Production and Characterization of a Novel Yeast Extracellular Invertase Activity Towards Improved Dibenzothiophene Biodesulfurization

ABSTRACT Erwinia rhapontici is able to convert sucrose into isomaltulose (palatinose, 6-O-α-d-glucopyranosyl-d-fructose) and trehalulose (1-O-α-d-glucopyranosyl-d-fructose) by the activity of a sucrose isomerase. These sucrose isomers cannot be metabolized by plant cells and most other organisms and therefore are possibly advantageous for the pathogen. This view is supported by the observation that in vitro yeast invertase activity can be inhibited by palatinose, thus preventing sucrose consumption. Due to the lack of genetic information, the role of sucrose isomers in pathogenicity has not been evaluated. Here we describe for the first time the cloning and characterization of the palatinose (pal) genes fromErwinia rhapontici. To this end, a 15-kb chromosomal DNA fragment containing nine complete open reading frames (ORFs) was cloned. The pal gene products of Erwinia rhapontici were shown to be homologous to proteins involved in uptake and metabolism of various sugars from other microorganisms. ThepalE, palF, palG, palH, palK, palQ, and palZgenes were oriented divergently with respect to the palRand palI genes, and sequence analysis suggested that the first set of genes constitutes an operon. Northern blot analysis of RNA extracted from bacteria grown under various conditions implies that the expression of the palI gene and the palEFGHKQZgenes is oppositely regulated at the transcriptional level. Genes involved in palatinose uptake and metabolism are down regulated by sucrose and activated by palatinose. Palatinose activation is inhibited by sucrose. Functional expression of palI andpalQ in Escherichia coli revealed sucrose isomerase and palatinase activity, respectively.

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A SUPPRESSOR OF snf1 MUTATIONS CAUSES CONSTITUTIVE HIGH-LEVEL INVERTASE SYNTHESIS IN YEAST

Genetics ◽

10.1093/genetics/107.1.19 ◽

1984 ◽

Vol 107 (1) ◽

pp. 19-32

Author(s):

Marian Carlson ◽

Barbara C Osmond ◽

Lenore Neigeborn ◽

David Botstein

Keyword(s):

Genetic Characterization ◽

Glucose Repression ◽

Invertase Activity ◽

Complementation Group ◽

Regulatory Function ◽

Wild Type ◽

Suppressor Mutations ◽

Complementation Groups ◽

High Level

ABSTRACT The SNF1 gene product of Saccharomyces cerevisiae is required to derepress expression of many glucose-repressible genes, including the SUC2 structural gene for invertase. Strains carrying a recessive snf1 mutation are unable to ferment sucrose. We have isolated 30 partial phenotypic revertants of a snf1 mutant that were able to ferment sucrose. Genetic characterization of these revertants showed that the suppressor mutations were all recessive and defined eight complementation groups, designated ssn1 through ssn8 (suppressor of snf1). The revertants were assayed for secreted invertase activity, and although activity was detected in members of each complementation group, only the ssn6 strains contained wild-type levels. Synthesis of secreted invertase in ssn6 strains was found to be constitutive, that is, insensitive to glucose repression; moreover, the ssn6 mutations also conferred constitutivity in a wild-type (SNF1) genetic background and are, therefore, not merely suppressors of snf1. Pleiotropic defects were observed in ssn6 mutants. Genetic analysis suggested that the ssn6 mutations are allelic to the cyc8 mutation isolated by R. J. Rothstein and F. Sherman, which causes increased production of iso-2-cytochrome c. The data suggest a regulatory function for SSN6.

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ISOLATION AND CHARACTERIZATION OF NEUROSPORA MUTANTS AFFECTED IN INVERTASE SYNTHESIS

Genetics ◽

10.1093/genetics/106.4.591 ◽

1984 ◽

Vol 106 (4) ◽

pp. 591-599

Author(s):

Deborah B Lee ◽

Stephen J Free

Keyword(s):

Neurospora Crassa ◽

Large Scale ◽

Invertase Activity ◽

Free Medium ◽

Wild Type ◽

Isolation And Characterization ◽

Normal Enzyme ◽

Enzyme Molecules ◽

Large Scale Screening

ABSTRACT We have outlined a procedure that allows the large-scale screening of mutagenized Neurospora crassa populations for invertaseless mutants. We have isolated and characterized three mutations, inv(DBL1), inv(DBL9) and inv(DBL14), which have been mapped at or near the invertase structural gene. One of these, inv(DBL1), is particularly interesting. Our experiments indicate that the reduced level of invertase activity in the inv(DBL1)-containing cell can be explained as the result of a reduced number of normal enzyme molecules. We also show that wild-type Neurospora is able to respond rapidly to a change of medium and can dramatically increase its production of invertase within 20 min after a transfer to a carbon-free medium.

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Cloning and Characterization of a Novel Invertase from the Obligate Biotroph Uromyces fabae and Analysis of Expression Patterns of Host and Pathogen Invertases in the Course of Infection

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-19-0625 ◽

2006 ◽

Vol 19 (6) ◽

pp. 625-634 ◽

Cited By ~ 67

Author(s):

Ralf T. Voegele ◽

Stefan Wirsel ◽

Ulla Möll ◽

Melanie Lechner ◽

Kurt Mendgen

Keyword(s):

Cell Wall ◽

Expression Analysis ◽

Biochemical Characterization ◽

Higher Plants ◽

Rust Fungus ◽

Expression Patterns ◽

Invertase Activity ◽

Pathogen Infection ◽

Uromyces Fabae

Invertases are key enzymes in carbon partitioning in higher plants. They gain additional importance in the distribution of carbohydrates in the event of wounding or pathogen attack. Although many researchers have found an increase in invertase activity upon infection, only a few studies were able to determine whether the source of this activity was host or parasite. This article analyzes the role of invertases involved in the biotrophic interaction of the rust fungus Uromyces fabae and its host plant, Vicia faba. We have identified a fungal gene, Uf-INV1, with homology to invertases and assessed its contribution to pathogenesis. Expression analysis indicated that transcription began upon penetration of the fungus into the leaf, with high expression levels in haustoria. Heterologous expression of Uf-INV1 in Saccharomyces cerevisiae and Pichia pastoris allowed a biochemical characterization of the enzymatic activity associated with the secreted gene product INV1p. Expression analysis of the known vacuolar and cell-wall-bound invertase isoforms of V. faba indicated a decrease in the expression of a vacuolar invertase, whereas one cell-wall-associated invertase exhibited increased expression. These changes were not confined to the infected tissue, and effects also were observed in remote plant organs, such as roots. These findings hint at systemic effects of pathogen infection. Our results support the hypothesis that pathogen infection establishes new sinks which compete with physiological sink organs.

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Invertase Production by Fungi, Characterization of Enzyme Activity and Kinetic Parameters

Revista de Chimie ◽

10.37358/rc.17.10.5856 ◽

2017 ◽

Vol 68 (10) ◽

pp. 2205-2208 ◽

Cited By ~ 1

Author(s):

Gabi Mirela Matei ◽

Sorin Matei ◽

Maria Pele ◽

Flavia Dumitrescu ◽

Adrian Matei

Keyword(s):

Aspergillus Niger ◽

Kinetic Parameters ◽

Invertase Activity ◽

Enzyme Concentration ◽

Maximal Velocity ◽

Aspergillus Wentii ◽

Penicillium Aurantiogriseum ◽

The Relationship ◽

Menten Constant

The present paper presents the results of research carried out on 14 fungal isolates of various origins aiming to select new efficient sources for invertase production for further biotechnological application. Aspergillus flavus presented the highest protein content and Aspergillus niger, the most intense invertase activity. The relationship between enzyme concentration and enzymatic activity at 0.25 mM mL-1sucrose as substrate assayed for successive decimal dilutions of Aspergillus niger enzyme ranging from 0.1 to 1mlLrevealed a linear correspondence between 0.1 and 0.5mL. The kinetic parameters Michaelis-Menten constant (Km) and maximal velocity (Vmax) for invertase activity of Aspergillus niger, Penicillium aurantiogriseum, Aspergillus wentii and Rhizopus stolonifer were calculated. The determination coefficients R2 calculated from Lineweaver-Burk plots presented values very close or equal to 1.

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Purification and characterization of β-Fructosidase with inulinase activity from Aspergillus niger - 245

Brazilian Archives of Biology and Technology ◽

10.1590/s1516-89131998000300003 ◽

1998 ◽

Vol 41 (3) ◽

pp. 288-295

Author(s):

Vinícius D'Arcadia Cruz ◽

Juliana Gisele Belote ◽

Claudia Dorta ◽

Luíza Helena Oliveira dos Santos ◽

Cláudia Regina Andriolo ◽

...

Keyword(s):

Aspergillus Niger ◽

Invertase Activity ◽

Inulinase Activity ◽

Km Value ◽

Good Potential ◽

Chicory Inulin ◽

Different Origins ◽

Hydrolysis Of ◽

Assay Conditions

Aspergillus niger - 245, a strain isolated from soil samples showed good β-fructosidase activity when inoculated in medium formulated with dahlia extract tubers. The enzyme was purified by precipitation in ammonium sulphate and percolated in DEAE-Sephadex A-50 and CM-cellulose columns, witch showed a single peack in all the purification steps, maintaining the I/S ratio between 0.32 to, 0.39. Optimum pH for inulinase activity (I) was between 4.0 - 4.5 and for invertase activity (S) between 2.5 and 5.0. The optimum temperature was 60O.C for both activities and no loss in activity was observed when it was maintained at this temperature for 30 min. The Km value was 1.44 and 5.0, respectively, for I and S and Vm value 10.48 and 30.55, respectively. The I activity was strongly inhibited by Hg2+ and Ag+ and 2 x 10-3 M of glucose, but not by fructose at the same concentration. The enzyme showed an exo-action mechanism, acting on the inulin of different origins. In assay conditions total hydrolysis of all the frutans was obtained, although it has shown larger activity on the chicory inulin than that one from artichoke Jerusalem and dahlia, in the first 30 min. The obtained results suggested that the enzyme presented good potential for industrial application in the preparing the fructose syrups

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Validation of a thin-layer chromatography/densitometry method for the characterization of invertase activity

Journal of Chromatography A ◽

10.1016/j.chroma.2016.11.049 ◽

2016 ◽

Vol 1477 ◽

pp. 108-113 ◽

Cited By ~ 7

Author(s):

Justine Ferey ◽

David Da Silva ◽

Sophie Bravo-Veyrat ◽

Pierre Lafite ◽

Richard Daniellou ◽

...

Keyword(s):

Thin Layer ◽

Thin Layer Chromatography ◽

Invertase Activity ◽

Layer Chromatography

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Physiological basis for enhanced sucrose accumulation in an engineered sugarcane cell line

Functional Plant Biology ◽

10.1071/fp10055 ◽

2010 ◽

Vol 37 (12) ◽

pp. 1161 ◽

Cited By ~ 15

Author(s):

Luguang Wu ◽

Robert G. Birch

Keyword(s):

Sucrose Synthase ◽

Sucrose Phosphate Synthase ◽

Saccharum Officinarum ◽

Invertase Activity ◽

Negative Regulator ◽

Sucrose Accumulation ◽

Physiological Basis ◽

Transgenic Sugarcane ◽

Extracellular Invertase ◽

Sucrose Phosphate

Transgenic sugarcane (Saccharum officinarum L. interspecific hybrids) line N3.2 engineered to express a vacuole-targeted sucrose isomerase was found to accumulate sucrose to twice the level of the background genotype Q117 in heterotrophic cell cultures, without adverse effects on cell growth. Isomaltulose levels declined over successive subcultures, but the enhanced sucrose accumulation was stable. Detailed physiological characterisation revealed multiple processes altered in line N3.2 in a direction consistent with enhanced sucrose accumulation. Striking differences from the Q117 control included reduced extracellular invertase activity, slower extracellular sucrose depletion, lower activities of symplastic sucrose-cleavage enzymes (particularly sucrose synthase breakage activity), and enhanced levels of symplastic hexose-6-phosphate and trehalose-6-phosphate (T6P) in advance of enhanced sucrose accumulation. Sucrose biosynthesis by sucrose phosphate synthase (SPS) and sucrose phosphate phosphatase (SPP) was substantially faster in assays conducted to reflect the elevation in key allosteric metabolite glucose-6-phosphate (G6P). Sucrose-non-fermenting-1-related protein kinase 1 (SnRK1, which typically activates sucrose synthase breakage activity while downregulating SPS in plants) was significantly lower in line N3.2 during the period of fastest sucrose accumulation. For the first time, T6P is also shown to be a negative regulator of SnRK1 activity from sugarcane sink cells, hinting at a control circuitry for parallel activation of key enzymes for enhanced sucrose accumulation in sugarcane.

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