| γ-glutamyltranspeptidase(γ-GTP) has been found in cells from bacteria to mammals and designated as the key enzyme in glutathione metabolism.With the development of biotechnology , preparationγ-Glutamyl compounds usingγ-GTP has become the research hot of biological catalysis field. Recently, our laboratory has screened a strain produced strong activity ofγ-GTP and has established a technology of processing L-theanine with microbial enzyme. On this basis, further investigation was exerted.ObjectiveThe optimum induction conditions of fermentation, the extraction technology ofγ-GTP , enzymology properties and L-theanine biosynthesis by immobilizedγ-GTP were investigated. The research has a great contribution to the industrial production L-theanine by immobilizedγ-GTP.Methods1.The induction conditions of fermentation:γ-GTP production of Bacillus licheniformis C12 was improved by substrates induction(Gly,Glu,Gln).2. Enzymology properties: Cells were disrupted with the methods of lysozyme , frozens and thawes and supersonic, respectively . And the crude enzyme extracts was extracted and purified by refrigerated centrifugation ,saturation ammonium sulfate and dialysis .In addition, enzymology properties ofγ-GTP was investigated by the traditional detection method .3. Immobilizedγ-GTP and catalytic condition:γ-GTP was immobilized by sodium alginate and the immobilized enzyme conditions were optimized by orthogonal test. L-theanine production was improved by adjusting substrate concentration, pH, temperature and adding enzyme accelerant. The best experiment program of biosynthesis L-theanine using immobilizationγ-GTP was investigated by orthogonal test. Results1. The induction conditions of fermentation:The optimal addition time and concentration of Gln were 2 hours and 5mmol/L, respectively. Under this condition, the maximum enzyme activity ofγ-GTP was 7.0U/ml. Enzyme activity was 2.26 times than the control group.2. Enzymology properties and crude enzyme extracts: The lysozyme was the best method of cell disruption. The optimal extracting conditions: the concentration of lysozyme was 2.5mg/g, 35℃and 20min .Enzyme extracts was purified after 80-90% saturation ammonium sulfate and dialysis. In addition, its enzymology properties was investigated. The results indicated that its optimum temperature and pH for biosynthesis L-theanine were 45℃and 8.0 respectively, Km was 0.73mmol/L. At the range of 25-35℃,γ-GTP was stable.γ-GTP was denaturalized at 50℃for 20min. The enzyme was activated by metal ions(Ba2+,Mg2+,Ca2+ ,5mmol/L ).3. Immobilizedγ-GTP and catalysis conditons:①I mmobilizedγ-GTP:The optimal immobilization conditions forγ-GTP were 3.0% sodium alginate , 0.25% acraldehyde, 6ml crude enzyme extracts, crosslinking for 2.5 hours. Under this condition, relative enzyme activity was 70.8%. The optimum temperature of immobilized enzyme was 37℃compared to 45℃of the free enzyme, declined 8℃, the optimum pH of immobilized enzyme was 9.0 compared to 10.0 of the free enzyme, increased 1.0 . Its thermal, basic resistant and acid resistant stability were improved.②Catalysis conditions: The optimum conditions of promoting biosynthesis L-theanine by orthogonal experiments were 2.0ml FDP fermentation, 3.0mmol/L Mg2+. Under this condition, the production of L-theanine was 9.5g/L, increased by 55.7% compared with the control group. The operation stabilities of immobilized enzyme was poor. After nine reactions, the production of L-theanine reduced to 31.1% compared with the first one. ConclusionIn this study, the fermentation condition has been optimized. The methods of extraction and purification ofγ-GTP from Bacillus licheniformis C12 has been established initially.γ-GTP immobilized in sodium alginate can be used to produce L-theanine. Of course ,this one research is an abecedarian only ,more work should be done to improve immobilized enzyme activity and operation stabilities.
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