ABSTRACT
Chitinases are hydrolases that degrade chitin, a polymer of N-acetylglucosamine linked β(1-4) present in the exoskeleton of crustaceans, insects, nematodes and fungal cell walls. A metagenome fosmid library from a wastewater-contaminated soil was functionally screened for chitinase activity leading to the isolation and identification of a chitinase gene named metachi18A. The metachi18A gene was subcloned and overexpressed in Escherichia coli BL21 and the MetaChi18A chitinase was purified by affinity chromatography as a 6xHis-tagged fusion protein. The MetaChi18A enzyme is a 92-kDa protein with a conserved active site domain of glycosyl hydrolases family 18. It hydrolyses colloidal chitin with an optimum pH of 5 and temperature of 50°C. Moreover, the enzyme retained at least 80% of its activity in the pH range from 4 to 9 and 98% at 600 mM NaCl. Thin layer chromatography analyses identified chitobiose as the main product of MetaChi18A on chitin polymers as substrate. Kinetic analysis showed inhibition of MetaChi18A activity at high concentrations of colloidal chitin and 4-methylumbelliferyl N,N′-diacetylchitobiose and sigmoid kinetics at low concentrations of colloidal chitin, indicating a possible conformational change to lead the chitin chain from the chitin-binding to the catalytic domain. The observed stability and activity of MetaChi18A over a wide range of conditions suggest that this chitinase, now characterized, may be suitable for application in the industrial processing of chitin.
Subject(s)
Chitinases/genetics , Chitin/genetics , Metagenome/genetics , Chitinases/chemistry , Chitin/chemistry , Chromatography, High Pressure Liquid , Escherichia coli , Gene Expression/genetics , Gene Library , Genetic Vectors , Hydrogen-Ion Concentration , Substrate SpecificityABSTRACT
O desenvolvimento de biomateriais com aplicações na área da saúde mostram-se cada vez mais importantes e a procura por novos polímeros com propriedades bioativas, biodegradabilidade, atoxicidade são o foco das principais pesquisas em diferentes aplicações médicas e odontológicas. Os materiais capeadores pulpares evoluíram rapidamente na ultima década, sendo que são disponibilizadas atualmente diversas alternativas para uso clínico odontológico. Este trabalho teve como objetivo o desenvolvimento de um novo produto bioestimulador e capeador dentino/pulpar que poderá ser base para o desenvolvimento e recobrimento de scaffolds para reparo das diferentes estruturas dentárias. O desenvolvimento das bandagens BBio e os resultados obtidos nos testes das propriedades físico-químicas (absorção de água, perda de massa e pH), bem como as análises biológicas da morfologia celular e viabilidade celular com MTT a BBio apresentaram dados favoráveis e desejáveis para sua aplicação clínica. A propriedade de liberação de cálcio foi bastante promissora, sendo esta uma condição que dará a diferenciação positiva da BBio como um produto bioestimulador pulpar. Com esses dados pode-se concluir que a mesma se encontra dentro dos parâmetros desejados para o produto final e com propriedades semelhantes aos produtos existentes no mercado, de qualidade e aprovados pelas agências reguladoras.(AU)
The development of biomaterials with applications in the health area are increasingly important and the search for new polymers with bioactive properties, biodegradability and toxicity are the focus of the main researches in different medical and dental applications. The pulp capping materials evolved rapidly in the last decade, and several alternatives are now available for clinical dental use. This project aimed to develop a new biostimulating and dentin / pulp capping product that could be the basis for the development and recoating of "scaffolds" for repair of different dental structures. The development of the BBio bandages and the results obtained in the physical-chemical properties tests (water absorption, loss of mass and pH), as well as the biological analyzes of the cellular morphology and cell viability with MTT to BBio presented favorable and desirable data for its clinical application. The calcium release property was quite promising, and this is a condition that will give BBio a positive differentiation as a pulp biostimulator product. With this data it can be concluded that it is within the parameters desired for the final product and with properties similar to the products on the market, of quality and approved by the regulatory agencies.(AU)
Subject(s)
Humans , Biocompatible Materials/chemistry , Dental Pulp/drug effects , Dentin/drug effects , Pulp Capping and Pulpectomy Agents/chemistry , Analysis of Variance , Biocompatible Materials/pharmacology , Biocompatible Materials/standards , Cell Survival , Chitin/chemistry , Chitosan/chemistry , Fibroblasts/drug effects , Materials Testing , Microscopy, Electrochemical, Scanning , Pulp Capping and Pulpectomy Agents/pharmacology , Pulp Capping and Pulpectomy Agents/standards , Reproducibility of Results , Time FactorsABSTRACT
In an attempt to isolate chitinase producers from soil, a streptomycete strain was found potent using natural chitin as the substrate. Chitinolytic activity was tested directly on agar plates, also with crude enzyme. Chitinase assay showed that the isolate could produce 0.8 U/ml of the enzyme. The morphological, cultural, physiological and biochemical characters of the isolate P10 were studied, and identified as Streptomyces venezuelae P10.
Subject(s)
Acetylglucosamine/chemistry , Agar/chemistry , Animals , Asparagine/chemistry , Brachyura , Chitin/chemistry , Chitinases/chemistry , Colloids/chemistry , Glucose/chemistry , Microscopy, Electron, Scanning , Streptomyces/enzymology , Time FactorsABSTRACT
Se estudiaron algunas propiedades físico/químicas de 3 derivados carboximetilados, sintetizados a partir de la quitina que se obtiene en Cuba de los carapachos de langostas. Se analiza el comportamiento hidrodinámico de las soluciones de estas sustancias, comprobándose las características de polielectrólito que presentan y se determinan los valores de la masa molar para cada una de ellas
Subject(s)
Chitin/analogs & derivatives , Chitin/chemistryABSTRACT
Se propuso combinar la gravimetría directa como método cuantitativo con la espectroscopia infrarroja como técnica cualitativa complementaria, con el objetivo de establecer la metodología para el seguimiento de la estabilidad química de la quitina como fármaco en diferentes formas farmacéuticas> suspensión, crema, supositorio. La determinación del contenido de quitina por técnicas gravimétricas se validó en cada caso según los parámetros> linealidad, precisión, exactitud y especificidad. El análisis cuantitativo mostró resultados satisfactorios con respecto al contenido de fármaco en el tiempo en todas las formulaciones evaluadas. Estos resultados fueron corroborados por los espectros infrarrojos, manifestándose que la quitina es el único producto retenido en los filtros y que desde el punto de vista químico no ha sufrido alteraciones transcurridos 2 a de estudio