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Optimal Conditions for Continuous Immobilization of Pseudozyma hubeiensis (Strain HB85A) Lipase by Adsorption in a Packed-Bed Reactor by Response Surface Methodology
Roberta Bussamara,Luciane Dall'Agnol,Augusto Schrank,Kátia Flávia Fernandes,Marilene Henning Vainstein
Enzyme Research , 2012, DOI: 10.1155/2012/329178
Abstract: This study aimed to develop an optimal continuous process for lipase immobilization in a bed reactor in order to investigate the possibility of large-scale production. An extracellular lipase of Pseudozyma hubeiensis (strain HB85A) was immobilized by adsorption onto a polystyrene-divinylbenzene support. Furthermore, response surface methodology (RSM) was employed to optimize enzyme immobilization and evaluate the optimum temperature and pH for free and immobilized enzyme. The optimal immobilization conditions observed were 150?min incubation time, pH 4.76, and an enzyme/support ratio of 1282?U/g support. Optimal activity temperature for free and immobilized enzyme was found to be 68°C and 52°C, respectively. Optimal activity pH for free and immobilized lipase was pH 4.6 and 6.0, respectively. Lipase immobilization resulted in improved enzyme stability in the presence of nonionic detergents, at high temperatures, at acidic and neutral pH, and at high concentrations of organic solvents such as 2-propanol, methanol, and acetone. 1. Introduction Biocatalyst enzymes play an important role in biotechnological applications due to their extreme versatility with respect to substrate specificity and stereoselectivity and exhibit many other features that render their use advantageous when compared to conventional chemical catalysts. As an example, fat and oil hydrolysis using NaOH as a catalyst requires high pressure and temperature to achieve high efficiency (97-98%); in contrast, the same process can be carried out effectively at normal temperature and pressure using lipases, with significant decrease in wastewater production. Lipases (triacylglycerol acylhydrolase; EC catalyze the hydrolysis of triglycerides to glycerol and fatty acids, as well as a variety of reactions in nonaqueous medium (e.g., transesterification, esterification, and interesterification). Such enzymatic properties allow a series of biotransformation reactions that lead to multiple industrial applications in foods, flavors, pharmaceuticals, detergent formulation, oil/fat degradation, cosmetics, and environmental remediation [1–4]. However, soluble enzymes usually exhibit lower stability than chemical catalysts and often cannot be recovered and reused. This severely hinders their application in practice. Nevertheless, this problem can be overcome by enzyme immobilization, which enhances thermal and operational stabilities, ease of handling, and prevention of aggregation and autolysis. Besides, immobilized lipases (IE) on solid support allow recoverability and reuse thus significantly
Determina??o do 5-hidroximetilfurfural em méis utilizando cromatografia eletrocinética capilar micelar
Silva, Sandra Jussara Nunes da;Schuch, Paula Zilles;Vainstein, Marilene Henning;Jablonski, André;
Ciência e Tecnologia de Alimentos , 2008, DOI: 10.1590/S0101-20612008000500008
Abstract: in this work, the occurrence of hmf (5-hydroxymethylfurfural) in honey marketed in porto alegre - rs was investigated using micellar electrokinetic capillary chromatography. the hmf, which is the product of the fructose condensation, is an indicator of honey quality and conservation. eleven types of honey commercialized in porto alegre were analyzed, and all of them contained hmf in a range from 0.191 to 6.206 mg.kg-1. in order to quantify the hmf present in the samples, the technique of standard addition was employed. the recovery was 98% and the detection limit was 0.025 mg.kg-1. the allowed limit of hmf in honey, according to the brazilian legislation, is 60 mg.kg-1.
Patulin in food: state-of-the-art and analytical trends
Silva, Sandra Jussara Nunes da;Schuch, Paula Zilles;Bernardi, Carmem Ronise;Vainstein, Marilene Henning;Jablonski, André;Bender, Renar Jo?o;
Revista Brasileira de Fruticultura , 2007, DOI: 10.1590/S0100-29452007000200043
Abstract: patulin is a mycotoxin produced by several fungal species of the genera penicillium and aspergillus, found on several fruit species and, remarkably, in apples and apple products. patulin has a broad spectrum of toxicity, including carcinogenicity and teratogenicity in animals. due to the stability of the molecule, considerable amounts of patulin still remain in apple products after processing. this paper reviews different analytical methods for patulin determination and methods to reduce levels of patulin in apple products as well.
In vitro susceptibility to antifungal agents of clinical and environmental Cryptococcus neoformans isolated in Southern of Brazil
ALVES, Sydney Hartz;OLIVEIRA, Loiva T.;COSTA, Jane M.;LUBECK, Irina;CASALI, Agnes Kiesling;VAINSTEIN, Marilene Henning;
Revista do Instituto de Medicina Tropical de S?o Paulo , 2001, DOI: 10.1590/S0036-46652001000500006
Abstract: the purpose of the present study was to compare the susceptibility to four antifungal agents of 69 cryptococcus neoformans strains isolated from aids patients with that of 13 c. neoformans strains isolated from the environment. based on the nccls m27-a methodology the minimal inhibitory concentrations (mics) obtained for amphotericin b, itraconazole and ketoconazole were very similar for clinical and environmental isolates. clinical isolates were less susceptible to fluconazole than environmental isolates. the significance of these findings and aspects concerning the importance, role and difficulties of c. neoformans susceptibility testing are also discussed.
Zap1 Regulates Zinc Homeostasis and Modulates Virulence in Cryptococcus gattii
Rafael de Oliveira Schneider, Natully de Souza Süffert Foga?a, Lívia Kmetzsch, Augusto Schrank, Marilene Henning Vainstein, Charley Christian Staats
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0043773
Abstract: Zinc homeostasis is essential for fungal growth, as this metal is a critical structural component of several proteins, including transcription factors. The fungal pathogen Cryptococcus gattii obtains zinc from the stringent zinc-limiting milieu of the host during the infection process. To characterize the zinc metabolism in C. gattii and its relationship to fungal virulence, the zinc finger protein Zap1 was functionally characterized. The C. gattii ZAP1 gene is an ortholog of the master regulatory genes zafA and ZAP1 that are found in Aspergillus fumigatus and Saccharomyces cerevisiae, respectively. There is some evidence to support an association between Zap1 and zinc metabolism in C. gattii: (i) ZAP1 expression is highly induced during zinc deprivation, (ii) ZAP1 knockouts demonstrate impaired growth in zinc-limiting conditions, (iii) Zap1 regulates the expression of ZIP zinc transporters and distinct zinc-binding proteins and (iv) Zap1 regulates the labile pool of intracellular zinc. In addition, the deletion of ZAP1 reduces C. gattii virulence in a murine model of cryptococcosis infection. Based on these observations, we postulate that proper zinc metabolism plays a crucial role in cryptococcal virulence.
Genetic Diversity of the Cryptococcus Species Complex Suggests that Cryptococcus gattii Deserves to Have Varieties
Popchai Ngamskulrungroj, Felix Gilgado, Josiane Faganello, Anastasia P. Litvintseva, Ana Lusia Leal, Kin Ming Tsui, Thomas G. Mitchell, Marilene Henning Vainstein, Wieland Meyer
PLOS ONE , 2009, DOI: 10.1371/journal.pone.0005862
Abstract: The Cryptococcus species complex contains two sibling taxa, Cryptococcus neoformans and Cryptococcus gattii. Both species are basidiomycetous yeasts and major pathogens of humans and other mammals. Genotyping methods have identified major haploid molecular types of C. neoformans (VNI, VNII, VNB and VNIV) and of C. gattii (VGI, VGII, VGIII and VGIV). To investigate the phylogenetic relationships among these haploid genotypes, we selected 73 strains from 2000 globally collected isolates investigated in our previous typing studies, representing each of these genotypes and carried out multigene sequence analyses using four genetically unlinked nuclear loci, ACT1, IDE, PLB1 and URA5. The separate or combined sequence analyses of all four loci revealed seven clades with significant support for each molecular type. However, three strains of each species revealed some incongruence between the original molecular type and the sequence-based type obtained here. The topology of the individual gene trees was identical for each clade of C. neoformans but incongruent for the clades of C. gattii indicating recent recombination events within C. gattii. There was strong evidence of recombination in the global VGII population. Both parsimony and likelihood analyses supported three major clades of C. neoformans (VNI/VNB, VNII and VNIV) and four major clades of C. gattii (VGI, VGII, VGIII and VGIV). The sequence variation between VGI, VGIII and VGIV was similar to that between VNI/VNB and VNII. MATa was for the first time identified for VGIV. The VNIV and VGII clades are basal to the C. neoformans or the C. gattii clade, respectively. Divergence times among the seven haploid monophyletic lineages in the Cryptococcus species complex were estimated by applying the hypothesis of the molecular clock. The genetic variation found among all of these haploid monophyletic lineages indicates that they warrant varietal status.
Virulence factors and antimicrobial resistance of escherichia coli isolated from urinary tract of swine in southern of Brazil
Costa, Mateus Matiuzzi da;Drescher, Guilherme;Maboni, Franciele;Weber, Shana;Botton, S?nia de Avila;Vainstein, Marilene Henning;Schrank, Irene Silveira;Vargas, Agueda Castagna de;
Brazilian Journal of Microbiology , 2008, DOI: 10.1590/S1517-83822008000400027
Abstract: the present study determined the molecular and resistance patterns of e. coli isolates from urinary tract of swine in southern of brazil. molecular characterization of urinary vesicle samples was performed by pcr detection of virulence factors from etec, stec and upec. from a total of 82 e. coli isolates, 34 (38.63%) harbored one or more virulence factors. the frequency of virulence factors genes detected by pcr were: pap (10.97%), hlya (10.97%), iha (9.75%), lt (8.53%), sta (7.31%) sfa (6.09%), f4 (4.87%), f5 (4.87%), stb (4.87%), f6 (1.21%) and f41 (1.21%). isolates were resistant to penicillin (95.12%), lincomycin (93.9%), erythromycin (92.68%), tetracycline (90.24%), amoxicillin (82.92%), ampicillin (74.39%), josamycin (79.26%), norfloxacin (58.53%), enrofloxacin (57.31%), gentamicin (39.02%), neomycin (37.8%), apramycin (30.48%), colistine (30.48%) and cefalexin (6.09%). a number of 32 (39.02%) e. coli isolates harbored plasmids.
Distinct chitinases are expressed during various growth phases of the human pathogen Paracoccidioides brasiliensis
Santana, Lidiane Aparecida da Penha;Vainstein, Marilene Henning;Tomazett, Patrícia Kott;Santos-Silva, Ludier Kesser;Góes, Alfredo Miranda;Schrank, Augusto;Soares, Célia Maria de Almeida;Pereira, Maristela;
Memórias do Instituto Oswaldo Cruz , 2012, DOI: 10.1590/S0074-02762012000300004
Abstract: the aim of this work was the partial purification and subsequent evaluation of chitinase expression during the various growth phases of paracoccidioides brasiliensis. initially, pbcts1r was expressed as a recombinant protein and displayed enzymatic activity against 4-mu-[n-acetylglucosamine (glcnac)]3 and 4-mu-(glcnac)2. two proteins, 45 kda and 39 kda in size, were partially purified from p. brasiliensis yeast crude extract using cation-exchange chromatography coupled with hplc and were characterised as pbcts1 and pbcts2, respectively. anti-pbcts1r antibody recognised two proteins in the crude extracts of yeast and the transitional stage between mycelial and yeast phases. in crude extracts of mycelium, only the 45 kda protein was detected. however, quantitative real-time polymerase chain reaction led to the detection of small quantities of pbcts2 transcript in the mycelial phase. in the yeast cell wall extract, only the 39 kda protein was detected. moreover, both proteins were secreted by the yeast parasitic phase, suggesting that these proteins participate in the modulation of the fungal environment. phylogenetic analysis of the predicted pbcts1 and pbcts2 proteins indicated that they code for distinct chitinases in p. brasiliensis. during evolution, p. brasiliensis could have acquired the paralogues pbcts1 and pbcts2 for growth and survival in diverse environments in both saprophytic and parasitic phases.
Comparative genome analysis of proteases, oligopeptide uptake and secretion systems in Mycoplasma spp
Staats, Charley Christian;Boldo, Juliano;Broetto, Leonardo;Vainstein, Marilene;Schrank, Augusto;
Genetics and Molecular Biology , 2007, DOI: 10.1590/S1415-47572007000200009
Abstract: mycoplasmas are very fastidious in their nutritional requirements for in vitro growth and have limited biosynthetic capacity, a reflection of their reduced genomes. as a result, these bacteria depend upon external metabolites for nutrition and growth and have developed dependence on their hosts for survival and maintenance. protein degradation and peptide importation play an important role in mycoplasma spp. nutrition, and proteases can play a role in host adaptation and pathogenicity. here, we present a general survey on the genes involved in protein degradation, secretion and importation, comparing all available mollicute genomes.
Fungal zinc metabolism and its connections to virulence
Charley C. Staats,Lívia Kmetzsch,Augusto Schrank,Marilene H. Vainstein
Frontiers in Cellular and Infection Microbiology , 2013, DOI: 10.3389/fcimb.2013.00065
Abstract: Zinc is a ubiquitous metal in all life forms, as it is a structural component of the almost 10% of eukaryotic proteins, which are called zinc-binding proteins. In zinc-limiting conditions such as those found during infection, pathogenic fungi activate the expression of several systems to enhance the uptake of zinc. These systems include ZIP transporters (solute carrier 39 family) and secreted zincophores, which are proteins that are able to chelate zinc. The expression of some fungal zinc uptake systems are regulated by a master regulator (Zap1), first characterized in the yeast Saccharomyces cerevisiae. In this review, we highlight features of zinc uptake and metabolism in human fungal pathogens and aspects of the relationship between proper zinc metabolism and the expression of virulence factors and adaptation to the host habitat.
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