Papain-Catalyzed Chemoenzymatic Synthesis of Telechelic Polypeptides Using Bis(Leucine Ethyl Ester) Initiator.

In order to construct unique polypeptide architectures, a novel telechelic-type initiator with two leucine ethyl ester units is designed for chemoenzymatic polymerization. Glycine or alanine ethyl ester is chemoenzymatically polymerized using papain in the presence of the initiator, and the propagation occurs at each leucine ethyl ester unit to produce the telechelic polypeptide. The formation of the telechelic polypeptides is confirmed by (1) H NMR and MALDI-TOF mass spectroscopies. It is revealed by AFM observation that long nanofibrils are formed from the telechelic polyalanine, whereas a conventional linear polyalanine with a similar degree of polymerization shows granule-like structures. The telechelic polyglycine and polyalanine show the crystalline structures of Polyglycine II and antiparallel ß-sheet, respectively. It is demonstrated that this method to synthesize telechelic-type polypeptides potentially opens up a pathway to construct novel hierarchical structures by self-assembly.

Development of papain containing pellets produced by extrusion-spheronization: an operational stage approach.

The performance of the standardized extrusion-spheronization technique, operational conditions, formulation parameters and storage of the final product over the bioactivity of papain containing pellets has been evaluated to obtain an insight into the potential of the technique for the manufacture of solid protein formulations. The pellets produced were assayed in terms of biological activity - monitored at each operational stage using N-benzoyl-dl-arginine ρ-nitroanilide as a substrate, and according to the physical properties - evaluated by means of size distribution, apparent density and friability. The produced pellets presented adequate physical and mechanical properties. Monitoring biological activity at each production stage revealed that the most critical steps corresponded to drying and storage, with bioactivity decay ranging from 5 to 30% and 5 to 20% for each process. Dry mixing and extrusion did not hold any influence over papain activity, while wet massing decreased the bioactivity by approximately 0-5% and the spheronization 0-2%. The results varied as a function of the experimental conditions and formulation components. In conclusion, the extrusion--spheronization technique was suitable to produce solid multiparticulate dosage forms for papain, considering the possibility to originate pellets with relatively low bioactivity decay. However, weak points of the technique corresponded to the wet massing and drying stages as well as storage.

Atividade antibacteriana in vitro de géis com diferentes concentrações de papaína / In vitro antibacterial activity of gels with different papain concentrations

A papaína é uma enzima proteolítica proveniente do mamão, Carica papaya Linn., e é muito empregada na indústria alimentícia, cosmética e farmacêutica. Objetivou-se determinar a atividade antibacteriana in vitro de géis com diferentes concentrações de papaína frente às bactérias padrão: Staphylococcus aureus (ATCC 6538) e Echerichia coli (ATCC 10538), bem como 4 Pseudomonas aeruginosa hospitalares. Alíquotas de 2,0ml dos inóculos bacterianos (metade da escala 1,0 de McFarland) foram semeadas em duplicata na superfície de placas de Petri. Os géis com as diferentes concentrações de papaína foram gotejados na superfície dos meios de cultura pela técnica de gotejamento e a incubação realizada a 35ºC por 24 horas. A leitura dos resultados da atividade antibacteriana foi efetuada pela mensuração dos halos de inibição ao redor das gotas dos géis de papaína. Apenas o gel de papaína a 10% foi capaz de inibir o crescimento do S. aureus e de 2 P. aeruginosa. Em conclusão, de acordo com a literatura a papaína apresenta ação proteolítica sobre os tecidos mortos, porém apenas a formulação em gel a 10% apresentou atividade antibacteriana in vitro. Assim, antes de utilizar alguma substância ou produto contra microrganismos é necessário saber se a mesma apresenta realmente atividade antimicrobiana.

The papain is a proteolytic enzyme from papaya, Carica papaya Linn., and it is so used in food, cosmetic and pharmaceutical industry. The aim of this study was to determine in vitro antibacterial activity of gels with different papain concentrations against standard bacteria: Staphylococcus aureus (ATCC 6538) and Echerichia coli (ATCC 10538), as well as 04 hospital Pseudomonas aeruginosa. Aliquots of 2.0ml from bacterial inocula (0.5 McFarland scale) were seeded in duplicate onto Petri plates? surface. The papain gels with different concentrations were dropped onto culture medium surface by the drop technique and the incubation realized at 35ºC for 24 hours. The results of antibacterial activity were obtained by measuring of growth inhibition halos around the drops of papain gels. Only the papain gel 10% was able to inhibit S. aureus and 2 P. aeruginosa growth. In conclusion, according to literature the papain has action on dead tissues, but only the gel formulation 10% presented in vitro antibacterial activity. So, before using any substance or product against microorganisms it is necessary to know if it really has antimicrobial activity.

La papaína es una enzima proteolitica de la papaya, Carica papaya Linn., y es muy utilizado en la industria del alimento, cosmética y farmacéutica. La puntería de este estudio era determinar la actividad anti-bacteriana in vitro de geles con diversas concentraciones de la papaína contra bacterias estándares: Staphylococcus aureus (ATCC 6538) y Echerichia coli (ATCC 10538), así como 4 Pseudomonas aeruginosa del hospital. Alícuotas de 2,0ml de inóculos bacterianos (0,5 de la escala McFarland) fueron sembradas por duplicado en la superficie de placas de Petri. Los geles con diferentes concentraciones de papaína fueron goteados en la superficie de los medios de cultura por la técnica de goteo y la incubación se celebró a 35ºC por 24 horas. La lectura de los resultados fue hecha por la medición de los halos de inhibición alrededor de las gotas de geles. Solamente el gel el 10% de la papaína fue capaz de inhibir inhibir el crecimiento de S. aureus y de 2 P. aeruginosa. En la conclusión, según la literatura la papaína tiene acción en tisúes muertos, pero solamente la formulación el 10% del gel presentó actividad anti-bacteriana in vitro. Así pues, antes de usar cualquier sustancia o producto contra microorganismos es necesario saber si realmente tiene actividad anti-microbiana.

Papain entrapment in alginate beads for stability improvement and site-specific delivery: physicochemical characterization and factorial optimization using neural network modeling.

This work examines the influence of various process parameters (like sodium alginate concentration, calcium chloride concentration, and hardening time) on papain entrapped in ionotropically cross-linked alginate beads for stability improvement and site-specific delivery to the small intestine using neural network modeling. A 3(3) full-factorial design and feed-forward neural network with multilayer perceptron was used to investigate the effect of process variables on percentage of entrapment, time required for 50% and 90% of the enzyme release, particle size, and angle of repose. Topographical characterization was conducted by scanning electron microscopy, and entrapment was confirmed by Fourier transform infrared spectroscopy and differential scanning calorimetry. Times required for 50% (T(50)) and 90% (T(90)) of enzyme release were increased in all 3 of the process variables. Percentage entrapment and particle size were found to be directly proportional to sodium alginate concentration and inversely proportional to calcium chloride concentration and hardening time, whereas angle of repose and degree of cross-linking showed exactly opposite proportionality. Beads with >90% entrapment and T(50) of <10 minutes could be obtained at the low levels of all 3 of the process variables. The inability of beads to dissolve in acidic environment, with complete dissolution in buffer of pH >or=6.8, showed the suitability of beads to release papain into the small intestine. The shelf-life of the capsules prepared using the papain-loaded alginate beads was found to be 3.60 years compared with 1.01 years of the marketed formulation. It can be inferred from the above results that the proposed methodology can be used to prepare papain-loaded alginate beads for stability improvement and site-specific delivery.