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Papers by Laura Lopez

Production, characterization, and identification using proteomic tools of a polygalacturonase from Fusarium graminearum

Journal of Basic Microbiology, 2014

Since enzymatic degradation is a mechanism or component of the aggressiveness of a pathogen, enzy... more Since enzymatic degradation is a mechanism or component of the aggressiveness of a pathogen, enzymatic activities from a Fusarium graminearum isolate obtained from infected wheat spikes of Argentina Pampa region were studied in order to understand the disease progression, tending to help disease control. In particular, the significance of the study of polygalacturonase activity is based on that such activity is produced in the early stages of infection on the host, suggesting a crucial role in the establishment of disease. In this sense, polygalacturonase activity produced by this microorganism has been purified 375 times from 2‐day‐old culture filtrates by gel filtration and ion‐exchange chromatography successively. The purified sample showed two protein bands in sodium dodecyl sulfate–polyacrylamide gels, with a molecular mass of 40 and 55 kDa. The protein bands were identified as an endopolygalacturonase and as a serine carboxypeptidase of F. graminearum, respectively, by peptide...

Applied biochemistry and biotechnology, Jan 6, 2016

The latex from the patagonic plant Philibertia gilliesii Hook. et Arn. (Apocynaceae) is a milky-w... more The latex from the patagonic plant Philibertia gilliesii Hook. et Arn. (Apocynaceae) is a milky-white suspension containing a proteolytic system constituted by several cysteine endopeptidases. A proteolytic preparation (philibertain g) from the latex of P. gilliesii fruits was obtained and characterized to evaluate its potential use in bioprocesses. Philibertain g contained 1.2 g/L protein and a specific (caseinolytic) activity of 7.0 Ucas/mg protein. It reached 80 % of its maximum caseinolytic activity in the pH 7-10 range, retained 80 % of the original activity after 2 h of incubation at temperatures ranging from 25 to 45 °C and could be fully inactivated after 5 min at 75 °C. Philibertain g retained 60 % of the initial activity even at 1 M NaCl and was able to hydrolyze proteins from stickwater one, of the main waste effluents generated during fishmeal production. Furthermore, as a contribution to the knowledge of the proteolytic system of P. gilliesii, we are reporting the purif...

Purificación de una nueva endopeptidasa aislada de frutos de Bromelia hieronymi Mez (Bromelia-ceae)

Acta Farmaceutica Bonaerense, 2002

Proteases present in latex from fruits of maclura pomifera raf. schneid. moraceae

Proteasas de plantas superiores ii. Ficina ( ec 3.4.22.32 )

New purified plant proteinases for the food industry

Acta Alimentaria, 1991

A crude enzyme preparation obtained from unripe fruits of Bromelia hieronymi Mez (Bromeliaceae) s... more A crude enzyme preparation obtained from unripe fruits of Bromelia hieronymi Mez (Bromeliaceae) shows a broad pH range of optimum proteolytic activity and a good thermal stability up to 45°C. Diafiltration followed by ion-exchange chromatography allows the separation of four proteolytic components of closely related molecular weights

RESUMEN. El presente trabajo es el primero de una serie destinada a actualizar el conocimiento de... more RESUMEN. El presente trabajo es el primero de una serie destinada a actualizar el conocimiento de las enzimas proteolíticas de plantas superiores. En esta oportunidad se brinda información sobre los sistemas de clasificación de las proteasas y de algunos aspectos nomenclaturales de las mismas, así como de las modificaciones que habrían sufrido sus estructuras a través de la evolución de los seres vivos. Se incluyen, además, algunas interpretaciones sobre el rol de las enzimas proteolíticas en los procesos fisiológicos primarios y auxiliares de los vegetales y una breve reseña de sus principales aplicaciones. SUMMARY. "Proteases of higller phnts. I. General features, physiological role and applications". The present is the fust paper of a series dealing with proteases of higher plants. General aspects of nomenclature, classification and evolution of proteolytic enzymes are here briefly described, together with their behaviour in primary and auxiliar physiological processes in plants. A short review on uses and applications of plant proteases is also given.

Proteasas de plantas superiores

Biotechnology Letters, 2019

Objective To determine the enzymatic properties of asclepain f, a plant cysteine protease isolate... more Objective To determine the enzymatic properties of asclepain f, a plant cysteine protease isolated and purified from the latex of Asclepias fruticosa, and to investigate its potential application to hydrolyze soybean proteins. Results Kinetic parameters were determined by hydrolysis of p-Glu-Phe-Leu-p-nitroanilide (PFLNA). The Km value for asclepain f was 6 to 8 times higher than those achieved for papain, bromelain and ficin, the main plant cysteine proteases. Asclepain f showed 12 cutoff points toward the oxidized B chain insulin, revealing that the enzyme possesses broad substrate specificity. The cut specificity was governed by the presence of hydrophobic residues (F, L, V) in the P2 position. Asclepain f was able to selectively hydrolyze soybean proteins at pH 10, employing an enzyme/substrate ratio of 0.2% (w/w). The enzymatic hydrolysis allowed a strong increase in the solubility, water and oil holding capacity. Conclusions Asclepain f was revealed as a successful enzyme for biocatalysis of protein hydrolysis processes at alkaline pH. This new plant protease has a broad substrate specificity and is capable of selectively degrading the fractions of soy proteins and improving its functional properties.

Applied biochemistry and biotechnology, Jan 15, 2018

The primary structure of macrodontain I, a peptidase from Pseudananas macrodontes fruits, was det... more The primary structure of macrodontain I, a peptidase from Pseudananas macrodontes fruits, was determined using Edman's degradation. The enzyme is a non-glycosylated peptidase composed by 213 amino acids with a calculated molecular weight of 23,486.18 Da, pI value 6.99, and a molar extinction coefficient at 280 nm of 61,685 M cm. The alignment of the sequence of macrodontain I with those cysteine peptidases from species belonging to the family Bromeliaceae showed the highest identity degree (87.74%) against fruit bromelain. A remarkable fact is that all these peptidase sequences show two Met contiguous residues (Met121 and 122) and the nonapeptide VPQSIDWRD located in the mature N-terminal region. Residues Cys26 and His159, which constitute the catalytic dyad in all cysteine peptidases, as well as active site residues Gln20 and Asn176, characteristic of Clan C1A, are conserved in macrodontain I. The 3-D model suggests that the enzyme belongs to the α + β class of proteins, with t...

Milk clotting activity and production of bioactive peptides from whey using Maclura pomifera proteases

LWT - Food Science and Technology, 2012

... Maria Alicia Corrons , E-mail The Corresponding Author , Juan Ignacio Bertucci , E-mail The C... more ... Maria Alicia Corrons , E-mail The Corresponding Author , Juan Ignacio Bertucci , E-mail The Corresponding Author , Constanza Silvina Liggieri , E-mail The Corresponding Author , Laura María Isabel López , E-mail The Corresponding Author , Mariela Anahí Bruno ...

Acta Farmacéutica …, 2002

A new plant endopeptidase has been obtained from unripe fruits of Bromelia hieronymi Mez (Bromeli... more A new plant endopeptidase has been obtained from unripe fruits of Bromelia hieronymi Mez (Bromeliaceae). Crude extracts were partially purified by organic solvents fractionation: best results (90% of proteins, 94% of total caseinolytic activity and only 9.9% of soluble sugars) were obtained by adding 4 volumes of cold acetone to the crude extract. This preparation (redissolved acetone precipitate, RAP) showed maximum activity (> 80%) at pH 7.3-10.7, and exhibited high thermal stability (80% of residual activity after heating for 30 min at 60 ºC). Low sodium chloride concentrations (0.2 M) does not affect caseinolytic activity, but diminishes with the increase of salt concentration (52% of residual activity at 2.5 M NaCl). RAP showed also clotting activity and a notably preference for κ κ casein over α α s1 , α αs 2 and β β caseins, properties that could be of interest in the cheese industry. The enzyme was completely inhibited by E-64 and iodoacetic and activated by the addition of cysteine; these results strongly suggest that the isolated protease should be included within the cysteine group, as all the other studied proteases belonging to the family Bromeliaceae. IEF-zymogram of RAP showed six bands (pI 5.9 to >9.3), most of them proteolytically actives, but only three of which (pI 6.4, 8.3 and >9.3) proved to be important. Cation exchange chromatography (FPLC) allowed the isolation of the main fraction, named hieronymain I (pI = >9.3, molecular weight = 25 kDa). RESUMEN. "Purificación de una nueva endopeptidasa aislada de frutos de Bromelia hieronymi Mez (Bromeliaceae)". Se ha aislado una nueva endopeptidasa a partir de frutos inmaduros de Bromelia hieronymi Mez (Bromeliaceae). Los extractos crudos fueron parcialmente purificados por fraccionamiento con solventes orgánicos, habiéndose obtenido los mejores resultados al precipitar el extracto crudo con 4 volúmenes de acetona fría (el precipitado retiene el 90% de las proteínas y el 94% de la actividad caseinolítica y contiene sólo 9,9% de azúcares solubles). El precipitado acetónico redisuelto (RAP) mostró máxima actividad (> 80%) a pH 7,3-10,7 y muestra gran estabilidad térmica (mantiene 80% de actividad residual luego de calentarlo a 60 ºC durante 30 min). Bajas concentraciones de cloruro de sodio (0,2 M) no afectan la actividad caseinolítica, pero ésta disminuye con el incremento de concentración salina (52% de actividad residual cuando la concentración de cloruro de sodio es 2,5 M). El RAP demostró poseer actividad coagulante de la leche y notable preferencia por la fracción κ caseína por encima de las α s1 , α s2 y β caseínas, características que pueden resultar de interés en la industria quesera. La enzima resultó totalmente inhibida por E-64 y ácido iodoacético y activada por cisteína, lo que sugiere que pertenece al grupo de la endopeptidasas cisteínicas, en el que se encuentran todas las proteasas estudiadas de la familia Bromeliaceae. El isoelectroenfoque seguido de zimograma revela la presencia de seis bandas (pI 5,9 a > 9,3), la mayoría de ellas proteolíticamente activas, pero sólo tres (pI 6,4, 8,3 y >9,3) de importancia. Por cromatografía de intercambio catiónico (FPLC) se logró aislar la fracción principal, denominada hieronymina I (pI >9,3 peso molecular 25 kDa).

Proteolytic extracts of three Bromeliaceae species as eco-compatible tools for leather industry

Environmental science and pollution research international, Jan 2, 2018

Most tanneries use high proportions of Na2S and CaO during the dehairing step, resulting in efflu... more Most tanneries use high proportions of Na2S and CaO during the dehairing step, resulting in effluents of high alkalinity and large amounts of suspended solid, besides the risk of liberating the toxic H2S. Solid waste rich in protein is another environmental problem of tanneries. Enzymes are an interesting technological tool for industry due to their biodegradability, nontoxic nature, and nonpolluting effluent generation. In the leather industry, proteases have been chosen as a promising eco-friendly alternative to Na2S/CaO dehairing. Extracts with high proteolytic activity have been obtained from fruits of Bromeliaceae species: Bromelia balansae Mez (Bb), Bromelia hieronymi Mez (Bh), and Pseudananas macrodontes (Morr.) Harms (Pm). In this work, Bb, Bh, and Pm have been studied for application in the leather industry, focusing in their dehairing properties. Enzymatic activities were measured against collagen, keratin, elastin, and epidermis while a dehairing assay was performed by em...

A Bowman-Birk protease inhibitor purified, cloned, sequenced and characterized from the seeds of Maclura pomifera (Raf.) Schneid

Planta, Jan 24, 2016

A new BBI-type protease inhibitor with remarkable structural characteristics was purified, cloned... more A new BBI-type protease inhibitor with remarkable structural characteristics was purified, cloned, and sequenced from seeds of Maclura pomifera , a dicotyledonous plant belonging to the Moraceae family. In this work, we report a Bowman-Birk inhibitor (BBI) isolated, purified, cloned, and characterized from Maclura pomifera seeds (MpBBI), the first of this type from a species belonging to Moraceae family. MpBBI was purified to homogeneity by RP-HPLC, total RNA was extracted from seeds of M. pomifera, and the 3'RACE-PCR method was applied to obtain the cDNA, which was cloned and sequenced. Peptide mass fingerprinting (PMF) analysis showed correspondence between the in silico-translated protein and MpBBI, confirming that it corresponds to a new plant protease inhibitor. The obtained cDNA encoded a polypeptide of 65 residues and possesses 10 cysteine residues, with molecular mass of 7379.27, pI 6.10, and extinction molar coefficient of 9105 M(-1) cm(-1). MpBBI inhibits strongly tryp...

Journal of protein chemistry, 2001

Asclepias fruticosa L. is a small shrub containing latex with proteolytic activity. The crude ext... more Asclepias fruticosa L. is a small shrub containing latex with proteolytic activity. The crude extract (latex diluted 1:250 and ultracentrifuged) contained 276 microg of protein/mL and the proteolytic activity reached 1.2 caseinolytic U/mL. This enzyme preparation was very stable even after 2 hours at 45 degrees C, but was quickly inactivated after 5 minutes at 80 degrees C. Chromatographic purification was achieved by FPLC using a cation exchanger (SP-Sepharose FF). Thus, a unique proteolitically active fraction could be isolated, being homogeneous by bidimensional electrophoresis and mass spectrometry (Mr = 23,652). The optimum pH range was achieved at 8.5-10.5. The enzyme activity was completely inhibited by specific cysteine peptidases inhibitors. Isoelectric focusing followed by zymogram showed the enzyme had a pI greater than 9.3. The N-terminus sequence (LPDSVDWREKGVVFPIRNQGK) shows a great deal of similarity to those of the other cysteine endopeptidases isolated from latices ...

Letters in Drug Design & Discovery, 2010

A new peptidic protease inhibitor (MpI) has been isolated from Maclura pomifera seeds, being the ... more A new peptidic protease inhibitor (MpI) has been isolated from Maclura pomifera seeds, being the first trypsin and chymotrypsin inhibitor from a species belonging to the family Moraceae. MpI was purified by acetone precipitation, gel filtration and ion exchange chromatography, successively, with purification factors of 112 and 109 for the aforementioned enzymes, which are infrequent high values for inhibitors isolated from seeds. MpI showed a unique band in SDS-Tricine PAGE (Mr 11 kDa) and isoelectric focusing (pI = 5.2), inhibited the serine proteases trypsin and-chymotrypsin (IC50 0.17 and 0.7 μg/ml, respectively), but not cathepsin B (cysteine protease), cathepsin D (aspartic protease) nor carboxypeptidase A (metallo protease). The N-terminal sequence was determined (AREPKFSTHCEEEESR) but no homology was detected with other peptide inhibitors isolated from seeds. Preliminary assays related to blood clotting reactions showed that the isolated inhibitor significatively increased the activated partial thromboplastin time (APTT), suggesting its potential use in the treatment of blood coagulation disorders.

Journal of protein chemistry, 2003

A new peptidase, named hieronymain I, was purified to homogeneity from unripe fruits of Bromelia ... more A new peptidase, named hieronymain I, was purified to homogeneity from unripe fruits of Bromelia hieronymi Mez (Bromeliaceae) by acetone fractionation followed by cation exchange chromatography (FPLC) on CM-Sepharose FF. Homogeneity of the enzyme was confirmed by mass spectroscopy (MALDI-TOF), isoelectric focusing, and SDS-PAGE. Hieronymain is a basic peptidase (pI > 9.3) and its molecular mass was 24,066 Da. Maximum proteolytic activity on casein (>90% of maximum activity) was achieved at pH 8.5-9.5. The enzyme was completely inhibited by E-64 and iodoacetic acid and activated by the addition of cysteine; these results strongly suggest that the isolated protease should be included within the cysteine group. The N-terminal sequence of hieronymain (ALPESIDWRAKGAVTEVKRQDG) was compared with 25 plant cysteine proteases that showed more than 50% of identity.

Planta, 2009

Asclepain f is a papain-like protease previously isolated and characterized from latex of Asclepi... more Asclepain f is a papain-like protease previously isolated and characterized from latex of Asclepias fruticosa. This enzyme is a member of the C1 family of cysteine proteases that are synthesized as preproenzymes. The enzyme belongs to the alpha + beta class of proteins, with two disulfide bridges (Cys22-Cys63 and Cys56-Cys95) in the alpha domain, and another one (Cys150-Cys201) in the beta domain, as was determined by molecular modeling. A full-length 1,152 bp cDNA was cloned by RT-RACE-PCR from latex mRNA. The sequence was predicted as an open reading frame of 340 amino acid residues, of which 16 residues belong to the signal peptide, 113 to the propeptide and 211 to the mature enzyme. The full-length cDNA was ligated to pPICZalpha vector and expressed in Pichia pastoris. Recombinant asclepain f showed endopeptidase activity on pGlu-Phe-Leu-p-nitroanilide and was identified by PMF-MALDI-TOF MS. Asclepain f is the first peptidase cloned and expressed from mRNA isolated from plant la...

The Protein Journal, 2006

From unripe fruits of Bromelia hieronymi Mez (Bromeliaceae), a partially purified protease prepar... more From unripe fruits of Bromelia hieronymi Mez (Bromeliaceae), a partially purified protease preparation was obtained by acetone fractionation of the crude extract. Purification was achieved by anionic exchange chromatography (FPLC) on Q-Sepharose HP followed by cationic exchange chromatography (SP-Sepharose HP). Homogeneity of the new enzyme, named hieronymain II, was confirmed by SDS-PAGE and mass spectroscopy (MALDI-TOF-TOF). The molecular mass of was 23,411 Da, and maximum proteolytic activity (more than 90% of maximum activity) was achieved at pH 7.5-9.0 on casein and at pH 7.3-8.3 on Z-Phe-Arg-p-nitroanilide. The enzyme was completely inhibited by E-64 and iodoacetic acid and activated by the addition of cysteine. The N-terminal sequence of hieronymain II (AVPQSIDWRVYGAV) was compared with those of 12 plant cysteine proteases which showed more than 70% of identity. Kinetic enzymatic assays were made on Z-Phe-Arg-pnitroanilide (K m ¼ 0:72 mM; k cat ¼ 1:82 seg À1 ; k cat / K m ¼ 2:54 seg À1 mM À1). No detectable activity could be found on PFLNA or Z-Arg-Arg-p-nitroanilide.