ABSTRACT
The permeabilization was used to transform microorganisms in cell biocatalysts with high enzymatic activity. The Saccharomyces fragilis IZ 275 yeast cells were permeabilized with ethanol, as permeabilizing agent. To optimize the permeabilization conditions were used the design of Box-Behnken 15 trials (3 central points). The independent variables and their levels were ethanol (29, 32 and 35%), temperature (15, 20 and 25°C) and time (15, 20 and 25 min). The answer (Y) function has beta-galactosidase activity (U mg-1). The optimum conditions for obtaining a high enzymatic activity were observed in 35% ethanol concentration, temperature 15ºC and 20 min. treatment time. The maximum activity of the enzyme beta-galactosidase obtained was 10.59 U mg-1. The permeabilization of the S. fragilis IZ 275 cells was efficient.
A permeabilização foi usada para transformar células de microrganismos em biocatalisadores com alta atividade enzimática. As células de levedura de Saccharomyces fragilis IZ 275 foram permeabilizadas com etanol, como agente permeabilizante. Para otimizar as condições de permeabilização foi utilizado o delineamento de Box-Behnken com 15 ensaios (3 repetições no ponto central) . As variáveis independentes e seus níveis foram etanol (29, 32 e 35%), temperatura (15, 20 e 25ºC) e tempo (15, 20 e 25 min.). A função resposta (Y) foi atividade de beta-galactosidase (U mg-1). As condições ótimas para a obtenção de uma alta atividade enzimática foram observadas em 35% de concentração de etanol, temperatura de 15°C e tempo de tratamento de 20 minutos. A máxima atividade da enzima beta-galactosidase obtida foi de 10.59 U mg-1. A permeabilização das células de S. fragilis IZ 275 foi eficiente.
Subject(s)
Saccharomyces , beta-Galactosidase , Permeability , Saccharomyces , Yeasts , Biotechnology , Biocatalysis , Hydrolysis , LactoseABSTRACT
GABA (γ-aminobutyric acid) is a four carbon non-protein amino acid that is widely distributed in plants, animals and microorganisms. As a metabolic product of plants and microorganisms produced by the decarboxylation of glutamic acid, GABA functions as an inhibitory neurotransmitter in the brain that directly affects the personality and the stress management. A wide range of traditional foods produced by microbial fermentation contain GABA, in which GABA is safe and eco-friendly, and also has the possibility of providing new health-benefited products enriched with GABA. Synthesis of GABA is catalyzed by glutamate decarboxylase, therefore, the optimal fermentation condition is mainly based on the biochemical properties of the enzyme. Major GABA producing microorganisms are lactic acid bacteria (LAB), which make food spoilage pathogens unable to grow and act as probiotics in the gastrointestinal tract. The major factors affecting the production of GABA by microbial fermentation are temperature, pH, fermentation time and different media additives, therefore, these factors are summarized to provide the most up-dated information for effective GABA synthesis. There has been a huge accumulation of knowledge on GABA application for human health accompanying with a demand on natural GABA supply. Only the GABA production by microorganisms can fulfill the demand with GABA-enriched health beneficial foods.
Subject(s)
gamma-Aminobutyric Acid/analysis , Glutamate Decarboxylase/analysis , Neurotransmitter Agents , Receptors, GABA/analysis , Methods , Retrospective StudiesABSTRACT
Single-Strand Conformation Polymorphism(SSCP) is an effect method for investigating environment microbial genetic polymorphism, with its characterization of rapidness, simplicity, and sensitivity. However, many factors can influence the results of SSCP in the analysis of complex environment samples, and its optimization is highly needed. In this paper, optimal PCR-SSCP conditions were discussed based on PAGE concentration, formamide deionized in denaturing loading buffer, electrophoresis time and temperature. The resluts showed that the optimal conditions were as follows: 16S rDNA V1~V3 was selected as the targeted gene, the ratio of acrylamide to N, N-dimethylacrylamide in 12% polyacrylamide gel electrophoresis(PAGE)gel was 49:1, the ratio of formamide deionized in denaturing loading buffer was 1:3, running the SSCP gel at 300 V for 18 h (under 4 ℃). Aside from this, the validations using samples from a simultaneous desulfurification and denitrification bioreactor were conducted under this optimal conditions.
ABSTRACT
OBJECTIVE: To find out the optimal conditions of human chromosomal analysis protocol in peripheral blood sample. METHODS: The experiments were made with the variations of phytohaemagglutinin, colcemid, ethidium bromide concentration and the variations of hypotonic solution exposure time. RESULTS: In the experiment on the optimal phytohaemagglutinin concentration, the highest mitotic index in the overall collected cells was obtained in phytohaemagglutinin concentration 15microL/ml. In the experiment on the concentration of mitotic arrestant colcemid, the proper chromosomal state that is meta phase stage and doesn't have many chromosomal crossings or tangles was obtained in colcemid concentration 0.05microg/ml. In the experiment on the optimal exposure time of hypotonic solution(0.075M KCl) treatment, the most suitable intervals between chromosomes were subtained in 20 minutes. In the experiment on the optimal concentration of ethidium bromide to obtain minute chromosomal bands, the best result was when ethidium bromide concentration 5microg/ml or 7.5microg/ml was addition to colcemid concentration 0.02microg/ml. CONCLUSION: The combination of phytohaemagglutinin 15microL/ml, colcemid 0.05microg/ml, hypotonic solution exposure time for 20 minutes is important to the collection of appropriate chromosome state in human chromosomal analysis using peripheral blood. In the case that needs to obtain minute bands, the elongated chromosomes are obtained when ethidium bromide 5microg/ml or 7.5microg/ml in addition to colcemid concentration 0.02microg/ml with the same conditions of phytohaemagglutinin and hypotonic solution.