Milk-clotting proteases from Pleurotus albidus: an innovative alternative for the production of Minas frescal cheese

Pleurotus albidus, a naturally growing species in the Amazon region, has been considered a promising source of milk-clotting proteases. The production of such enzymes using lignocellulosic residues is a sustainable alternative to replace mammalian rennet. The application of P. albidus milk-clotting proteases in cheese making has not yet been reported in the scientific literature. The aim of this study was to characterize the milk-clotting proteases of P. albidus and use these enzymes in the production of Minas frescal cheese. For the production of coagulating proteases, the mushroom was grown in açaí seeds supplemented with rice bran (10%, w/w). The parameters affecting the production of coagulant, such as inoculum size, fermentation time, initial pH of cultivation medium and age of the inoculum were evaluated. The coagulant extract obtained under optimal production conditions was evaluated for optimal pH and temperature, pH and temperature stability, effect of ions and inhibitors. Significant production of coagulating proteases was obtained under the following conditions: inoculum size (2.5%), fermentation time (10 days), initial pH of the cultivation medium (6), and inoculum age (10 days). The coagulant exhibited significant catalytic activity in pH 5.0 at 55°C, with stability at 45°C and was completely inhibited by iodoacetic acid. The milk-clotting proteases of P. albidus were efficient for making Minas frescal cheese that presented 55.0% of moisture, 20.0% of lipids and 17.20% of protein. Pleurotus albidus is a potential source of milk-clotting proteases that can be applied in dairy industry for production of fresh Minas frescal cheese.

Proteolytic Enzymes Screening for Enzymolysis Preparation Technology of Active Parts of Periplaneta americana against Liver Fibrosis / 中国药房

OBJECTIVE： To optimize the proteolytic enzymes for enzymolysis technology of degreasing ointment from Periplaneta americana， and to improve the extraction rate and activity of anti-liver fibrosis active part from P. americana. METHODS： Using degreasing ointment of P. americana as control， ninhydrin method and folin-ciocalteu method were used to investigate the hydrolysis degree of trypsin （TR）， pepsin （PE）， alkaline protease （AL）， papain （PA） and neutral protease （NE） to the degreasing ointment. Macroporous resin isolation and purification method was used to investigate the yield of elution part from hydrolyzate， with 50％， 60％， 70％， 95％ ethanol as eluting solvents. Inhibition test in vitro of rat hepatic stellate cells HSC-T6 was performed， and anti-liver fibrosis activity of elution part from hydrolyzate was investigated. RESULTS： The hydrolysis degree of PA and NE were 14.15％ and 15.70％， showing strong enzymatic hydrolysis ability. The yield of 95％ ethanol elution part from PA， NE and AL hydrolyzate were （0.73±0.04）％，（0.65±0.01）％ and（0.64±0.05）％， improving 30.36％， 16.07％， 14.29％ compared with degreasing ointment without enzyme. Results of inhibition test in vitro showed that inhibitory rate of 50％， 60％， 70％ ethanol elution parts isolated and  purified from hydrolyzate had a low inhibition rate or a growth-promoting effect on HSC-T6 cells. Inhibition rates of 95％ ethanol elution parts to HSC-T6 cells were all more than 20％. IC50 of 95％ ethanol elution part isolated and purified from PA and NE hydrolyzate for 24-72 h were 94.5-112.3 and 117.1-120.0 μg/mL， which were lower than that （116.1-123.0 μg/mL） of degreasing ointment without enzyme. CONCLUSIONS： PA is the best hydrolyzate for enzymolysis technology of active parts against liver fibrosis in degreasing ointment from P. americana， followed by NE and AL； PE and TR， which have poor effect， are not suitable for the enzymatic hydrolysis technology.

Operatoria dental y endodoncia 5: protección de la interfase resina-dentina mediante inhibidores de las enzimas colagenolíticas / Operative dentistry and endodontics 5: protection of the resin-dentin interface by colagenolytic enzyme inhibitors

La adhesión entre las resinas hidrófilas y la dentina se encuentra sometida a una degradación permanente cuya intensidad aumenta en función del tiempo transcurrido. Esto es producto de la actividad de las metaloproteinasas, de las catepsinas y otras enzimas colagenolíticas, responsables de la destrucción paulatina de las fibras colágenas de la capa híbrida. La mayoría de las estrategias para inhibir estas enzimas han sido ensayos de laboratorio, mientras que las investigaciones clínicas son escasas. En este trabajo se analiza la literatura relacionada con las estrategias sugeridas para prevenir la degradación del colágeno de la capa híbrida en la interfaz resina-dentina (AU)

The bonds between hidrophylic resins and dentin are subjected to a time-dependent collagenolytic degradation. Endogenous enzymes such as matrix metalloproteinases, catepsins and other enzymes are responsible for the hybrid layer destruction. The majority of strategies developed to inhibit these enzymes are laboratory evaluations while clinical studies are scarce. This review examines the literature related to the strategies suggested to prevent collagen degradation of the hybrid layer in resin-dentin interfaces (AU)

Pathophyslology of Bone Metastsis And Its Implications - A Review.

In tumors with a propensity to spread to bone a significant proportion of patients who present with cancer that appears to be localized will eventually develop incurable metastatic disease. Bone metastases are common in many advanced cancers and are an avoidable yet, annoying source of skeletal morbidity. The bone mineral matrix contains numerous growth factors that are released during normal bone remodeling, providing a fertile microenvironment for tumor cell colonization and proliferation. Tumor cells then release a variety of growth factors that promote bone resorption and increase the risk of skeletal complications. Metastasis of tumor cells to bone requires a complex cascade of events involving detachment from the primary tumor site, invasion of the vasculature, migration and adherence to distant capillaries of the bone, extravasation, and proliferation. Metastatic bone lesions are classified as osteolytic or osteoblastic, based on their radiographic appearance. Bone is the third most common site of metastatic disease .Carcinomas are much more likely to metastasize to bone than sarcomas. Routine use of whole body PET/CT in restaging HNSCC can therefore, facilitate early detection of occult bone metastases and this detection often influences therapeutic decision making.

The use of Fluorescence Resonance Energy Transfer (FRET) peptidesfor measurement of clinically important proteolytic enzymes

Proteolytic enzymes have a fundamental role in many biological processes and are associated with multiple pathological conditions. Therefore, targeting these enzymes may be important for a better understanding of their function and development of therapeutic inhibitors. Fluorescence Resonance Energy Transfer (FRET) peptides are convenient tools for the study of peptidases specificity as they allow monitoring of the reaction on a continuous basis, providing a rapid method for the determination of enzymatic activity. Hydrolysis of a peptide bond between the donor/acceptor pair generates fluorescence that permits the measurement of the activity of nanomolar concentrations of the enzyme. The assays can be performed directly in a cuvette of the fluorimeter or adapted for determinations in a 96-well fluorescence plate reader. The synthesis of FRET peptides containing ortho-aminobenzoic acid (Abz) as fluorescent group and 2, 4-dinitrophenyl (Dnp) or N-(2, 4-dinitrophenyl)ethylenediamine (EDDnp) as quencher was optimized by our group and became an important line of research at the Department of Biophysics of the Federal University of São Paulo. Recently, Abz/Dnp FRET peptide libraries were developed allowing high-throughput screening of peptidases substrate specificity. This review presents the consolidation of our research activities undertaken between 1993 and 2008 on the synthesis of peptides and study of peptidases specificities.

As enzimas proteolíticas têm um papel fundamental em muitos processos biológicos e estão associadas a vários estados patológicos. Por isso, o estudo da especificidade das peptidases pode ser importante para uma melhor compreensão da função destas enzimas e para o desenvolvimento de inibidores. Os substratos com supressão intramolecular de fluorescência constituem uma excelente ferramenta, pois permitem o monitoramento da reação de forma contínua, proporcionando um método prático e rápido para a determinação da atividade enzimática. A hidrólise de qualquer ligação da cadeia peptídica entre o grupo doador e o grupo supressor gera fluorescência que permite detectar concentração nanomolar de enzima. Os ensaios podem ser acompanhados diretamente na cubeta ou adaptados para determinações de fluorescência em leitoras de placa. A síntese dos peptídeos com supressão intramolecular de fluorescência contendo o grupo fluorescente Abz (orto-aminobenzóico) e o grupo supressor EDDnp (N-[2, 4-dinitrofenil]-etilenodiamino ou Dnp (2, 4-dinitrophenyl) foi otimizada pelo nosso grupo e tornou-se uma importante linha de pesquisa no Departamento de Biofísica da Universidade Federal de São Paulo. Recentemente, foram desenvolvidas bibliotecas de peptídeos fluorogênico contendo Abz/Dnp como grupo doador/supressor trazendo um grande avanço no estudo de especificidade das peptidases. Esta revisão apresenta o trabalho desenvolvido pelo nosso grupo entre 1993 e 2008 sobre a síntese de peptídeos e o estudo da especificidade de peptidases.

Efeitos da aplicação de transglutaminase na fabricação do pão de forma / Effects of transglutaminase application on breadmaking

Este trabalho teve o objetivo de avaliar o efeito da adição da transglutaminase microbiana (MTGase) na fabricação de pão de forma, através do desenvolvimento de formulação ideal, com combinações de aditivos e enzima, e da avaliação do efeito da enzima nas proteínas, na massa crua, na massa após a fermentação e no produto final. Para comparar a qualidade dos pães produzidos com ou sem enzima, foram testadas três formulações: a básica, livre de aditivos (pão Zero); com a adição de emulsificante e ácido ascórbico (pão Controle); e a preparada com a formulação básica adicionada de enzima (pão MTGase). A avaliação da qualidade dos pães foi feita por meio de medidas físicas e instrumentais. A análise de textura foi realizada pelo método TPA (Texture Profíle Analysis), cujas respostas de firmeza, elasticidade, mastigabilidade e gomosidade podem ser correlacionadas com análises sensoriais. Paralelamente, de amostras de farinha, de massa e de pão foram obtidas as frações protéicas de gliadinas, gluteninas e os resíduos de extração. As gliadinas e as gluteninas foram analisadas por cromatografia líquida de alta eficiência em fase reversa e por eletroforese em gel de poliacrilamida contendo SDS. Os resultados de volume e de firmeza dos diferentes pães apresentaram diferenças significativas a nível de 5%, em que as respostas do pão MTGase foram melhores que as do pão Zero, porém ainda inferiores às do Controle. A melhor formulação foi obtida por meio de um planejamento composto central, com variações nas concentrações de emulsificante, ácido ascórbico e enzima, com os resultados avaliados pela metodologia de superfície de resposta. Exceto para a coesividade, todos os outros parâmetros de volume, dureza (TA), firmeza, elasticidade, mastigabilidade e gomosidade (TPA) apresentaram resultados positivos pela ação da transglutaminase a 0,6%, combinada com 0,2 % de emulsificante e 70 ppm de ácido ascórbico. Os resultados sugerem que a enzima foi capaz de modificar as propriedades químicas das proteínas, o comportamento reológico da massa e as propriedades funcionais do pão, melhorando a força da massa, a textura e o volume dos pães. As análises das frações gliadínicas apresentaram cerca de 3% de Nitrogênio total, em base seca, e as frações glutenínicas apresentaram entre 2 e 5% de Nitrogênio total. Os perfis cromatográficos e eletroforéticos dessas frações sugerem que as gliadinas não foram afetadas pela presença da enzima, que envolveram sobretudo as gluteninas. O conjunto de resultados indica que a aplicação de MTGase em associação com aditivos convencionais pode ser uma alternativa à panificação, embora os mecanismos de sua ação na massa não estejam completamente esclarecidos

The application of microbial transglutaminase on weak gluten flour used in breadmaking was studied over the process. To verify the enzyme effects, three formulations were tested: Base formulation, characterized by the absence of enzyme and emulsifying agents; Control formulation, comprised by the presence of emulsifying agents and ascorbic acid and MTGase formulation, with the enzyme. Samples of flour, dough and bread were analyzed. The effect of enzyme on bread quality was estimated by parameters of Texture Analysis, Texture Profile Analysis and specific volume. The protein contents from those samples were determined by the total nitrogen in glutenin and gliadin fractions, that were also analyzed by RP-HPLC (reversed phase high performance liquid chromatography) and by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). Although the MTGase bread did not reach the same quality parameters as those achieved by the Control samples, it showed as an alternative formulation to reduce the quantity of emulsifying agents and ascorbic acid as compared to the Control. The results indicate that the enzyme modified chemical and functional properties of glutenin fraction, improving dough strength and bread volume. Results of total nitrogen content, and electrophoretic and chromatographic profiles of the protein fractions suggest that while glutenin proteins were modified by enzyme, gliadin proteins were not affected

Defense and carnivory: Dual role of bracts in Passiflora foetida.

Members of the genus Passiflora are reported to have evolved modifications which kill insects; they have however never been tested for carnivorous syndrome. The flowers of Passiflora foetida consists of highly reticulate bracts which cover and grow along with the buds and fruits. Removal of bracts from developing bud and fruit resulted in higher predatory damage compared to those where the bracts were intact. These bracts also possess a large number of minute glands which ooze sticky secretion. A variety of tiny insects were found trapped by the secretion of the bracts. The secretion of these glands show high proteases and acid phosphatase activity, two common digestive enzymes found in traps of true carnivorous plants. A high quantity of aminoacids were released from freshly freeze killed ants when incubated in buffer extract of bracts· [14C] phenylalanine smeared on the glandular surface of bracts was recovered from ovules suggesting potential for absorption of aminoacids. These results suggest a novel role for bracts where primary function is to minimize predatory damage to developing flowers and fruits. The bracts serve as insect traps and also possess the mechanism to digest the trapped insects to obtain free aminoacids.

ANALYSIS OF KINDS AND ACTIVITY OF PROTEOLYTIC ENZYMES IN DIGESTIVE SYSTEM OF BUTEO BUTEO / 解剖学报

Objective The kinds and activities of proteolytic enzymes of tongue,esophagus,craw,muscular stomach,glandular stomach,duodenum,large intestine,pancreas,liver in digestive system of Buteo buteo were studied to provide basic information for taxonomy and evolvement study of wild birds. Methods The experiment was carried out at 4℃ by using gelatine polyacrylamide gel electrophoresis(G-PAGE) ameliorated by Xu Cunshuan. Results 1.At least 26 kinds of proteolytic enzymes were found in digestive system of Buteo buteo.2.The kinds and activities of proteolytic enzymes in the digestive system of Buteo buteo were affected by pH.The activities of proteolytic enzymes was the weakest in the acid environment and the strongest in the alkaline environment,showing that the activity of the alkaline proteolytic enzymes were stronger than that of the neutral proteolytic enzymes,and that of the acid proteolytic enzymes were weakest.3.Duodenum and pancreas had more kinds and stronger activities of proteolytic enzymes than those in tongue,esophagus and craw in any pH value condition.4.The proteolytic enzymes with 75 and 70?kD molecular weight existed in every organ of digestive system except pancreas and liver in any pH condition.Conclusion The similarity of the kinds and the activities of proteolytic enzymes exist in the organs with similar structure and similar functions. The proteolytic enzyme of 75 and 70?kD molecular weight,whose pH-dependent activity is not obvious,exists widely in digestive system of Buteo buteo.The activity of proteolytic enzyme with 198?kD molecular weight is found in every organ of digestive system under pH 8.5 condition.

STUDY ON KINDS AND ACTIVITIES OF PROTEOLYTIC ENZYMES IN THE DIGESTIVE SYSTEM OF GRUS GRUS / 解剖学报

Objective The kinds and activities of proteolytic enzymes of organs in the digestive system of Grus grus were studied to provide basic information for the taxonomy and evolvement study of wild birds. Methods The experiment was carried out at 4℃ by using gelatine polyacrylamide gel electrophoresis(G-PAGE) ameliorat. Results 1.Altogether 26 kinds of proteolytic enzymes were found in the digestive system of Grus grus in all pH value conditions.2.The kinds and activities of proteolytic enzymes in the digestive system of Grus grus were affected by pH.The activities of proteolytic enzymes were weakest in the acid environment and strongest in the neutral environment,which shows that the activities of the neutral proteolytic enzymes were stronger than those of the alkaline proteolytic enzymes,and those of the acid proteolytic enzymes were weakest.3.The kinds and activities of proteolytic enzymes of the same organ varied tremendously in different pH value conditions.The proteolytic enzymes of different organs had different optimal pH values.4.Proteolytic enzymes with 19?kD molecular weight existed in every organ of the digestive system except glandular stomach in all pH conditions.Proteolytic enzyme with 66 and 22?kD molecular weight was found in every organ of the digestive system in the neutral environment.Conclusion The optimal pH value of proteolytic enzymes in the digestive system of Grus grus is 7.0.The distribution,activity and pH-dependency of proteolytic enzymes of 66?kD,22?kD,19?kD molecular weight are the important characteristic of proteolytic enzymes in the digestive system of Grus grus.