RESUMO
Spanish dry-cured ham is an uncooked meat product highly appreciated due to its characteristics flavour. In this study, we examined the accuracy of biochemical tests and 16S rDNA sequencing in the identification of 56 staphylococcal strains isolated during industrial Spanish dry-cured ham processes. Important differences were observed comparing genotypic and phenotypic data. Staphylococcus xylosus was the prevalent species identified by biochemical methods (87.5%), however, sequencing of the 16S rDNA resulted in an unambiguous identification of Staphylococcus equorum (73.2%) and Staphylococcus vitulinus (8.9%) strains. Reliable identification of meat staphylococci, mainly among S. xylosus and S. equorum strains could be also achieved by means of recA gene sequence comparison. Two degenerate primers previously described for lactic acid bacteria were used to amplify an internal fragment of the recA gene. This fragment was amplified from twelve staphylococcal type strains representing frequent meat species. The results indicated that recA sequencing is an adequate method to discriminate among meat staphylococci. In addition, S. xylosus and S. equorum strains could be more accurately discriminated by recA sequencing than 16S rDNA or sodA sequencing. The S. equorum sequence diversity showed at the intra-species level by recA gene sequencing confirmed the high heterogeneity described among S. equorum strains.
Assuntos
Proteínas de Bactérias/genética , Técnicas de Tipagem Bacteriana/métodos , Produtos da Carne/microbiologia , Recombinases Rec A/genética , Staphylococcus/genética , Staphylococcus/isolamento & purificação , Primers do DNA/genética , Análise de Sequência de DNA , Staphylococcus/classificaçãoRESUMO
Immobilised derivatives of tannase from Lactobacillus plantarum were able to catalyse the transesterification of tannic acid by using moderate concentrations of 1-propanol in aqueous media. Transesterification of tannic acid was very similar to transesterification of methyl gallate. The synthetic yield depended on the pH and concentration of 1-propanol, although it did not vary much when using 30% or 50% 1-propanol. Synthetic yields of 45% were obtained with 30% of 1-propanol at pH 5.0. The product was chromatographically pure, and the reaction by-product was 55% pure gallic acid. On the other hand, immobilised tannase was fairly stable under optimal reaction conditions.
RESUMO
This study describes the application of a multilocus sequence typing (MLST) analysis for molecular discrimination at the strain level of Spanish wine yeast strains. The discrimination power of MLST is compared to mitochondrial RFLP analysis. Fragments of the ADP1, ACC1, RPN2, GLN4, and ALA1 genes were amplified by PCR from chromosomal DNA of 18 wine Saccharomyces cerevisiae strains. Ten polymorphic sites were found in the five loci analyzed showing 13 different genotypes, with 11 of them represented by only one strain. RFLP analysis of the same 18 wine yeast strains showed seventeen different mitochondrial patterns. Phylogenetic relationships among the strains analyzed, inferred by MLST data, showed wine isolates of S. cerevisiae as a rather homogeneous group. The discrimination potential of mitochondrial RFLP analysis was superior to the MLST scheme used in this work. However, MLST analysis allowed an easy construction of reliable phylogenetic trees. MLST analysis offers the possibility of typing wine S. cerevisiae strains simultaneously to the study of the genetic relationship among them.
Assuntos
DNA Fúngico/genética , Técnicas de Tipagem Micológica/métodos , Polimorfismo Genético/genética , Saccharomyces cerevisiae/classificação , Saccharomyces cerevisiae/genética , Vinho/microbiologia , Biodiversidade , DNA Mitocondrial/genética , Microbiologia de Alimentos , Amplificação de Genes , Genótipo , Filogenia , Polimorfismo de Fragmento de Restrição/genética , Análise de Sequência de DNARESUMO
A study was carried out to determine the in vitro interaction between ochratoxin A (OTA) and wine lactic acid bacteria (LAB). Fifteen strains belonging to five relevant oenological LAB species were grown in liquid synthetic culture medium containing OTA. The portion of OTA removed during the bacterial growth was 8 to 28%. The OTA removed from the supernatants was partially recovered (31 to 57%) from the bacterial pellet. Cell-free extracts of three representative strains were produced by disrupting cells in a French pressure cell. The ability of crude cell-free extracts to degrade OTA was studied. OTA was not degraded by cell-free extracts of wine LAB strains, and no degradation products of OTA were detected in the high-performance liquid chromatograms of the methanol extract of the bacterial pellet. On the basis of these results, we conclude that OTA removal by wine LAB is a cell-binding phenomenon. The chemistry and the molecular basis of OTA binding to wine LAB remains unknown.
Assuntos
Descontaminação/métodos , Contaminação de Alimentos/análise , Lactobacillus/fisiologia , Ocratoxinas/análise , Vinho , Humanos , Microbiologia Industrial/métodos , Lactobacillus/metabolismo , Vitis/química , Vinho/análise , Vinho/microbiologiaRESUMO
The potential to produce biogenic amines was investigated for 56 coagulase-negative staphylococci isolated during industrial Spanish dry-cured ham processes. The presence of biogenic amines from bacterial cultures was determined by thin-layer chromatography. The percentage of strains that decarboxylated amino acids was very low (3.6%). The only staphylococci with aminogenic capacity were an histamine-producing Staphylococcus capitis strain, and a Staphylococcus lugdunensis strain that simultaneously produced putrescine and cadaverine. In both strains, PCR was used to confirm the presence of the genes encoding the amino acid decarboxylases responsible for the synthesis of these amines. This study reveals that production of biogenic amines is not a widely distributed property among the staphylococci isolated from Spanish dry-cured hams.
RESUMO
Putrescine has a negative effect on health and is also used as an indicator of quality on meat products. We investigated the genes involved in putrescine production by Serratia liquefaciens IFI65 isolated from a spoiled Spanish dry-cured ham. We report here the genetic organization of its ornithine decarboxylase encoding region. The 5506-bp DNA region showed the presence of three complete and two partial open reading frames. Putative functions have been assigned to several gene products by sequence comparison with proteins included in the databases. The second gene putatively coded for an ornithine decarboxylase. The functionality of this decarboxylase has been experimentally demonstrated by complementation to an E. coli defective mutant. Based on sequence comparisons of some enterobacterial ornithine decarboxylase regions, we have elaborated a hypothetical pathway for the acquisition of putrescine biosynthetic genes in some Enterobacteriaceae strains.
Assuntos
Clonagem Molecular , Produtos da Carne/microbiologia , Ornitina Descarboxilase , Serratia liquefaciens/enzimologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Conservação de Alimentos , Dados de Sequência Molecular , Ornitina Descarboxilase/genética , Ornitina Descarboxilase/metabolismo , Putrescina/biossíntese , Análise de Sequência de DNA , Serratia liquefaciens/genética , Espanha , SuínosRESUMO
This study describes an easy PCR method for the detection of foodborne bacteria that potentially produce histamine, tyramine, putrescine, and cadaverine. Synthetic oligonucleotide pairs for the specific detection of the gene coding for each group of bacterial histidine, tyrosine, ornithine, or lysine decarboxylases were designed. Under the conditions used in this study, the assay yielded fragments of 372 and 531 bp from histidine decarboxylase-encoding genes, a 825-bp fragment from tyrosine decarboxylases, fragments of 624 and 1,440 bp from ornithine decarboxylases, and 1,098- and 1,185-bp fragments from lysine decarboxylases. This is the first PCR method for detection of cadaverine-producing bacteria. The method was successfully applied to several biogenic amine-producing bacterial strains.
Assuntos
Aminas Biogênicas/isolamento & purificação , Contaminação de Alimentos/análise , Bactérias Gram-Negativas/metabolismo , Bactérias Gram-Positivas/metabolismo , Reação em Cadeia da Polimerase/métodos , Técnicas Bacteriológicas , Aminas Biogênicas/análise , Aminas Biogênicas/biossíntese , Cadaverina/análise , Cadaverina/biossíntese , Cadaverina/isolamento & purificação , Fragmentação do DNA , Microbiologia de Alimentos , Bactérias Gram-Negativas/isolamento & purificação , Bactérias Gram-Positivas/isolamento & purificação , Histamina/análise , Histamina/biossíntese , Histamina/isolamento & purificação , Humanos , Putrescina/análise , Putrescina/biossíntese , Putrescina/isolamento & purificação , Especificidade da Espécie , Tiramina/análise , Tiramina/biossíntese , Tiramina/isolamento & purificaçãoRESUMO
The potential of several alternative genetic engineering based strategies in order to accelerate Saccharomyces cerevisiae autolysis for wine production has been studied. Both constitutively autophagic and defective in autophagy strains have been studied. Although both alternatives lead to impaired survival under starvation conditions, only constitutively autophagic strains, carrying a multicopy plasmid with the csc1-1 allele under the control of the TDH3 promoter, undergo accelerated autolysis in the experimental conditions tested. Fermentation performance is impaired in the autolytic strains, but industrial strains carrying the above-mentioned construction are still able to complete second fermentation of a model base wine. We suggest the construction of industrial yeasts showing a constitutive autophagic phenotype as a way to obtain second fermentation yeast strains undergoing accelerated autolysis.
Assuntos
Expressão Gênica , Genes Fúngicos , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Alelos , Aminoácidos/análise , Contagem de Colônia Microbiana , Meios de Cultura/química , Etanol/metabolismo , Fermentação , Microbiologia de Alimentos , Glucose/metabolismo , Microbiologia Industrial/métodos , Plasmídeos , Regiões Promotoras Genéticas , Vinho/microbiologiaRESUMO
Yeast autolysis is the source of several molecules responsible for the quality of wines aged in contact with yeast cells. However, the mechanisms of yeast autolysis during wine aging are not completely understood. All descriptions of yeast autolysis in enological conditions emphasize the disturbance of cell organization as the starting event in the internal digestion of the cell, while no reference to autophagy is found in wine-related literature. By using yeast mutants defective in the autophagic or the Cvt pathways we have demonstrated that autophagy does take place in wine production conditions. This finding has implications for the genetic improvement of yeasts for accelerated autolysis.
Assuntos
Autofagia , Saccharomyces cerevisiae/fisiologia , Vinho , Western Blotting , FermentaçãoRESUMO
Five mutants (obtained by UV mutagenesis) and the parent strain were selected to produce sparkling wines following the traditional or champenoise method. The wines were aged with the yeast for 9 months, with samples being taken each month for analytical and sensory determinations. The wines elaborated with mutant strain IFI473I demonstrated an accelerated release of protein, amino acids, and polysaccharides. An analysis of the secreted polysaccharides revealed that mannose was the major sugar present. The effects of the products released by yeasts on the foaming properties of the wines were determined by both sensory and instrumental analysis. In all cases, the wines elaborated with mutant strain IFI473I showed improved foaming properties as compared to wines fermented without this strain. Similar results were obtained at a decreased aging time of 6 months, thereby confirming the capacity of IFI473I strain to carry out an accelerated autolysis. These results demonstrate that mutant strain IFI473I can significantly reduce production times of high-quality sparkling wines.
Assuntos
Saccharomyces cerevisiae/metabolismo , Vinho/análise , Bebidas Gaseificadas/análise , Fenômenos Químicos , Físico-Química , Fermentação , Manipulação de Alimentos/métodos , Humanos , Mutação , Polissacarídeos/análise , Saccharomyces cerevisiae/genética , Sensação , Fatores de TempoRESUMO
This study describes a simple, rapid, and inexpensive method to determine the ability to produce biogenic amines (BA) by bacteria in liquid culture media containing the corresponding amino acid precursor. In view of their role as starters in food fermentation, BA formation by these microorganisms has to be taken into consideration by selecting appropriate strains. For the standardization of the assay pure BA were mixed. The method avoids a prior extraction step of the amines and allows the separation and identification of the amines histamine, tyramine, putrescine, and phenylethylamine using thin-layer chromatography. The method was successfully applied to several BA-producer bacterial strains. This method constitutes a simple solution to the previous reports describing false-positive reactions in routine screening procedures generally involving the use of a differential medium containing a pH indicator.
Assuntos
Aminoácidos/metabolismo , Bactérias/metabolismo , Aminas Biogênicas/análise , Cromatografia em Camada Fina/métodos , Cromatografia em Camada Fina/economia , Fermentação , Microbiologia de Alimentos , Concentração de Íons de Hidrogênio , Fatores de TempoRESUMO
In Albariño white wines, aging of wines on lees is a technique not used or only used empirically by some producers to obtain a distinctive character in the final wine. This study analyzes the influence of a short aging on lees on the chemical and sensorial parameters of this young white wine. Albariño grape must was inoculated with a locally selected yeast (Saccharomyces cerevisiae 1) and the effect of a short aging on lees was studied during different times (10, 20, 30, 40, and 50 d). Mannoprotein content and the aromatic profile were determined and a sensorial analysis of the wines was conducted. Results showed that aging time was correlated with the concentration of some key aroma compounds and mannoproteins in Albariño wines. The best sensorial character was obtained in wines aged 20 d on lees. Further aging times decreased the sensorial quality of Albariño wine and modified its volatile profile and mannoprotein concentration.
Assuntos
Manipulação de Alimentos , Glicoproteínas de Membrana/análise , Odorantes/análise , Saccharomyces cerevisiae/química , Vitis/química , Compostos Orgânicos Voláteis/análise , Vinho/análise , Microbiologia de Alimentos , Frutas , Humanos , Especificidade da Espécie , Vitis/classificação , Vinho/microbiologiaRESUMO
Ion-exchange chromatography using commercial ionic supports is a commonly used technique for protein purification. However, selective adsorption of a target protein from a given extract onto commercial ion exchangers seems to be quite complex since they are designed to adsorb the maximum percentage of proteins with the opposite charge. In this paper, ion-exchanger supports with different activation degrees (from 1 to 40 micromol of amino groups per g of agarose) have been prepared and used for the purification of large proteins. These kinds of proteins have large surfaces to interact by many points with the support. Therefore, it was possible to purify large proteins as beta-galactosidase from Thermus sp. strain T2 from a crude extract from Escherichia coli or bovine liver catalase from a commercial preparation, with tailor-made ion-exchanger supports. A simple step of adsorption/desorption on lowly activated supports rendered both enzymes rather pure as confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Moreover, this strategy makes also easy the desorption step that requires rather low NaCl concentrations, which may become a serious problem for desorption of large proteins when using conventional supports, due to their ability of generating a very strong adsorption.
Assuntos
Cromatografia por Troca Iônica/métodos , Proteínas/isolamento & purificação , Cromatografia em Gel/métodos , Eletroforese em Gel de Poliacrilamida , Thermus/enzimologia , beta-Galactosidase/químicaRESUMO
A strategy to selectively adsorb large proteins on immobilized metal ion affinity chromatography supports is presented. It is based on the fact that large proteins have a large surface that permits the long distance interaction with groups placed quite far apart (very dispersed onto the support surface) in the support, therefore, even using lowly activated supports, these proteins may be able to yield multiple interactions with the support, which is not possible for smaller proteins. This has been shown using a crude extract from Escherichia coli, where only large proteins were adsorbed on supports having 0.25 micromol of metallic groups/g of support. Then, these lowly activated supports have been used for purifying multimeric enzymes from thermophilic organisms (alpha- and beta-galactosidases from Thermus sp. strain T2) cloned and over-expressed in mesophilic ones. A previous heating step of the crude extract destroyed the quaternary structure of all multimeric enzymes from the host (E. coli). Thus, the only large protein remaining in the supernatant of this heated extract are the cloned multimeric thermophilic enzymes, permitting their very simple purification by using only one chromatographic step.
Assuntos
Cromatografia de Afinidade/métodos , Metais/química , alfa-Galactosidase/isolamento & purificação , beta-Galactosidase/isolamento & purificação , Adsorção , Eletroforese em Gel de Poliacrilamida , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Thermus/enzimologia , alfa-Galactosidase/química , beta-Galactosidase/químicaRESUMO
This work exemplifies the advantages of using a battery of new heterofunctional epoxy supports to immobilize enzymes. We have compared the performance of a standard Sepabeads-epoxy support with other Sepabeads-epoxy supports partially modified with boronate, iminodiacetic, metal chelates, and ethylenediamine in the immobilization of the thermostable beta-galactosidase from Thermus sp. strain T2 as a model system. Immobilization yields depended on the support, ranging from 95% using Sepabeads-epoxy-chelate, Sepabeads-epoxy-amino, or Sepabeads-epoxy-boronic to 5% using Sepabeads-epoxy-IDA. Moreover, immobilization rates were also very different when using different supports. Remarkably, the immobilized beta-galactosidase derivatives showed very improved but different stabilities after favoring multipoint covalent attachment by long-term alkaline incubation, the enzyme immobilized on Sepabeads-epoxy-boronic being the most stable. This derivative had some subunits of the enzyme not covalently attached to the support (detected by SDS-PAGE). This is a problem if the biocatalysts were to be used in food technology. The optimization of the cross-linking with aldehyde-dextran permitted the full stabilization of the quaternary structure of the enzyme. The optimal derivative was very active in lactose hydrolysis even at 70 degrees C (over 1000 IU/g), maintaining its activity after long incubation times under these conditions and with no risk of product contamination with enzyme subunits.
Assuntos
Aldeídos/química , Dextranos/química , Compostos de Epóxi/química , Lactose/química , Thermus/enzimologia , beta-Galactosidase/química , Adsorção , Dimerização , Ativação Enzimática , Estabilidade Enzimática , Enzimas Imobilizadas/química , Hidrólise , Polímeros/química , Conformação Proteica , Estrutura Quaternária de Proteína , Especificidade da Espécie , Thermus/classificaçãoRESUMO
The heating of protein preparations of mesophilic organism (e.g., E. coli) produces the obliteration of all soluble multimeric proteins from this organism. In this way, if a multimeric enzyme from a thermophilic microorganism is expressed in these mesophilic hosts, the only large protein remaining soluble in the preparation after heating is the thermophilic enzyme. These large proteins may be then selectively adsorbed on lowly activated anionic exchangers, enabling their full purification in just these two simple steps. This strategy has been applied to the purification of an alpha-galactosidase and a beta-galactosidase from Thermus sp. strain T2, both expressed in E. coli, achieving the almost full purification of both enzymes in only these two simple steps. This very simple strategy seems to be of general applicability to the purification of any thermophilic multimeric enzyme expressed in a mesophilic host.
Assuntos
Cromatografia por Troca Iônica/métodos , Escherichia coli/enzimologia , Temperatura Alta , Complexos Multiproteicos/isolamento & purificação , Thermus/enzimologia , alfa-Galactosidase/isolamento & purificação , beta-Galactosidase/isolamento & purificação , Resinas de Troca Aniônica , Dimerização , Escherichia coli/genética , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Engenharia de Proteínas/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/efeitos da radiação , Thermus/genética , alfa-Galactosidase/química , alfa-Galactosidase/genética , alfa-Galactosidase/efeitos da radiação , beta-Galactosidase/química , beta-Galactosidase/genética , beta-Galactosidase/efeitos da radiaçãoRESUMO
The degradation of natural phosphorylated compounds (galactose-1-phosphate, N-acetyl-glucosamine-1-phosphate, glycerophosphoethanolamine, and glycerophosphocholine) and added phosphorylated compounds (diphosphate) in milk was investigated by phosphorus 31 nuclear magnetic resonance on the incubation of a sterile milk with Pseudomonas fluorescens CECT381, Lactococcus lactis CECT539, and Kluyveromyces marxianus CECT10584. This preliminary study showed that the degradation of these compounds was dependent on the compound, microorganism, and temperature of incubation. K. marxianus CECT10584 did not show any capability to degrade these compounds, and L. lactis CECT539 was only able to degrade diphosphate at its optimum growth temperature. P. fluorescens CECT381 was the most active strain and possessed more hydrolytic capabilities at 10 degrees C than at its optimum growth temperature. It is suggested that cold-induced enzymes are involved in the ability of P. fluorescens CECT381 to hydrolyze the natural phosphorylated compounds in milk. Consequent potential alterations of dairy products are discussed.
Assuntos
Kluyveromyces/metabolismo , Lactococcus lactis/metabolismo , Leite/microbiologia , Polifosfatos/administração & dosagem , Pseudomonas fluorescens/metabolismo , Animais , Laticínios/análise , Laticínios/microbiologia , Difosfatos , Microbiologia de Alimentos , Imageamento por Ressonância Magnética , Fosforilação , Temperatura , Fatores de TempoRESUMO
BACKGROUND: Owing to its antimicrobial properties dietary tannins may alter the functional efficacy of probiotic lactobacilli in the gastrointestinal (GI)-tract influencing their growth, viability and molecular adaptation to the intestinal environment. METHODS AND FINDINGS: The effects of tannic acid on Lactobacillus plantarum WCFS1 were studied by in vitro growth monitoring and visualizing the morphological alteration on the cell wall using transmission electron microscopy. Growth upon tannic acid was characterized by dose-dependent reductions of initial viable counts and extended lag phases. Lag phase-cells growing upon 0.5 mM tannic acid were abnormally shaped and experienced disturbance on the cell wall such as roughness, occasional leakage and release of cell debris, but resumed growth later at tannic acid concentrations high as 2.5 mM. To gain insight on how the response to tannic acid influenced the molecular adaptation of L. plantarum to the GI-tract conditions, gene expression of selected biomarkers for GI-survival was assessed by RT-qPCR on cDNA templates synthetized from mRNA samples obtained from cells treated with 0.5 or 2 mM tannic acid. Tannic acid-dependent gene induction was confirmed for selected genes highly expressed in the gut or with confirmed roles in GI-survival. No differential expression was observed for the pbp2A gene, a biomarker negatively related with GI-survival. However PBP2A was not labeled by Bocillin FL, a fluorescent dye-labeled penicillin V derivative, in the presence of tannic acid which suggests for enhanced GI-survival reportedly associated with the inactivation of this function. CONCLUSIONS: Probiotic L. plantarum WCFS1 is able to overcome the toxic effects of tannic acid. This dietary constituent modulates molecular traits linked to the adaptation to intestinal environment in ways previously shown to enhance GI-survival.
Assuntos
Trato Gastrointestinal/microbiologia , Lactobacillus plantarum/efeitos dos fármacos , Lactobacillus plantarum/metabolismo , Taninos/farmacologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Humanos , Lactobacillus plantarum/genética , Lactobacillus plantarum/ultraestrutura , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The preparation of novel immobilized and stabilized derivatives of trypsin is reported here. The new derivatives preserved 80% of the initial catalytic activity toward synthetic substrates [benzoyl-arginine p-nitroanilide (BAPNA)] and were 50,000-fold more thermally stable than the diluted soluble enzyme in the absence of autolysis. Trypsin was immobilized on highly activated glyoxyl-Sepharose following a two-step immobilization strategy: (a) first, a multipoint covalent immobilization at pH 8.5 that only involves low pK(a) amino groups (e.g., those derived from the activation of trypsin from trypsinogen) is performed and (b) next, an additional alkaline incubation at pH 10 is performed to favor an intense, additional multipoint immobilization between the high concentration of proximate aldehyde groups on the support surface and the high pK(a) amino groups at the enzyme surface region that participated in the first immobilization step. Interestingly, the new, highly stable trypsin derivatives were also much more active in the proteolysis of high molecular weight proteins when compared with a nonstabilized derivative prepared on CNBr-activated Sepharose. In fact, all the proteins contained a cheese whey extract had been completely proteolyzed after 6 h at pH 9 and 50°C, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Under these experimental conditions, the immobilized biocatalysts preserve more than 90% of their initial activity after 20 days. Analysis of the three-dimensional (3D) structure of the best immobilized trypsin derivative showed a surface region containing two amino terminal groups and five lysine (Lys) residues that may be responsible for this novel and interesting immobilization and stabilization. Moreover, this region is relatively far from the active site of the enzyme, which could explain the good results obtained for the hydrolysis of high-molecular weight proteins.
Assuntos
Biocatálise , Enzimas Imobilizadas/síntese química , Proteínas/metabolismo , Tripsina/química , Tripsina/metabolismo , Sítios de Ligação , Enzimas Imobilizadas/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Conformação Proteica , Estabilidade ProteicaRESUMO
Rhizomucor miehei lipase (RML) is greatly hyperactivated (around 20- to 25-fold toward small substrates) in the presence of sucrose laurate. Hyperactivation appears to be an intramolecular process because it is very similar for soluble enzymes and covalently immobilized derivatives. The hyperactivated enzyme was immobilized (in the presence of sucrose laurate) on cyanogen bromide-activated Sepharose (very mild covalent immobilization through the amino terminal residue), on glyoxyl Sepharose (intense multipoint covalent immobilization through the region with the highest amount of Lys residues), and on different anion exchangers (by multipoint anionic exchange through the region with the highest density of negative charges). Covalent immobilization does not promote the fixation of the hyperactivated enzyme, but immobilization on Sepharose Q retains the hyperactivated enzyme even in the absence of a detergent. The hydrolysis of fish oils by these hyperactivated enzyme derivatives was sevenfold faster than by covalently immobilized derivatives and three and a half times faster than by the enzyme hyperactivated on octyl-Sepharose. The open structure of the hyperactivated lipase is fairly exposed to the medium, and no steric hindrance should interfere with the hydrolysis of large substrates. These new hyperactivated derivatives seem to be more suitable for hydrolysis of oils by RML immobilized inside porous supports. In addition, the hyperactivated derivatives are fairly stable against heat and organic cosolvents.