Biomedical Engineering Reference
In-Depth Information
chemical approach and for their remarkable characteristics. Biocatalysis in non-conventional
(non-aqueous) media has expanded the spectrum of application of proteases to those reactions
that cannot proceed effectively in aqueous environments, this is, the synthesis of peptide
bonds instead of their hydrolysis [12-15].
Enzymatically synthesized small peptides (usually di or tripetides) are being used
successfully for human and animal nutrition and also as pharmaceuticals and agrochemicals.
Some relevant examples are the synthesis of the leading non-caloric sweetener aspartame, the
lysine sweet peptide, kyotorphin, angiotensin, enkephalin and dynorphin [16-22], and some
nutritional dipeptides and tripeptides [23-25]. Some small peptides have been synthesized at
commercial scale in continuously operated enzyme reactors [26, 27]. Kimura et al. [23] have
proposed various kinds of reactors to synthesize peptides from essential aminoacids using
papain, α-chymotripsin, and thermolysin.
The aim of this work was to study the performance of proteolytic enzymes of
Acacia
caven
(Mol.) Molina pollen for its potential application as an additive in various laundry
detergents formulations and as catalyzer of the peptide synthesis in aqueous-organic media.
Materials and Methods
Plant Material
Pollen grains of flowers of
Acacia caven
(Mol.) Molina
Fabaceae
(argentine folk names:
“espinillo”) was obtained from San Luis range, Argentina.
Acacia caven
plant was classified
at Universidad Nacional de San Luis and registered as Voucher Nº 462, III, 1998. The pollen
sample was maintained safe from the light, dried to room temperature and stored in
dessicator.
Elution of the Enzyme from the Pollen Wall
Pollen grains (35 mg/ml) were suspended in 0.1M Tris-HCl buffer pH 7.4 and slowly
shaken for 2 h at 25° C. Then, the slurry was centrifugated for 30 min at 8000 rpm. The
supernatant (crude enzyme extract, CE) was tested in proteolytic activity and protein content
by Bradford's method [28].
Chemical Reagents
Synthetic substrates L-phenylalanine methyl ester (Phe-OMe.HCl), N--
benzyloxycarbonyl-L-amino acids (Z-Ala, Z-Gln and Z-Phe) and N--benzyloxycarbonyl-L-
Alanine p-nitrophenyl ester (Z-Ala-pNO) were supplied by Bachem (California, USA). The
synthetic substrate N--benzoyl-DL-Arginine 4-nitroanilide (BAPNA) and HPLC-grade
organic solvents were purchased from Sigma (St. Louis, USA).
