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La bromelina: una proteasa de interés comercial

July 1996
Ciencia y Tecnologia Alimentaria 1(2)

Authors:

Universidade da Coruña

Dentro de los derivados procedentes de las plantas, encontramos ciertas enzimas proteolíticas interesantes por sus aplicaciones alimentarias y terapéuticas. Entre ellas, la bromelina extraída de la planta y del fruto de la piña tropical es objeto de esta revisión. La principal ventaja de esta proteasa frente a otras como la papaina o la ficina es que se extrae fácilmente a partir de los residuos, tanto de las plantaciones como de las fábricas de enlatado de piña.ABSTRACTAmong the secundary products from vegetables, there are several proteolitic enzymes with interest dued to their food and therapeutic applications. Among these enzymes, this revision paper deals with Bromelain extracted from pine. The major advandage of this protease is the easy extraction process from the waste, as from the plantation as from the canned pine manufactures.RESUMEDentro dos derivados procedentes das plantas, atopamos certas enzimas proteoliticas interesantes polas súas aplicacións alimentares e terapéuticas. Entre elas, a bromelina extraída da planta e do froito da ananá tropical é obxecto desta revisión. A principal aventaxe desta proteasa cara a outras coma a papaína ou a ficina. é que se extrae doadamente a partires dos refugallos, tanto das plantacións como das fábricas de enlatado de ananá.

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Ciencia y Tecnología Alimentaria

Sociedad Mexicana de Nutrición y Tecnología de Alimentos

somenta@gmail.com

ISSN (Versión impresa): 1135-8122

ISSN (Versión en línea): 1696-2443

MÉXICO

1996

I. López Lago / J. Díaz Varela / F. Merino de Cáceres

LA BROMELINA: UNA PROTEASA DE INTERÉS COMERCIAL

Ciencia y Tecnología Alimentaria, junio, año/vol. 1, número 002

Sociedad Mexicana de Nutrición y Tecnología de Alimentos

Reynosa, México

pp. 17-22

Red de Revistas Científicas de América Latina y el Caribe, España y Portugal

Universidad Autónoma del Estado de México

http://redalyc.uaemex.mx

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The cysteine proteinases of the pineapple plant

Article

Full-text available

Apr 1990

The pineapple plant (Ananas comosus) was shown to contain at least four distinct cysteine proteinases, which were purified by a procedure involving active-site-directed affinity chromatography. The major proteinase present in extracts of plant stem was stem bromelain, whilst fruit bromelain was the major proteinase in the fruit. Two additional cysteine proteinases were detected only in the stem: these were ananain and a previously undescribed enzyme that we have called comosain. Stem bromelain, fruit bromelain and ananain were shown to be immunologically distinct. Enzymic characterization revealed differences in both substrate-specificities and inhibition profiles. A study of the cysteine proteinase derived from the related bromeliad Bromelia pinguin (pinguinain) indicated that in many respects it was similar to fruit bromelain, although it was found to be immunologically distinct.

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Jul 1989

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Jul 1957

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Article

Jul 1976

Fruit bromelain FA2, the main proteinase component of the juice of pineapple fruit, has been purified and characterized. 1.Efficient extraction of this enzyme from the crude material was possible using “ Cellulosin AP, ” a microbial polysaccharidase preparation containing cellulase, hemi-cellulase, and pectinase. The enzyme was purified mainly by successive applications of anion-exchange chromatography, yielding an apparently homogeneous protein as judged by several physical, chemical, and immunochemical criteria. Properties of FA2 include: molecular weight, 31, 000; isoelectric point, pH 4.6; absorbance at 280 nm of a 1% solution at pH 7.0 per cm, 19.2. 2. FA2 gave only alanine phenylthiohydantoin upon amino-terminal group analysis by the Edman procedure. Stepwise degradation yielded the amino-terminal sequence Ala-Val-Pro-Gln-Ser-Ile-Asp-Trp-Arg-Asp-Tyr-Gly-Ala. The amino acid composition of FA2 was not markedly different from that of stem bromelain, except for a much smaller lysine content and a smaller alanine content relative to glycine in FA2. FA2 contained neither amino sugars nor neutral carbohydrates as determined by several methods, so FA2 is not a glycoprotein. 3. By labeling the reactive cysteine residue (CYS) with [¹⁴C]iodoacetate, the following partial amino acid sequence has been determined. Asn-Glx-Asn-Pro-Cys-Gly-Ala-CYS 4. FA2 showed pH optima of 8.0 and 8.3 toward hemoglobin and casein, respectively. The Michaelis constant for α-N-benzoyl-L-arginine ethyl ester was 43 mM at pH 6.0 and 25°, FA2 cleaved bradykinin between Gly⁴-Phe⁵ and between Phe⁵-Ser⁶ at comparable rates; it hydrolyzed angiotensin II preferentially between Tyr⁴-lle⁵. 5. Similarities and differences found thus far between fruit bromelain FA2, stem bromelain SB1, and papain are tabulated.

Bromelain enzyme

Article

Feb 1976
METHOD ENZYMOL

Takashi Murachi

Bromelain enzymes are found in tissues of the plant family Bromeliaceae of which pineapple, Ananas comosus (L), is the best known. The proteolytic enzyme found in the juice of pineapple stem is called stem bromelain and the enzyme in the fruit was first described under the name of bromelin 1 and is now called fruit bromelain. Systematic number EC 3.4.22.4 is given to stem bromelain, and EC 3.4.22.5 to fruit bromelain. The purpose of the present chapter is to review the recent information and accumulated data about these two enzymes. The chapter lists the general purification methods, as well as the specific purification method of Murachi, which consists of six stages. It also elaborates on the alternative purification n procedure for the stem enzyme. It lists the physical and chemical properties of the enzymes. The Specificity, Kinetic Properties, and Enzymic Mechanism have also been discussed. The chapter also discusses the Fractionation Procedure of purification for the fruit enzyme and its properties.

Reinvestigation of Fractionation and Some Properties of the Proteolytically Active Components of Stem and Fruit Bromelains

Article

Aug 1985

To check whether crude stem and fruit bromelains can be fractionated further or not, systematic separation procedures were applied to both enzymes. Six proteolytically active components, which were designated as SBB 1-5 and SBA, were fractionated from crude stem bromelain by successive use of gel filtration on Sephadex G-75, and chromatographies on CM-Sephadex and DEAE-Sephacel. One main and one minor active components, designated as FBA and FBB, respectively, were also separated from crude fruit bromelain by chromatographies on DEAE-Sephacel and then CM-Sephadex. Some of the physico-chemical and enzymatic properties of these eight components were compared. Each component migrated as a single band on SDS-polyacrylamide gel electrophoresis. Molecular weights determined by the same electrophoresis were about 27,000 for SBB 1-3 and FBB, and about 23,000 for the other four components. In terms of amino acid composition, FBB resembled SBB 1-3, which were remarkably similar to each other. FBA was also similar to SBA in amino acid composition, and contained much less basic amino acids than SBB 1 through 5. The principal amino-terminal residues determined by the cyanate method were valine in SBB 1-5 and SBA, and alanine in FBA and FBB. The principal carboxyl-terminal residues determined by the hydrazinolysis method were glycine in SBB 1-3, SBA and FBA, and serine in SBB 4-5 and FBB. However, fractional amounts of a few other amino- and carboxyl-terminal residues were also detected. As regards enzymatic activities, FBA and SBB 4 and 5 were much more active than the other five components against casein and some synthetic substrates [Bz-Arg-amide (at pH 6.1), Z-Gly-X, and Z-Ala-X (at pH 3.5)] with the notable exception that FBA was much less active than SBB 4 and 5 toward tripeptides (X-Gly-Gly).

[38] Pineapple cysteine endopeptidases

Article

Feb 1994
METHOD ENZYMOL

This chapter focuses on pineapple cysteine endopeptidases. The pineapple plant contains at least four distinct cysteine endopeptidases. The major endopeptidase present in extracts of plant stem is stem bromelain, and fruit bromelain is the major endopeptidase in the fruit. Two additional cysteine endopeptidases, ananain and comosain, are detected only in the stem. Both stem and fruit bromelain are assayed with protein substrates such as casein and hemoglobin. Substrates containing longer peptide sequences and more sensitive leaving groups are employed in assay of stem bromelain. The substrate, Z-Arg-Arg-NHMec, which is scarcely fluorescent, is hydrolyzed to liberate 7-amino-4-methylcoumarin, and this is quantified fluorimetrically once the enzyme is inactivated with chloroacetate. The fluorescence of the free aminomethylcoumarin is determined by excitation at 360 nm and emission at 460 nm. All enzymes purified by active-site-directed affinity chromatography are reversibly blocked by mercuric chloride or a disulfide. As no further activation is performed and disulfide is routinely added to column fractions, the enzymes remain in the reversibly blocked state following freeze-drying.

Isolation and properties of bromelin protease

Article

Apr 1969
FEBS LETT

Preparation and Chemical Properties of Purified Stem and Fruit Bromelains *

Article

Mar 1964

The sulfhydryl-activated proteases of the stem and the fruit of the pineapple plant have been isolated in chromatographically purified form and some of the chemical properties of the preparations have been compared. One main proteolytically active component has been obtained from stem bromelain by gel filtration on Sephadex G-75 and chromatography on sulfoethyl-Sephadex at pH 6. The enzyme present in the pineapple fruit has been purified by acetone precipitation followed by chromatography on DEAE-cellulose. Both these preparations give single zones upon chromatography and electrophoresis. However, end-group analysis by the cyanate method indicates that the preparations are not homogeneous. The principal NH2-terminal residue in the stem enzyme is valine (0.9 residue per molecule of mw 36,000), but fractional amounts of other NH2-terminal residues (alanine 0.2, glycine 0.06) were also found. The principal NH2-terminal residue in the fruit enzyme is alanine (0.9 residue per molecule of mw 30,000), but once again additional NH2-terminal residues were noted (valine 0.3, serine 0.2, glycine 0.1). The use of phenylmercuric acetate as an inhibitor of autodigestion during the purification procedures led to the smallest quantities of additional NH2-terminal residues. The stem enzyme is basic and amino acid analyses show a relatively high content of lysine and arginine. The enzyme from the fruit is an acidic protein. The carbohydrate analyses support the recent conclusion of Murachi and associates that the stem enzyme is a glycoprotein. The fruit enzyme also contains carbohydrate that cannot be removed by the procedures used thus far. The purified enzymes differ in their relative activities toward casein and benzoyl-L-argininamide.