| The y-Amino Butyric Acid (GABA) is a natural and widely distributed in organisms.It can be detected in the body animals, plants and other organisms. Many important physiological functions of the y-Amino Butyric Acid have been founded by a study. The presence of the y-Amino Butyric Acid is found in the brain and spinal cord of mammals, and produced a variety of effects. The effect of contraction and relaxation on the inner blood vessel in the tissue can be improved. It can work on the role of the nervous system to be specific for the treatment of certain types of neurological diseases. In short by using the y-Amino Butyric Acid to treat some complaints, the effects were better.GABA plays an important role in the regulation and secretion of the endocrine system; the liver function and renal function can be effective regulation and improved by the Υy-Amino Butyric Acid. In practical application, through adding a certain amount of the Υ-Amino Butyric Acid in feeding to make livestock and poultry has a certain tolerance for the surrounding environment of the stimulus. This was the generally described the anti-stress effect. Under the effect of this, poultry can be effective in the growth and development and the growth of the body was promoted. Based on the special role of GABA, the people will be more in-depth studies about it.In the future it will become a hot spot about the drug development, food and feed additives and so on. Glutamate decarboxylase(GAD) has been acted on the glutamate substrate.And the product of the glutamate substrate catalyzed was GABA. The essence of GAD is a kind of protein enzyme.The enzyme as a kind of catalyst was high efficiency and specificity. GAD also has these enzyme glutamic acid decarboxylase characteristic. Due to the high reaction efficiency of GAD, the study of cell endogenous glutamate decarboxylase be used to prepare the GABA.The foundation of this study was based on finding the gene fragment which controlled the gene of GAD expression, and the molecular biology technology can be used to the target species construction and recombination.The content of this paper can be divided into the following parts, the expression product of GAD1 gene constructed by the above in the yeast was activated, inducted, separated and purified. The characteristics of the enzyme activity of the product were studied. The regulation mechanism of the activity of the recombinant yeast was given in the reaction of the transformation method the purpose of it is looking for the most favorable conditions for the transformation. In order to make the GAD of recombinant yeast enriched and tried to increase production of GABA.The purpose of this paper is to study the following aspects:Through the method of inducing culture or transformation makes the expression level of GAD in the recombinant yeast promoting. The most suitable conditions was chose by the recombinant yeast transforming.Making the GAD as the substrate of easier to convert the decarboxylation reaction GABA as the goal. In order to achieve the above objectives, first of all the control expression of gene fragment cloned and recombined in BG1805 simple vector cloning manipulation of glutamic acid decarboxylase expression gene fragment and cultivate the expression. This preliminary work is done by the Japanese friends.In this paper, different inducing agents were added to the culture of the recombinant yeast, after culture, the product was separated and refined. The influence about GAD in different induction conditions was analyzed.By controlling the external factor.The effect of temperature and some additives can make some impacts on the expression level of the enzyme. The conditions which can improve the yield and activity of recombinant yeast were chose. There were some advantages and disadvantages between the fermentation and transformation methods. By controlling the conditions of artificial controlled culture, the most suitable conditions are controlled to achieve the maximum conversion of the recombinant yeast to the production of gamma amino acid to provide theoretical basis for large-scale production.Using modified paper chromatography is the criterion of judgment for measure the content of GABA. In the process of transformation the content of GABA and glutamate can be make sure, calculated the value of molar conversion and negative flow.The results are as follows:Results for GAD abduction separation, purification:With different concentrations of raffinose and galactose, after a certain period of time the gene vector recombinant GAD1 bacteria by shaking medium The OD of SC-URA 2% raffinose medium is 3.65, The OD of SC-URA 2% galactose medium is 0.21. In the process of induction of cultured,the OD of 3×YP+6% raffinose medium is 16.5 the OD of 3 x YP+6% galactose medium is 8.1. After ultrasonic broken,the medium of the yeast via the protein isolatio of Profinia TM automated purification system, run the SDS-PAGE electrophoresis gel, The results showed that:The isolated glutamic acid decarboxylase in the 3 x YP+6% raffinose medium is 0.008g/mL. The isolated glutamic acid decarboxylase in the 3 x YP+6% galactose medium is 0.06g/mL, molecular weight of 67kD. Addition of raffinose medium can cultivate the number of bacteria although more, but the effect of expression of GAD is not ideal.So we select the 3 x YP+6% galactose medium as an inducer of the recombinant yeast to produce glutamic acid decarboxylase inducing conditions.The measurement results of the enzyme activity:The purified glutamate decarboxylase added to a culture medium which with containing glutamate in the substrate, After3 hours, the Υ-Amino Butyric Acid production was detected by using a modified paper chromatography. The results show that in the 3 x YP+6% galactose medium each 1.75mg/4mL glutamic acid decarboxylase is reduced to 0.25mg/ 4mL,The y-Amino Butyric Acid made 0 to 1.35mg/4mL. And in the 3×YP+6% raffinose medium, each 1.75mg/4mL glutamic acid decarboxylase is reduced to 1.5mg /4mL,The y-Amino Butyric Acid made 0 to 0.25mg/4mL. The results show that YP+ 6% galactose to the culture conditions induced by glutamate decarboxylase activity is better, capable of catalyzing produced glutamate.The measurement results about factors affecting the activity of glutamate decarboxylase. To compare with PLP, pyridoxine, pyridoxal,these three kind of things how to influence the activity of GAD. The best activity when determining the amount of added PLP in temperature, pH, reaction time optimal conditionsy-Amino Butyric Acid the optimum amount consumed when the PLP. The results showed that an amount of consumption PLP 2.4 μmol/4ml. The most pH optimum measurement result is about 4.8. The optimum temperature affects the enzyme activity about 40℃.And the result of added some kinds of active agent and chemical substances, that showed us isoamyl alcohol and Mn2+can be increased activity of glutamate decarboxylase.Using a conversion method glutamic acid-producing y-Amino Butyric Acid conversion conditions were studied, the most suitable temperature for transforming is 37℃pH is 5.0,The optimal buffer system is acetic acid-sodium acetate buffer, The optimum substrate was added 0.1 mol/L, The optimum cell concentration was added at 6%, the speed 200r/min, the optimum conversion time of 12h,. Under these conditions the most effect, the ability of cells transformed to basically stable. And less chance of side reactions, The increase GABA production can be realized. |
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