the degradation of RNA by the Bacillus enzyme system. The activity of RNA-degrading enzyme system of Bacillus subtilis contained in the supernatant of culture fluid was found to be lost at 700°C but remained to certain extent at 100°C, a possible mechanism for the phenomenon being discussed. Usability of the Bacillus enzyme system in the practical production of 5′-nucleotides under the condition of high RNA concentration was discussed.     

Identification of some industrially importantActinoplanes species

Summary Three actinomycetes designated A294, A385 and A448 all produced novel inhibitors of an enzyme involved in the regulation of mammalian cholesterol metabolism. The organisms were identified as members of the genusActinoplanes using morphological and chemotaxonomic characters. Numerical phenetic data obtained for clusters defined at the 77.5% S_sm similarity level in the taxonomic study ofActinoplanes and related taxe by Goodfellow et al. [6], was used to construct a probabilistic identification matrix for the genusActinoplanes. This was in an attempt to characterise the producing strains to species level. Identification scores for the three organisms, together with two type strains, were determined using the MATIDEN program of Sneath [36]. Strains A294 and A385 did not identify correctly with any cluster. Strain A448 was identified asActinoplanes derwentensis. The two type strains identified within acceptable statistical limits to their correct clusters. Due to the absence of other taxonomic criteria with which to speciate members of the genusActinoplanes we recommend that the two unidentified producing strains should be considered novel until such time as surther techniques become available.     

Degradation of Barley Glucan and Lichenan by a Bacillus pumilus Enzyme

A bacterial strain was isolated from soil, which rapidly degraded purified barley β-glucan as well as lichenan. The strain belonged to Bacillus pumilus, and some authentic strains of this species were also shown to hydrolyze the gluean. An enzyme active on the above substrates but not on laminaran and on CM-cellulose was partially purified from the culture fluid. This enzyme, about 27,000 in molecular weight, was found to cleave a β-(1 → 4) linkage adjacent to a β-(1 → 3) in the polymers. It was suggested that only an enzyme of this type should be called a ‘lichenanase’ and discriminated from cellulases and laminaranases.     

Partial Purification,of Extracellular Cellulase from Pyricularia oryzae and Comparison of the Elution Patterns among Various Strains

In order to explain the difference in extracellular cellulase activities (C₁ and C_x enzyme activities) among various strains of P. oryzae, the elution patterns from the column were compared among various strains, following each step of the partial purification.

The crude enzymes, prepared by ammonium sulfate fractionation (0.2~0.8 sat.) from the culture filtrates, which were obtained from various strains of P. oryzae cultured on rice plant powder as the carbon source, were fractionated by DEAE-Sephadex A–50 chromatography into two components; the passing-through fraction (I) and the fraction (II) adsorbed and eluted from the column with 0.5 M NaCl The percentage of the enzyme activity (C_x enzyme activity) in fraction I to that of the crude extract was found to vary chracteristically according to the strain, and the variation was in a good correlation to that of the extracellular cellulase activities.

Fractions I and II were then separated by Sephadex G–100 into two (peaks a and b) and at least five (peaks c, d, e, f and g) components, respectively. The activities in peaks a, b and g were found to vary according to the strain, while those of peaks c and e were common among various strains.

The cell wall fraction prepared from C–3 strain, which was previously shown to be low in enzyme activity and thus out of the correlation between the degree of pathogenicity and extracellular cellulase activity, was found to exhibit higher cellulase activities (C₁ and C_x enzyme activities) than those of other strains examined. Thus, the low extracellular cellulase activity in the case of C–3 strain was suggested to be due to the abnormality in the mechanism of enzyme excretion.     

Localization and stability of hydrogenases from aerobic hydrogen bacteria

Alcaligenes eutrophus strains H 16, B 19, G 27 and N9A contained two different hydrogenases. One enzyme catalyzed the reduction of NAD by hydrogen and was strictly localized in the soluble cell fraction, while the second enzyme was found to be particulate and unable to react with NAD.All other tested strains, Alcaligenes paradoxus SA 29, Pseudomonas facilis, P. palleronii RH 2, Pseudomonas sp. strain GA 3, Paracoccus denitrificans, Aquaspirillum autotrophicum SA 32, and Corynebacterium autotrophicum 14g and 7C contained only a single enzyme exclusively bound to membranes. This was established using fractional centrifugation, indicator enzyme systems, gentle methods of cell disintegration and discontinuous sucrose density gradient centrifugation. In cell-free extracts obtained by rough disruption (sonication) of cells, hydrogenase was associated to particles of different size and sedimentation velocity. A partial solubilization of hydrogenase caused by sonication was observed with P. facilis.Without exception, the particulate hydrogenases were found (1) to be unable to reduce pyridine nucleotides, and (2) to reduce methylene blue at an extremely high activity. The eminent reaction rate of 34 moles H₂ oxidized per min and mg protein has been determined in particle suspensions of Pseudomonas sp. strain GA 3. All hydrogenases were stable during storage under hydrogen atmosphere, except the soluble enzyme from A. eutrophus H 16 which was shown to be more stable under aerobic conditions.     

The distribution of transglucosyl-amylase amongCandida yeasts

Candida transglucosyl-amylase (Sawai, 1958, 1960; Sawai and Hehre, 1962) was found to be produced intracellularly by three of 28 species within theCandida genus, i.e. by 29 of 35C. tropicalis strains, all of 15C. albicans strains and oneC. claussenii. The novel capacity of this enzyme to catalyze transglucosylation from starch was substantiated by the observation that preparations from all the positive strains were highly effective in causing such transfer to glycerol, and by the fact that isomaltose was synthesized from dextrin by the action of all preparations checked for this capacity (21 strains). Two strains ofC. pelliculosa produced an amylase of a different kind, while representatives of the remaining 24Candida species tested (one or two strains of each species) gave no evidence of amylase production. In view of the narrow and apparently specific distribution of transglucosyl-amylase within the genus, it seems possible that production of this enzyme might be useful as an aid in the taxonomic differentiation ofCandida yeasts.A preliminary account of this work was presented at the 60th National Meeting of the Society of American Bacteriologists, held at Philadelphia, Pa., U.S.A. (Sawai and Hehre, 1960).     

Molecular Characterization of <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> Strains Isolated from <Emphasis Type="Italic">Agave fourcroydes</Emphasis> (Lem.) in Yucatan,Mexico

The molecular characterization of 14 strains of Kluyveromyces marxianus isolated from Agave fourcroydes (Lem.) in Yucatan, Mexico, was performed by AP-PCR analysis, PCR-RFLP of 5.8S-ITS, and complete NTS regions. A sequence analysis of the D1/D2 domain of the 26S rDNA was also carried out in six selected strains. The AP-PCR approach had the highest discrimination power for the molecular characterization of new henequen K. marxianus strains. PCR-RFLP of 5.8S-ITS regions did not reveal polymorphisms in this group of strains. The restriction enzyme digestion analysis of NTS region enables the separation among strains which coincides with ascospore shape groups. The molecular tools used in this article may be useful to confirm a preliminary screen of yeasts isolated from henequen without the use of growth characteristics or morpho-physiological tests.