Biomedical subjects

P Galzy

Publications and source records attributed to P Galzy.

At least 19 recordsLinked to original sources

PubMed1997

Fatty hydroxamic acid biosynthesis in aqueous medium in the presence of the lipase-acyltransferase from Candida parapsilosis.

The lipase-acyltransferase from Candida parapsilosis has been shown to catalyze fatty hydroxamic acid biosynthesis in a biphasic lipid/aqueous medium. The substrates of the reaction were an acyl donor (fatty acid or fatty acid methyl ester) and hydroxylamine. The transfer of acyl groups from a donor ester to hydroxylamine (aminolysis) was catalyzed preferentially to the reaction of free fatty acids. The highest synthesis activity was obtained in the presence of 1 M hydroxylamine at 45 degrees C and pH 6. This work confirmed the originality of the enzyme from Candida parapsilosis, which acts more like an acyltransferase than an hydrolase. This feature makes it an enzyme of choice for the direct bioconversion of oils in aqueous medium.

Acyltransferases↗

PubMed1997

Glucose metabolism in the yeast Schwanniomyces castellii: role of phosphorylation site I and an alternative respiratory pathway.

Glucose metabolism in a Crabtree-negative yeast, Schwanniomyces castellii, and a cytochrome b-deficient mutant of this strain was investigated in chemostat culture. The wild-type and mutant strains exhibited the same behavior. Oxidative metabolism was observed when the substrate uptake rate (qS) was low. Fermentative metabolites were excreted when the qS value was higher than 0.40 g.g-1.h-1, indicating the occurrence of a respirofermentative metabolism; however, the respiratory quotient (RQ) remained near 1. When fermentation occurred, the cytochrome pathway was repressed but not the salicylhydroxamic acid (SHAM)-sensitive pathway. The presence of an alternative SHAM-sensitive respiratory pathway and the presence of phosphorylation site I in all metabolic conditions explained the RQ value of 1 and accounted for high biomass yields in oxidative metabolism conditions (0.62 g.g-1 for the wild-type strain and 0.31 g.g-1 for the cytochrome b-deficient mutant strain).

Biomass↗

PubMed1997

Contribution to the study of surface microflora on Cantal cheese.

Bacteria and mould were isolated from microflora of Cantal cheese rind. They were identified and the influence of pH, temperature and water activity on growth rate were studied. An equation for growth rate mu(h-1) relative to temperature is described according to a mathematical model.

Bacteria↗

PubMed1996

Study of the amidase signature group.

Computer methods for database search, multiple alignment and cluster analysis indicated significant homology between amino-acid sequences of 21 amidases or amidohydrolases (EC 3.5). All of them were found to be involved in the reduction of organic nitrogen compounds and ammonia production. A conserved motif was found which may be important in amide binding and in catalytic mechanisms. Homology studies between these amidases and some ureases, nitrilases and acyl-transferases or enzymes with unknown functions provided new insight into the evolution of these proteins. Dissemination of these genes seemed to be facilitated by transfer of genetic elements such as transposons and plasmids.

Amidohydrolases↗

PubMed1996

Amide metabolism: a putative ABC transporter in Rhodococcus sp. R312.

The DNA sequence has been determined upstream of the amiE structural gene in the amidase operon of Rhodococcus sp. R312 and a new ORF (amiS2) identified. The amiS2 gene encodes a potential 206 amino acid (aa) protein containing a high proportion of hydrophobic residues. The AmiS2 protein possesses high homology to the ORFP3, amiS and ureI gene products from the Mycobacterium smegmatis (Ms) acetamidase operon, Pseudomonas aeruginosa (Pa) amidase operon and Helicobacter pylori (Hp) urease operon, respectively. Hydropathic analysis and secondary structure prediction of AmiS2 suggested the presence of seven potential transmembrane (TM) alpha-helices. Sequence analysis of the amiB2 gene, located downstream of the Rhodococcus sp. R312 amiE gene, showed that it encoded a 351-aa protein containing a potential ATP-binding motif. AmiB2 showed significant homology with the ATP-binding subunit of the bacterial Clp protease and high homology with the amiB product located within the Pa amidase operon. AmiB2 and AmiS2 appear to be two components of a recently identified novel family of ABC transporters (Wilson et al., 1995) and might be responsible for the adsorption of amidase substrates or release of their hydrolysis products.

ATP-Binding Cassette Transporters↗

PubMed1996

Study of the delta 12-desaturase system of Lipomyces starkeyi.

The specific activity of the microsomal delta 12-desaturase system, which transforms oleic acid into linoleic acid, was about 16 pmol/min/mg protein. However, most of the total activity was nonsedimentable even after a 200000 x g centrifugation for 100 min. The study of various physicochemical parameters showed that this enzymatic complex, functioning optimally between pH 7 and 8, had low thermal stability. Ca2+ which may cause an aggregation of the microsomes, and Hg2+ completely inhibited the activity, whereas Mg2+, Mn2+, and Zn2+ were activators. The delta 12-desaturase system was relatively specific toward oleic acid, though isomers of this fatty acid also had an action, either as substrates or as competitive inhibitors, on the activity of the system. The study of the effect of the exogenous oleoyl-CoA and elaidoyl-CoA on the specific activity of the delta 12-desaturase system showed a preference toward oleoyl-CoA.

Acyl Coenzyme A↗

PubMed1995

Cloning and sequencing of the beta-glucosidase-encoding gene from Candida molischiana strain 35M5N.

We have isolated a gene (bgln) encoding beta-glucosidase (beta Glu) from a cosmid library of the yeast, Candida molischiana 35M5N. The nucleotide sequence of bgln and its flanking regions was determined. This gene was found to be composed of 2289 bp and 763 amino acid (aa) residues encoding an 83.3-kDa protein. The aa sequence shared eleven putative N-glycosylation sites. Homology comparisons showed that this enzyme can be considered as a new member of the family-3 glycosyl hydrolases. Multiple alignment experiments revealed four conserved regions on aa sequences from beta Glu of this family.

Amino Acid Sequence↗

PubMed1995

Factors influencing ester synthesis catalysed in aqueous media by the lipase from Candida deformans (Zach) Langeron and Guerra.

The purified lipase from Candida deformans was shown to catalyse ester production in aqueous media by esterification of free fatty acids but not by alcoholysis. As the enzyme also catalysed ester hydrolysis, the influence of various physico-chemical factors on ester hydrolysis and synthesis was studied and compared. Substrate specificities were also studied. Both activities had the same pH and temperature optima, and did not require a metal cofactor. Tyrosine appeared to be one of the amino acids of the enzyme required by both catalytic activities, whereas serine-reactive reagents inhibited synthesis only. The highest synthesis and hydrolysis activities were obtained with mono-, di- and tri-unsaturated fatty acids containing a [cis] delta-9 unsaturation and 16 to 18 carbon atoms. Only esters of primary alcohols were hydrolysed and synthesised. For alcohols ranging from methanol to butanol, synthesis activity increased with the length of the alcohol whereas hydrolysis activity of the corresponding esters decreased. The presence of alcohol inhibited hydrolysis. An optimum concentration was found for each primary alcohol at which ester synthesis was maximum and hydrolysis was low.

Alcohols↗

PubMed1995

Glucose metabolism, enzymic analysis and product formation in chemostat culture of Hanseniaspora uvarum.

The physiology of Hanseniaspora uvarum K5 was studied in glucose-limited chemostat cultures and upon glucose pulse. Up to a dilution rate of 0.28 h-1, glucose was completely metabolized in biomass and CO2. Above this value, increase in the dilution rate was accompanied by sequential production of metabolites (glycerol, acetate and ethanol) and decrease in cell yield. Similar results were observed upon glucose pulse. From the enzyme activities (pyruvate dehydrogenase, pyruvate decarboxylase, NAD and NADP-dependent acetaldehyde dehydrogenases, acetyl coenzyme A synthetase and alcohol dehydrogenase) and substrate affinities, the following conclusions were drawn with respect to product formation of cells: (1) pyruvate was preferentially metabolized via pyruvate dehydrogenase, when biomass and CO2 were the only products formed; (2) acetaldehyde formed by pyruvate decarboxylase was preferentially oxidized in acetate by NADP-dependent aldehyde dehydrogenase; acetate accumulation results from insufficient activity of acetyl-CoA synthetase required for the complete oxidation of acetate; (3) acetaldehyde was oxidized in ethanol by alcohol dehydrogenase, in addition to acetate production.

Acetates↗

PubMed1995

Brevibacterium linens pBL33 and Rhodococcus rhodochrous pRC1 cryptic plasmids replicate in Rhodococcus sp. R312 (formerly Brevibacterium sp. R312).

The replication of two cryptic plasmids from Brevibacterium linens ATCC 9174 (pBL33) and Rhodococcus rhodochrous ATCC 4276 (pRC1) was investigated in Rhodococcus sp. R312 (formerly Brevibacterium sp. R312). The recombinant plasmids pSP33 (pBL33 derivative) and pSPC1 (pRC1 derivative) were found to be suitable for establishing new host-vector systems for Rhodococcus sp. R312. They all carry the Tn903 neomycin-resistance-encoding gene (aphI).

Bacterial Proteins↗

PubMed1995

Functioning and regioselectivity of the lipase of Candida parapsilosis (Ashford) Langeron and Talice in aqueous medium. New interpretation of regioselectivity taking acyl migration into account.

The lipase of Candida parapsilosis catalyses the formation of esters in aqueous media. In addition, the hydrolytic activity of the enzyme has been described in a recent publication as being selective for the 2 position, which is extremely rare. These features led to deeper investigation of the functioning and regioselectivity of the lipase in a biphasic aqueous medium. It is shown that, in addition to hydrolysis, the lipase of C. parapsilosis catalyses an alcoholysis reaction in the strict sense of the term, i.e. the transfer of fatty acyls groups from acylglycerols to various alcohols without direct involvement of water. In the presence of alcohol in the aqueous medium, alcolysis occurred preferentially to hydrolysis. The enzyme thus displays transferase activity in which the acyl acceptor may be either water or alcohol. This activity is not stereospecific to either the acyl donor or acceptor. Hydrolysis and alcoholysis of monooleoylglycerols, dioleoylglycerols and trioleoylglycerols were studied successively. Investigation of the regiospecificity of alcoholysis in the presence of the lipase of C. parapsilosis showed that the selectivity of hydrolysis for the 2 position was, in fact, only apparent. In certain cases, and particularly when the initial substrate was a triacylglycerol, the similtaneous functioning of the two hydrolysis and alcoholysis reactions led to the appearance of equivalent quantities of 1,2(2,3)-diacylglycerol and 1,3-diacylglycerol in the reaction mixture; this proportion might then be interpreted as the result of selectivity of the hydrolysis reaction for position 2 of the triacylglycerol.

Alcohols↗

PubMed1995

Karyotype studies on different strains of Candida molischiana by pulsed-field gel electrophoresis.

We used pulsed-field gel electrophoresis to compare the electrophoretic karyotype of a Candida molischiana mutant, de-repressed for beta-glucosidase production, with a wild-type strain and a reference strain. The chromosomal organization in this mutant yeast was found to be quite different. Hybridization patterns and the relative fluorescence of all bands indicated eight chromosomes in the mutant strain and seven in the other two. All three strains seemed to be haploid, with an estimated genome size of 12 Mb; the beta-glucosidase gene was on the same chromosome in all of them and the SfiI restriction patterns of this chromosome indicated that it is not affected by the mutation.

Candida↗

PubMed1995

Sizing of the Rhodococcus sp. R312 genome by pulsed-field gel electrophoresis. Localization of genes involved in nitrile degradation.

The two restriction enzymes AsnI and DraI were found to produce DNA fragment sizes that could be used for mapping the Rhodococcus sp. R312 (formerly Brevibacterium sp. R312) genome by pulsed-field gel electrophoresis. AsnI produced 24 fragments (4 to 727 kb) and DraI yielded 15 fragments (8.5 to 2400 kb). The fragment lengths in each digest were summed, indicating that the size of the chromosome ranged from 6.31 to 6.56 Mb, with a mean of 6.44 Mb. In addition, the wide-spectrum amidase gene (amiE) and the operon containing the enantiomer-selective amidase gene (amdA) and the nitrile hydratase structural gene (nthA, nthB) were localized on the AsnI and DraI fragments.

Amidohydrolases↗

PubMed1995

Fatty acid and carotenoid composition of Rhodotorula strains.

Lipid content and composition of fatty acids with 6-25 carbon atoms were studied on strains of the 13 pink or red yeast species belonging to the genus Rhodotorula. The total amount of lipid represented an average of 13% of the dry weight. The neutral and polar lipid fractions were analyzed separately. For all the strains studied, the major fatty acids in both fractions were oleic, linoleic and palmitic acids, which formed 80% of the total number of fatty acids. A notable amount of arachidonic acid, a precursor of eicosanoid hormones, was found in R. acheniorum, R. aurantiaca and R. bacarum. Depending on the strain, 1-10 carotenoid pigments were detected; beta-carotene was always the major carotenoid present.

Carotenoids↗

PubMed1995

Substrate specificity of the lipase from Candida parapsilosis.

Substrate specificity of the acyltransferase activity of the lipase (EC 3.1.1.3) from Candida parapsilosis CBS 604 was studied in aqueous media. The specificity toward both acid and alcohol parts of a large number of acylglycerols and aliphatic esters was investigated. This lipase showed a high activity in the presence of esters with long-chain fatty acids and particularly unsaturated fatty acids with a cis-delta 9 double bond. It was observed that the activity profile depended not only on the alcohol part of the acyl ester, but also on the temperature of the reactant medium. The best lipid substrates had their melting point between -40 to +20 degrees C, 14 to 18 carbon atoms in the acyl group and 1 to 4 carbon atoms in the alkyl group. The enzyme, defined as an acyltransferase in a previous paper, showed a high affinity for primary and secondary alcohols with a short carbon chain (1 to 5 carbon atoms) as acyl acceptors. The influence of free alcohols in the reactant medium on the hydrolysis and alcoholysis activities of the enzyme is discussed. Two phenomena seem to be involved, depending on the alcohol: competition with water for the acyltransfer reaction and lipid substrate dilution when the alcohol places at the oil/water interface.

Alcohols↗

PubMed1995

A very stable beta-glucosidase from a Candida molischiana mutant strain: enzymatic properties, sequencing, and homology with other yeast beta-glucosidases.

We purified a beta-glucosidase from the mutant strain Candida molischiana 35M5N. Analysis of the kinetic properties of this enzyme did not show any differences between the previously purified wild-type enzyme and that of the mutant. Nevertheless, a study of the stability of the enzyme at different pH levels and temperatures showed the increase resistance of this protein. This enzyme was found to be stable at pH 5 for 145 h and retained 78% of its initial activity after the same time at pH 3.5 (optimal pH) and 30 degrees C. This difference between the wild-type and the mutant enzyme could be explained by differences in the quantity or quality of glycosylation. This glycoprotein showed different forms after deglycosylation. Some peptides from this protein were also sequenced. An homology analysis found similarities between this beta-glucosidase and beta-glucosidases of Candida pelliculosa and Schizophyllum commune.

Amino Acid Sequence↗

PubMed1995

Remarks on the appearance of "violine" on Cantal cheese (a note).

A mould, Scopulariopsis brevicaulis was isolated from a Cantal cheese. This mould was responsible for the "violine" phenomenon. It's optimum pH growth was about 7 and the mould was able to grow suitably at 10 degrees C, the temperature of cellar for cheese ripening. It was inhibited by high sodium chloride contents or low water activities.

Cheese↗

PubMed1994

Cloning of the wide spectrum amidase gene from Brevibacterium sp. R312 by genetic complementation. Overexpression in Brevibacterium sp. and Escherichia coli.

The amiE gene of Brevibacterium sp. R312 encoding wide spectrum amidase was isolated by complementation of a Brevibacterium sp. mutant using a plasmid gene bank of chromosomal DNA. The amiE structural gene and its promoter were localized on a 1.8-kb fragment by subsequent subcloning and complementation studies. Another promoter localized in the pSR 1 fragment of the cloning vector was shown to be able to control amiE gene expression. In Brevibacterium sp., the investigation of amidase activities related to one copy of the gene suggested that the regulation of the amiE gene expression was under negative control. High expression levels have been obtained in Brevibacterium sp. and, after substitution of the amiE promoter by the tac promoter, in Escherichia coli.

Amidohydrolases↗